RESEARCH PAPERS

INDEX
Page
Sr.No. Research Paper
No.

Hypertension Research Papers

Acute Hemodynamic Effects of Remote Ischemic Preconditioning on Coronary Perfusion

1. Pressure and Coronary Collateral Blood Flow in Coronary Heart Disease – by Taiwan Society of 001
Cardiology
Chronic remote ischemic preconditioning-induced increase of circulating hSDF-1α level and its
2. relation with reduction of blood pressure and protection endothelial function in hypertension – by 009
Journal of Human Hypertension

Remote Ischemic Preconditioning Acutely improves coronary Microcirculatory Function – by

3. 016
American Heart Association

Diabetic Research Papers

Research on Intermitted cycles of remote ischemic preconditioning augment Diabetic Foot Ulcer
4. 030
Healing – by National Institutes of Health

Research Paper on Seven-day remote ischaemic preconditioning improves endothelial function

5. 036
in patients with type 2 diabetes mellitus: a randomized pilot study – by Research Gate

Research Paper on The Effect Of Remote Ischemic Preconditioning In Diabetic Patients After
6. 046
Elective Percutaneous Coronary Intervention – by JNHA

Research Paper on Summary on Remote ischaemic conditioning in the context of type 2

7. diabetes and neuropathy: the case for repeat application as a novel therapy for lower extremity 053
ulceration – by Cardiovascular Diabetology

Endothelial Function Research Papers

Remote Ischemic Preconditioning Protects Against Endothelial Dysfunction in a Human Model

8 of Systemic Inflammation: A Randomized Clinical Trial – by Arteriosclerosis, Thrombosis, and 066
Vascular Biology

Long-term, regular remote ischemic preconditioning improves endothelial function in patients

9 077
with coronary heart disease – by Brazilian Journal of Medical & Biological Research

Seven-Day Remote Ischemic Preconditioning Improves Local and Systemic Endothelial

10. 085
Function and Microcirculation in Healthy Humans – by American Journal of Hypertension

Ischemic Preconditioning Improves Microvascular endothelial function in Remote Vasculature by

11. 093
Enhanced Prostacyclin Production – by American Heart Association
 Acute Hemodynamic Effects of Remote Ischemic
Preconditioning on Coronary Perfusion Pressure and
Coronary Collateral Blood Flow in Coronary Heart Disease

Background:

The aim of this study was to assess the acute hemodynamic effects of remote ischemic preconditioning
(RIPC) on coronary perfusion pressure and coronary collateral blood flow.

Methods:

A total of 17 patients with coronary heart disease with severe (70%-85%) stenosis in one or two vessels
confirmed by angiography were enrolled into this study. They were randomly divided into the RIPC group
(9 patients) and the control group (8 patients). Distal pressure of coronary artery stenosis before balloon
dilation (non-occlusive pressure, Pn-occl) and distal coronary artery occlusive pressure (Poccl) during
balloon dilation occlusion were measured in all patients. The patients in the RIPC group received three
cycles of limb ischemia-reperfusion preconditioning. For controls, the cuff was not inflated. After this
process, Pn-occl and Poccl were measured again in each patient.

Results:

There were no significant differences in angiographic characteristics between the two groups (all p >
0.05). Troponin I (TNI) levels after percutaneous coronary intervention (PCI) were lower in the RIPC
group than in the control group (p = 0.004). In the RIPC group, mean Pn-occl and Poccl were significantly
increased after RIPC compared to before RIPC [(72.78 10.10) mmHg vs. (79.67 9.79) mmHg, p = 0.002,
(20.89 8.61) mmHg vs. (26.78 10.73) mmHg, p = 0.001, respectively].

Conclusions:

RIPC improve distal coronary perfusion pressure and rapidly increase distal coronary occlusive
pressure thereby improving coronary collateral blood flow.

001
 Acta Cardiol Sin 2018;34:299-306
Original Article doi: 10.6515/ACS.201807_34(4).20180317A

Coronary Artery Disease

Acute Hemodynamic Effects of Remote Ischemic

Preconditioning on Coronary Perfusion Pressure
and Coronary Collateral Blood Flow in
Coronary Heart Disease
Yuansheng Xu,# Qinkai Yu,# Jianmin Yang, Fang Yuan, Yigang Zhong, Zhanlin Zhou and Ningfu Wang

Background: The aim of this study was to assess the acute hemodynamic effects of remote ischemic preconditioning
(RIPC) on coronary perfusion pressure and coronary collateral blood flow.
Methods: A total of 17 patients with coronary heart disease with severe (70%-85%) stenosis in one or two vessels
confirmed by angiography were enrolled into this study. They were randomly divided into the RIPC group (9 patients)
and the control group (8 patients). Distal pressure of coronary artery stenosis before balloon dilation (non-occlusive
pressure, Pn-occl) and distal coronary artery occlusive pressure (Poccl) during balloon dilation occlusion were measured
in all patients. The patients in the RIPC group received three cycles of lower limb ischemia-reperfusion preconditioning
(5 minutes inflation of a blood pressure cuff, followed by 5 minutes reperfusion). For controls, the cuff was not
inflated. After this process, Pn-occl and Poccl were measured again in each patient.
Results: There were no significant differences in angiographic characteristics between the two groups (all p > 0.05).
Troponin I (TNI) levels after percutaneous coronary intervention (PCI) were lower in the RIPC group than in the
control group (p = 0.004). In the RIPC group, mean Pn-occl and Poccl were significantly increased after RIPC compared
to before RIPC [(72.78 ± 10.10) mmHg vs. (79.67 ± 9.79) mmHg, p = 0.002, (20.89 ± 8.61) mmHg vs. (26.78 ± 10.73)
mmHg, p = 0.001, respectively].
Conclusions: RIPC can improve distal coronary perfusion pressure and rapidly increase distal coronary occlusive
pressure thereby improving coronary collateral blood flow.

Key Words: Collateral · Coronary heart disease · Ischemic preconditioning

INTRODUCTION described in a canine experimental model in 1986. In

Myocardial protection by preconditioning was first
Received: June 22, 2017 Accepted: March 17, 2018
Department of Cardiology, The Affiliated Hangzhou Hospital of Nanjing
Medical University, Hangzhou First People’s Hospital, Hangzhou,
310006, China.
Corresponding author: Dr. Ningfu Wang, Department of Cardiology,
The Affiliated Hangzhou Hospital of Nanjing Medical University,
Hangzhou First People’s Hospital, No. 261, Huansha Road, Hangzhou,
310006, China. Tel: 86-571-88017958; Fax: 86-571-87914773; E-mail:
ningfuwang.zcmu@hotmail.com
#
Y. Xu and Q. Yu contributed equally to this work.
that study, several brief periods of myocardial ischemia
(ischemic preconditioning) were found to substantially
reduce the extent of infarction after restoration of blood
flow.1 The most powerful intervention in animals to aug-
ment the benefits of reperfusion is remote ischemic
conditioning (RIC), which consists of cycles of brief is-
chemia and reperfusion to an organ or tissue remote
from the heart.2
Due to the reduced myocardial injury during coro-
nary angioplasty and thrombolytic therapy, RIC has de-
monstrated clinical value. Remote preconditioning has
also been recently introduced. Transient episodes of

Acta Cardiol Sin 2018;34:299-306

002
 Yuansheng Xu et al.
ischemia and reperfusion of organs or tissues far from
the heart (such as kidneys, intestine and skeletal mus-
cles) can not only reduce the subsequent long period of
ischemic injury of the organ or tissue itself, but also pro-
tect the remote heart.3 This new concept expands the
clinical application and suggests a more significant clini-
cal role, since ischemia and reperfusion could be per-
formed on limbs noninvasively. This study aimed to as-
sess the acute hemodynamic effects of remote ischemic
preconditioning (RIPC) on coronary perfusion pressure
and coronary collateral blood flow.
MATERIALS AND METHODS
Study population
Between June 2015 and October 2015, patients with
coronary heart disease confirmed by angiography were
enrolled into this study. The inclusion criteria were as
follows: one or two major vessels with stenosis of 70%-
85% confirmed by coronary angiography, with one ves-
sel with type A or type B lesion chosen as the target ves-
sel. The exclusion criteria were: acute myocardial infarc-
tion within 1 month, severe stenosis of the main stem (>
50%), target vessel lesion stenosis > 85%, distal target
vessel diffused lesion or severe three-vessel lesions, and
patients with infectious diseases, tumors, hematological
system diseases, significant congestive heart failure, se-
rious arrhythmia, uncontrolled hypertension, and dia-
betic mellitus. The eligible patients were randomly di-
vided into the RIPC group and control group. This proto-
col was approved by the Ethics Committee of our hospi-
tal before implementation. Written informed consent to
participate in this study was obtained from each partici-
pant.
Biochemical measurements
Venous blood samples were collected on the morn-
ing of admission to the hospital and again the next morn-
ing after a percutaneous coronary intervention (PCI) to
measure levels of serum troponin I (TNI). Serum levels
of alanine aminotransferase (ALT), total cholesterol (TC),
triglyceride (TGs), low-density lipoprotein cholesterol
(LDL-C), high-density lipoprotein cholesterol (HDL-C), and
creatinine were measured using a clinical automated
biochemistry analyzer (Hitachi HCP-7600, Japan).
Acta Cardiol Sin 2018;34:299-306
003
Study designand procedures
Coronary angiography and PCI data were obtained
using a digital angiography machine (INOVA 2000, GE
Company, USA), and coronary blood pressure was re-
corded using a multichannel electrophysiology recorder
(Cardiolab 4000 EP Recording System, GE Company, USA).
At the same time, aortic blood pressure and distal coro-
nary blood pressure were assessed using a coronary
guiding catheter (Cordis Company, USA) and 2F micro-
catheter (Terumo Company, Japan), respectively. Of
note, the 2F micro-catheter had its tip bent into a J
shape using steam and a 0.016-inch steerable guide
wire. In addition, a Doppler blood flow survey meter
(BIDOP ES-100V3, Hayashidenko, Japan) was used to de-
tect blood flow in the dorsalis pedis artery.
An injection of 8000U heparin was administrated in-
travenously to the patients before the PCI procedures.
Two 0.016-inch guidewires were the placed into the
same target vessel, one of which was used to transport
a 2F micro-catheter to the area distal to stenosis for cor-
onary blood pressure measurements and the other to
transport a balloon to the area proximal to stenosis for
vessel transient occlusion. Electrocardiography (ECG),
blood pressure and arterial oxygen saturation (SaO 2 )
were recorded continually and no drugs were injected
during the procedure.
For each patient, a guiding catheter was used to de-
termine the aortic blood pressure, and a 2F micro-ca-
theter was also used to determine the coronary arterial
blood pressure distal to stenosis (Pn-occl). A balloon, which
had the same diameter as the target vessel, was then
placed proximal to the coronary stenosis and dilated for
30 seconds to occlude blood flow completely. Coronary
arterial blood pressure distal to the balloon transient oc-
clusion (Poccl) was recorded.
RIPC and control interventions
Before the PCI procedure, the RIPC and control in-
terventions were completed in under 2 hours. During
this process, the patients in the RIPC group received
ischemic preconditioning induced by oppression of a
blood-pressure cuff inflated above the left thigh to to-
tally occlude blood flow in the dorsalis pedis artery (vas-
cular Doppler revealed no blood flow) for 5 minutes, fol-
lowed by 5 minutes of deflation to allow reperfusion.
This procedure was repeated 3 times. For the patients in
 Remote Ischemic Preconditioning of Coronary Arteries

the control group, they had a similar cuff placed around Table 1. Clinical and angiographic characteristics of patients in
the left thigh which was not inflated. After this proce- two groups
dure, the aortic pressure (Pao), Pn-occl and Poccl were re- RIPC Control
p
corded again for all patients. During the measurements (n = 9) (n = 8)
of Pn-occl, Poccl, and Pao, the heart rate was also recorded. Age, years 65.88 ± 4.99 65.25 ± 5.52 0.805
Male, n (%) 3 (33.3) 3 (37.5) 1.000
Statistical analysis BMI, kg/m2 23.44 ± 2.24 23.75 ± 3.33 0.825
SPSS 19.0 (SPSS Inc., Chicago, USA) was used to ana- Heart rate, beats/min 71.00 ± 8.53 70.38 ± 8.70 0.883
Blood examination
lyze data. Continuous variables were reported as mean
ALT, U/L 14.78 ± 7.01 13.50 ± 5.42 0.683
± SD, and the paired t-test was used to compare data TC, mmol/L 04.46 ± 0.89 04.30 ± 1.10 0.752
before and after preconditioning. Data from different TG, mmol/L 01.83 ± 0.40 01.92 ± 0.45 0.663
groups were compared using the two-sample t-test. Cat- LDL-C, mmol/L 02.41 ± 0.48 02.46 ± 0.65 0.855
egorical variables were analyzed using the chi-square HDL-C, mmol/L 01.18 ± 0.27 01.01 ± 0.23 0.200
test or Fisher’s exact test. A p value < 0.05 was consid- Creatinine, mmol/L 82.33 ± 7.43 82.13 ± 8.61 0.958
TNI, ng/mL 0 0 -
ered to be statistically significant.
TNI, ng/mL* 00.21 ± 0.16 00.62 ± 0.31 0.004
Medical histories
Current smokers, n (%) 1 (11.1) 1 (12.5) 1.000
RESULTS Hypertension, n (%) 6 (66.7) 6 (75.0) 1.000
Diabetic mellitus, n (%) 1 (11.1) 2 (25.0) 0.576
Patients Medication, n (%)
Aspirin 09 (100.0) 08 (100.0) 1.000
The study flowchart is shown in Figure 1. Of 40 pa-
Statins 09 (100.0) 08 (100.0) 1.000
tients with coronary heart disease confirmed by angio- ACEI/ARB 8 (88.9) 08 (100.0) 1.000
graphy, 17 patients were included (9 in the RIPC group Beta-blockers 7 (77.8) 6 (75.0) 1.000
and 8 in the control group). Affected Vessels, n (%) 0.153
Table 1 shows the clinical and angiographic charac- 1 3 (33.3) 6 (75.0)
teristics of the patients in the two groups. No adverse 2 6 (66.7) 2 (25.0)
Lesion type, n (%) 1.000
events occurred during the whole trial, and all 17 pa-
Type A 5 (55.6) 4 (50.0)
tients received PCI successfully. No significant differ- Type B 4 (44.4) 4 (50.0)
ences in age, gender, risk factors, anthropometric data, Target vessel, n (%) 0.453
laboratory data, medication use, and angiographic vari- LAD 5 (55.6) 6 (75.0)
Cx 2 (22.2) 1 (12.5)
RC 2 (22.2) 1 (12.5)
Segment, n (%) 0.743
Proximal 3 (33.3) 2 (25.0)
Mid 5 (55.6) 4 (50.0)
Distal 1 (11.1) 2 (25.0)
Reference lumen diameter, mm 02.92 ± 0.42 02.95 ± 0.37 0.889
Stenosis, % 77.89 ± 3.72 78.75 ± 3.92 0.649
Lesion length, mm 16.33 ± 3.35 15.75 ± 3.33 0.724
Data are presented as mean ± SD or n (%).
* Serum TNI levels after PCI.
ACEI, angiotensin-converting enzyme inhibitor; ALT, alanine
aminotransferase; ARB, angiotensin II receptor blocker; BMI,
body mass index; Cx, circumflex; HDL-C, high-density
lipoprotein cholesterol; LAD, left anterior descending; LDL-C,
low-density lipoprotein cholesterol; RC, right coronary; RIPC,
Figure 1. Flowchart of the study population and design. CHD, coro- remote ischemic preconditioning; TC, total cholesterol; TG,
nary heart disease; RIPC, remote ischemic preconditioning. total triglycerides; TNI, troponin I.

Acta Cardiol Sin 2018;34:299-306

004
 Yuansheng Xu et al.

ables were observed before PCI (all p > 0.05) between
the two groups. After PCI, the levels of TNI were lower
in the RIPC group than in the control group (p < 0.01).
Distal coronary non-occlusive pressure in the RIPC
and control groups
Coronary arterial blood pressure distal to the steno-
sis, termed distal coronary Pn-occl, was determined in the
two groups. There was no significant differences in
baseline pressure between the two groups (p > 0.05).
After the RIPC, the systolic blood pressure, diastolic
blood pressure and mean arterial blood pressure distal
to stenosis of the RIPC group were significantly elevated
(p < 0.05). In addition, the diastolic blood pressure and
mean arterial blood pressure distal to stenosis were sig-
nificantly decreased in the patients in the control group
(p < 0.05, Table 2). At the same time, during all mea-
surements of Pn-occl, Poccl, and Pao, there were no signifi-
cant changes in heart rate (all p > 0.05, Table 2, Table 3,
Table 4).
Distal coronary occlusive pressure in the RIPC and
control groups
Coronary arterial blood pressure distal to balloon

Table 2. Pn-occl in two groups

RIPC Control
p p
Pre-RIPC Post-RIPC Pre-Interval* Post-Interval*
cSBP, mmHg 87.67 ± 12.00 95.44 ± 13.68 0.005 92.63 ± 21.25 90.88 ± 21.26 0.082
cDBP, mmHg 65.22 ± 11.32 75.44 ± 15.15 0.021 53.63 ± 20.85 51.75 ± 19.70 0.011
cMAP, mmHg 72.78 ± 10.10 79.67 ± 9.79 0.002 66.63 ± 16.12 64.82 ± 15.69 0.000
Heart rate, bpm 72.78 ± 9.900 71.89 ± 8.33 0.839 75.13 ± 10.59 71.00 ± 10.52 0.447
Data are presented as mean ± SD.
* The patients in the control group had a similar cuff placed around the left thigh which was not inflated. The interval time was 3
cycles of lower limb ischemia-reperfusion preconditioning (30 minutes).
The baseline pressure in the two groups was not significantly different. Distal coronary non-occlusive pressure in RIPC and control
group.
cDBP, coronary arterial diastolic blood pressure; cMAP, coronary arterial mean blood pressure; cSBP, coronary arterial systolic
blood pressure; Pn-occl, distal coronary non-occlusive pressure.

Table 3. Poccl in two groups

RIPC Control
p p
Pre-RIPC Post-RIPC Pre-Interval Post-Interval
cSBP, mmHg 026.67 ± 11.05 33.56 ± 10.26 < 0.001 26.00 ± 21.41 26.50 ± 18.79 0.787
cDBP, mmHg 18.00 ± 8.22 26.11 ± 10.31 0.006 20.13 ± 18.06 22.00 ± 17.91 0.224
cMAP, mmHg 20.89 ± 8.61 26.78 ± 10.73 0.001 22.08 ± 19.14 23.50 ± 18.17 0.371
Heart rate, bpm 73.33 ± 8.80 74.89 ± 9.770 0.727 72.00 ± 8.550 77.13 ± 7.750 0.241
Data are presented as mean ± SD.
The baseline pressure in the two groups was not significantly different. Distal coronary occlusive pressure in RIPC and control group.
cDBP, coronary arterial diastolic blood pressure; cMAP, coronary arterial mean blood pressure; cSBP, coronary arterial systolic
blood pressure; Poccl, distal coronary occlusive pressure.

Table 4. Aortic pressure in two groups

RIPC Control
p
Pre-RIPC Post-RIPC Pre-Interval Post-Interval
SBP, mmHg 132.22 ± 20.73 132.22 ± 23.34 140.38 ± 32.15 137.38 ± 20.32 0.875
DBP, mmHg 071.67 ± 10.14 075.78 ± 10.49 72.50 ± 9.86 75.38 ± 8.05 0.764
MAP, mmHg 091.85 ± 12.71 094.59 ± 13.28 095.13 ± 15.08 096.04 ± 11.29 0.921
Heart rate, bpm 71.44 ± 8.59 73.00 ± 9.60 71.38 ± 6.16 072.13 ± 11.76 0.981
Data are presented as mean ± SD.

Acta Cardiol Sin 2018;34:299-306

005
 Remote Ischemic Preconditioning of Coronary Arteries
occlusion, termed Poccl, was determined in the two groups.
Similarly, there was no significant difference in baseline
pressure (p > 0.05) between the two groups. After RIPC,
the corresponding pressures were significantly elevated
(all p < 0.01), while for patients in the control group, the
differences were not significant (all p > 0.05, Table 3).
Aortic pressure in the RIPC and control groups
Table 4 shows P ao in two groups. Among the two
groups before and after the interval, there were no sig-
nificant differences in systolic blood pressure, diastolic
blood pressure and mean arterial blood pressure in Pao
(one-way analysis of variance, all p > 0.05).
DISCUSSION
In this preliminary study, we demonstrated that RIPC
could significantly improve distal coronary Pn-occl and dis-
tal coronary Poccl. Interestingly, Pn-occl in the control group
was significantly decreased. This may have been be-
cause balloon dilation for 30 seconds in the control
group (to measure Poccl before the interval) may have in-
duced myocardial ischemia, which further caused a sig-
nificant drop in blood pressure. We speculate that the
cardiac function was depressed after heart ischemia,
and that this may have been due to decreased Pn-occl.
Experimental and human studies have shown the ef-
fectiveness of transient limb ischemia to induce distant
organ protection 4-8 and TNI release was substantially
lower in the group receiving RIPC.9 Patients who receive
intermittent limb ischemia in an ambulance during trans-
fer for angioplasty have been shown to have a signifi-
cant increase in myocardial salvage as assessed by nu-
clear scintigraphy.8 The effect of either ischemic precon-
ditioning or RIPC on coronary pressures distal to steno-
sis has also been investigated recently. Lambiase et al.10
reported that exercise-induced myocardial ischemia had
an enhanced resistance to further ischemia caused by
exercise. The cardioprotection from preconditioning was
assessed by collateral blood flow, and was found to be
positively associated with collateral blood pressure. Fur-
thermore, Rentrop et al. used repeated coronary artery
occlusion to assess the contribution of ischemic and col-
lateral recruitment to the development of tolerance
against myocardial ischemia in patients with angiogra-
006
phically poor collaterals.11 This method demonstrated
significantly increased pressure-derived collateral flow
during repeated coronary occlusion.12 The underlying
mechanisms of myocardial protection by RIPC remain to
be confirmed, and the release of neurogenic and circu-
lating factors may be involved in this process.13,14 In ad-
dition, some researchers have investigated the protec-
tive effect on coronary arteries by RIPC.8,9,13,14 However,
the exact mechanisms are still unclear as to whether
RIPC has a positive impact on the recruitment of coro-
nary collateral circulation.
Improvements in distal coronary Pn-occl and Poccl, which
confirm a progressive adaptation to RIPC, stimulate the
recruitment of collateral channels in humans12 A previ-
ous study used trans-stenotic pressure to assess the
function of stenosis in a few centers15 Many of the phys-
iological phenomena underlying coronary flow regula-
tion have been studied in conscious and unconscious
animal preparations where there is great freedom in in-
strumentation and intervention.16 The pathophysiologi-
cal roles of P n-occl and P occl may explain why pressure
measurements are useful when assessing coronary flow.
In functional terms, the two major determinants of cor-
onary flow are coronary arterial pressure and myocar-
dial oxygen consumption.17 It has been reported that at
constant oxygen consumption, coronary flow is rela-
tively independent of arterial pressure, which is referred
to as coronary autoregulation.18 Of note, coronary auto-
regulation is not perfect, and it can correspond to hori-
zontal plateaus in the autoregulation range. Therefore,
coronary arterial pressure can be used as a parameter
for predicting coronary flow. Occluded coronary arteries
treated by PCI may be crossed with an over-the-wire
balloon, which may allow for measurements of Pn-occl
and Poccl. The pressure distal to the occluded segment of
the culprit coronary artery (Poccl) is generated by collat-
eral circulation from the feeding coronary artery sup-
plied by the systemic circulation.19 Thus, Poccl is similar
to the pressure at the distal end of the collateral vessels.
Intracoronary distal pressure measurements during
vessel occlusion could be used to quantitatively assess
coronary collateral circulation. Pijls et al. suggested that
the collateral flow index could be measured by the phys-
iologic derivation of collateral flow from coronary Poccl.19
Distal coronary pressure during balloon occlusion is sim-
ilar to the pressure at the distal end of the collateral
Acta Cardiol Sin 2018;34:299-306
 Yuansheng Xu et al.
vessels, and Pn-occl reflects the resistance to flow along
the epicardial vessel and can be used to assess arteriolo-
sclerosis and microcirculation dysfunction.20 Poccl pro-
vides retrograde flow that partly nourishes the ischemic
myocardium subtended by the occluded culprit artery in
short-term effects, and it may also provide supporting
evidence according to significant differences in troponin
index. Furthermore, Pn-occl-guided PCI has been shown
to improve clinical outcomes and procedural cost effec-
tiveness. A low Pn-occl may reflect a no-reflow phenome-
non,which can guide the progress of various therapies
to improve the microcirculation.21
RIPC (four cycles of 5 min of ischemia followed by 5
min of reperfusion of the arm) can protect against endo-
thelial dysfunction induced by subsequent long-lasting
ischemia in the other arm.5-8 The endothelial protection
effect of preconditioning has also been reported in other
studies.13,14,22-24 The underlying mechanism may involve
many signaling processes. This pertains to humoral fac-
tors, blood cells, neurohumoral mediators and presum-
ably a complex interplay. Hypoxic NO signaling may be
associated with the course of RIPC, and this may require
an S-nitrosation modification of mitochondrial elements
in particularly. Naturally, many previously proposed me-
chanisms will work alongside the proposed mechanism
based on NO.25 In addition, in a porcine model, RIPC, in-
duced by transient limb ischemia, could reduce coro-
nary resistance and increase coronary blood flow,26 and
this effect has been detected in healthy volunteers.22
However, another study found that although myocardial
salvage was increased in the intervention group com-
pared to the control group, a significant difference in
corrected TIMI frame count between the two groups
was not detected.8 As an important index to assess blood
flow of the main stem of coronary arteries, corrected
TIMI frame count cannot represent blood flow of the
collateral circulation. Moreover, with regards to time,
transient limb ischemia in this study was induced after
ST-elevation myocardial infarction (after myocardial is-
chemia), which was termed preconditioning. The mech-
anisms and effectiveness of coronary artery protection
by ischemic stimuli remain to be clarified in future stud-
ies.
Collateral artery growth has been considered to be
a potent natural defense mechanism to prevent death
and myocardial infarction in occlusive artery disease,
Acta Cardiol Sin 2018;34:299-306
007
and the stimulation of collateral circulation has been ap-
plied to clinical applications.26-28 Coronary collateral cir-
culation plays an important role in patients who lost in-
dications for PCI and surgery.29-31 We first showed that
the RIPC induced by lower limb ischemia and reperfu-
sion had an acute effect to elevate Poccl, which included
systolic blood pressure, diastolic blood pressure and
mean arterial blood pressure in the patients with coro-
nary artery disease. Meanwhile, Pao was not affected by
RIPC. RIPC may become a new topic in the treatment of
coronary artery disease since stimulation of collaterals
has generated such interest.29
This study also showed that RIPC could increase dis-
tal coronary perfusion pressure (Pn-occl). Reduced coro-
nary resistance and increased coronary blood flow by
RIPC has been reported previously,13,22-29 and these find-
ings may explain the increased distal coronary perfusion
pressure in this study. Based on our preliminary data,
RIPC appeared to have a protective effect on coronary
perfusion pressure and coronary collateral circulation.
Whether these effects are associated with the final myo-
cardial effects remains unclear. 29,30 Timely treatment
with thrombolytic drugs for acute coronary syndrome
has been developed based on the observation that early
reperfusion is crucial to reduce damage. The evidence
of coronary collateral circulation responsiveness to RIPC
suggests that, despite severe stenosis, coronary vaso-
active phenomena related to preconditioning are pre-
served. Some studies have evaluated the role of RIC in
reducing ischemia/reperfusion injury. Indeed, our re-
sults are similar to those reported by Lansky et al., 32
who only managed to show a significant short-term ef-
fect on post-PCI troponin release. In addition, Wang et
al.33 recently showed that RIPC reduced the incidence of
MI type 4a in patients undergoing elective PCI, but failed
to show a significant effect on CRP level or subsequent
cardiovascular events. The potential clinical use of RIPC
seems to be positive. High-quality random clinical trials
with a larger sample size are needed to verify the clini-
cal usefulness of RIPC.
Study limitations
Since this study was a small sample trial, the results
and conclusions may be limited. In addition, pressure
wire, which is much thinner than the 2F micro-catheter,
was not used to measure intracoronary pressures. The
 Remote Ischemic Preconditioning of Coronary Arteries

size of a 2F micro-catheter could partly obstruct the ves-

sel and underestimate the true intracoronary pressure.
Furthermore, we observed a decrease in Pn-occl in the
controls. This phenomenon maybe explained by signifi-
cant heart ischemia induced by prolonged balloon dila-
tion. The underlying mechanism should be explored in
greater detail in future studies.

CONCLUSIONS

In summary, this study showed that RIPC induced by

an inflated blood pressure cuff improves distal cor-
onary perfusion pressure and rapidly increase distal cor-
onary occlusive pressure thereby improving
coronary collateral blood flow. Distal coronary
perfusion pressure and coronary collateral flow were
found to be effective in the application of RIPC in
humans.

CONFLICTS OF INTEREST

The authors have declared that no conflicts of inter-

est exist.

Acta Cardiol Sin 2018;34:299-306

008
 Chronic remote ischemic preconditioning-induced increase of circulating
hSDF-1α level and its relation with reduction of blood pressure and protection
endothelial function in hypertension

Background:
Although previous data showed that remote ischemic preconditioning (RIPC) has beneficial
effect on blood pressure (BP) reduction, the efficacy of RIPC-induced decline in BP and the
favorable humoral factors in hypertension is elusive.

Aim:
This present study is performed to evaluate whether RIPC reduces BP, improves
microvascular endothelial function and increases circulating hSDF-1α generation in
hypertension.

Method:
Fifteen hypertensive patients received 3 periods of 5-min RIPC on the upper arm daily for 30
days.

Assessment:

● Clinic and 24-h ambulatory blood pressure monitoring (ABPM) were examined before
and after the end of this procedure.

● Microvascular endothelial function was measured by finger reactive hyperemia index

(RHI) using the Endo-PAT 2000 device.

● The circulating hSDF-1α level was tested by ELISA.

Results:

● RIPC significantly decreased systolic BP (139.13 ± 6.68 versus 131.45 ± 7.45 mmHg)
and diastolic BP (89.67 ± 4.98 versus 83.83 ± 6.65 mmHg), meanwhile 24-h
ambulatory systolic and diastolic BP dropped from 136.33 ± 9.10 mmHg to
131.33 ± 7.12 mmHg and 87.60 ± 6.22 mmHg to 82.47 ± 4.47 mmHg respectively.

● RHI was improved (1.95 ± 0.34 versus 2.47 ± 0.44). Plasma hSDF-1α level was
markedly increased after RIPC (1585.86 ± 167.17 versus 1719.54 ± 211.17 pg/ml).

● The increase in hSDF-1α level was associated with the fall in clinic and 24-h ABPM
and rise in RHI.

Conclusion:
The present data suggests that RIPC is a novel alternative or complementary intervention
means to treat hypertension and protect endothelial function.

009
 Journal of Human Hypertension
https://doi.org/10.1038/s41371-018-0151-1

ARTICLE

Chronic remote ischemic preconditioning-induced increase of

circulating hSDF-1α level and its relation with reduction of blood
pressure and protection endothelial function in hypertension
Xin-zhu Tong1 Wan-fu Cui1 Yan Li1 Chen Su1 Yi-jia Shao1 Jia-wen Liang1 Zi-ting Zhou1 Chan-juan Zhang1
● ● ● ● ● ● ● ●

Jian-ning Zhang1 Xiao-yu Zhang1 Wen-hao Xia1 Jun Tao1

● ● ●

Received: 18 September 2018 / Revised: 16 November 2018 / Accepted: 3 December 2018

© Springer Nature Limited 2019

Abstract
Although previous data showed that remote ischemic preconditioning (RIPC) has beneficial effect on blood pressure (BP)
reduction, the efficacy of RIPC-induced decline in BP and the favorable humoral factors in hypertension is elusive. This
present study is performed to evaluate whether RIPC reduces BP, improves microvascular endothelial function and increases
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circulating hSDF-1α generation in hypertension. Fifteen hypertensive patients received 3 periods of 5-min inflation/deflation
of the forearm with a cuff on the upper arm daily for 30 days. Clinic and 24-h ambulatory blood pressure monitoring
(ABPM) were examined before and after the end of this procedure. Microvascular endothelial function was measured by
finger reactive hyperemia index (RHI) using the Endo-PAT 2000 device. The circulating hSDF-1α level was tested by
ELISA. RIPC significantly decreased systolic BP (139.13 ± 6.68 versus 131.45 ± 7.45 mmHg) and diastolic BP (89.67 ±
4.98 versus 83.83 ± 6.65 mmHg), meanwhile 24-h ambulatory systolic and diastolic BP dropped from 136.33 ± 9.10 mmHg
to 131.33 ± 7.12 mmHg and 87.60 ± 6.22 mmHg to 82.47 ± 4.47 mmHg respectively. RHI was improved (1.95 ± 0.34 versus
2.47 ± 0.44). Plasma hSDF-1α level was markedly increased after RIPC (1585.86 ± 167.17 versus 1719.54 ± 211.17 pg/ml).
The increase in hSDF-1α level was associated with the fall in clinic and 24-h ABPM and rise in RHI. The present data
suggests that RIPC is a novel alternative or complementary intervention means to treat hypertension and protect endothelial
function.

Introduction clinic significance aimed at lowering high incidence of

Hypertension is one of the most common risk factors for the
pathogenesis of atherosclerotic cardiovascular diseases
(ASCVD) and become public health problem all over the
world [1]. Despite antihypertensive medication therapy
achieves great advances, searching for innovative and cost-
less intervention means to treat hypertension has important
These authors contributed equally: Xin-Zhu Tong, Wan-fu Cui
Supplementary information The online version of this article (https://
doi.org/10.1038/s41371-018-0151-1) contains supplementary
material, which is available to authorized users.
* Jun Tao
taojungz123@163.com
1 for these secreted materials still needed to be elucidated.
Department of Hypertension and Vascular Disease, The First
Affiliated Hospital, Sun Yat-Sen University, Guangzhou 510080,
China
hypertension-associated ASCVD caused by blood pressure
(BP) elevation.
Remote ischemic preconditioning (RIPC) is a non-
pharmacological strategy inducing transient episodes of
ischemia by the occlusion of blood flow in non-target tissue
before a subsequent ischemia-reperfusion injury occurs in a
more distant organ [2]. Previous reports showed that RIPC
has a blood pressure lowing effect and improves endothelial
function in persons [3–7]. Despite the encouraging data of
RIPC in some human observations [6, 8–10], there are
growing appeals for carrying out the further clinic investi-
gation to prove the efficacy of RIPC on the reduction of BP
in patients with hypertension. Furthermore, whether a
RIPC-mediated release of humoral factors might play a
crucial role in the protection of human microvascular
endothelial function and which are the potential new target
It has been reported that patients with hypertension are
associated with abnormalities in endothelial structure and

010

Fig. 1 Protocol of the study. Every RIPC session consisted of 3 per-
iods of 5-min inflation/deflation of a blood pressure cuff. Clinic BP,
ABPM, RHI and plasma were performed before RIPC intervention
(T0; prior to 1 day of the first RIPC), and immediately after 1 month
function [7]. Our previous studies [11–13] have demonstrated
that endothelial progenitor cells (EPCs) is closely linked with
endothelial dysfunction in patients with hypertension. Stromal
cell-derived factor-1 alpha (SDF-1α), as a critical regulator for
normal development of the cardiovascular systems [14], has
been shown to play a key role in promoting EPCs homing and
improving endothelial repair capacity [12, 13]. Although a
50% plasma increment of SDF-1α level was detected in rats
subjected to a RIPC protocol compared to control [15].
However, there is no any study to demonstrate this is the case
also in hypertensive patients.
To answer these questions, hypertensive patients were
subjected to RIPC therapy daily for 1 month. We evaluated
the alterations in BP by clinic and 24-h ambulatory blood
pressure monitoring (ABPM), finger reactive hyperemia for
the microvascular endothelial function and circulating humoral
SDF-1α levels. This present study may provide a novel evi-
dence for the clinical application of RIPC intervention and its
potential novel targets in the management of hypertension.
Materials and methods
Patients
This is a pilot study with a small sample size, self-control,
single-blind (outcomes assessors were unaware of the
011
Xin-zhu Tong et al.
(T1; the next day after the 30-day RIPC). BP blood pressure, ABPM
ambulatory blood pressure monitoring, RHI reactive hyperemia index,
hSDF-1α human stromal cell-derived factor-1 alpha, RIPC remote
ischemic preconditioning, H hypoxia, d day
treatment assignments). Fifteen newly diagnosed, never-
treated, office systolic/diastolic BP ≥ 130/80 mmHg primary
hypertensives were enrolled into this study. Of course, all
the patients did not undergo simultaneous medical treat-
ment. Major exclusion criteria included age ≤ 18 years,
presence or past history of coronary artery disease, heart
valve disease, symptomatic valvular disease, heart failure,
diabetes, peripheral artery disease, active inflammatory
disease, malignancy, and other serious medical conditions
(acute liver, renal failure and other diseases).
All subjects enrolled gave written informed consent prior
to study participation. Approved by the ethical committee of
the First Affiliated Hospital, Sun Yat-Sen University
(Guangzhou, China), the consent procedure has been per-
formed in accordance with the 1964 Declaration of Helsinki
and its later amendments.
Study protocol
After all subjects were enrolled, RIPC was performed as 3
cycles of 5 min ischemia alternating with 5 min reperfusion
intervals on the upper-arm per day, lasting for 1 month.
Blood pressure underwent strict measurements by clinic and
24-h ABPM before (T0; prior to 1 day of the first RIPC) and
after (T1; the next day after the 30-day RIPC) the long-term
RIPC procedure. Also, RHI and blood samples were col-
lected in the same time points. The plasma was centrifuged
Chronic remote ischemic preconditioning-induced increase of circulating hSDF-1α level and its relation. . .

Table 1 Characteristics of the hypertensives before and after long-term (10:00 pm to 6:59 am). The ABPM readings were kept
RIPC therapy
working the whole day, and the subjects were instructed to
Parameters Before After P value engage in ordinary activities and avoid intense exercise. Valid
device readings must meet a series of pre-set quality control
N 15 – –
criteria, including number of recordings ≥40 times and
Age (years) 44.33 ± 8.99 – –
effective recordings ≥80% of BP during the day and night.
Female 33% – –
BMI (kg/m2) 25.07 ± 2.42 – –
Reactive hyperemia index (RHI)
HR (bpm) 71.27 ± 6.20 69.87 ± 6.09 0.314
Clinic systolic blood 139.13 ± 6.68 131.45 ± 7.45 0.002*
Endothelial function was measured by RHI, non-invasive
pressure (mmHg)
peripheral artxerial tonometry, using the Endo-PAT 2000
Clinic diastolic blood 89.67 ± 4.98 83.83 ± 6.65 0.004*
pressure (mmHg) device (Itamar Medical Inc., Caesarea, Israel) which is
24-h systolic blood 136.33 ± 9.10 131.33 ± 7.12 0.019* cleared by Food and Drug Administration. The technique
pressure (mmHg) detects digital flow-mediated dilation from both fingers
24-h diastolic blood 87.60 ± 6.22 82.47 ± 4.47 0.002* (control and occluded side) to assess endothelial responding
pressure (mmHg) vasodilatation during reactive hyperemia. RHI measure-
Daytime systolic blood 138.33 ± 9.40 132.33 ± 7.98 0.012* ments were performed before and after 1month-RIPC
pressure (mmHg) treatment in the morning.
Daytime diastolic blood 88.60 ± 7.15 83.07 ± 5.47 0.002*
pressure (mmHg)
Measurement of human stromal cell-derived factor-
Nighttime systolic blood 128.47 ± 8.42 125.40 ± 8.63 0.096 1 alpha (SDF-1α)
pressure (mmHg)
Nighttime diastolic blood 79.53 ± 7.99 78.67 ± 7.27 0.632
pressure (mmHg) The concentrations of human SDF-1α in RIPC-plasma were
Uric acid (µmol/L) 403.13 ± 99.93 381.60 ± 91.36 0.274
determined using specific ELISA kit (Sigma-Aldrich, MO,
Fasting plasma glucose 4.90 ± 0.40 5.11 ± 0.48 0.132
USA). Each blood samples could be tested at least 2 re-
(mmol/L) holes. Absorbance was measured at 450 nm using a
Total cholesterol (mmol/ 4.78 ± 0.91 4.64 ± 1.14 0.626 microplate reader (Tecan, Crailsheim, Germany) and SDF-
L) 1α concentrations were calculated from the standard curve.
Triglyceride (mmol/L) 1.69 ± 0.76 1.46 ± 0.68 0.251
HDL-C (mmol/L) 1.11 ± 0.19 1.09 ± 0.19 0.399 Statistical analysis
LDL-C (mmol/L) 3.08 ± 0.80 3.07 ± 0.90 0.969
RHI 1.95 ± 0.34 2.47 ± 0.44 0.006* Statistics were using the SPSS statistical software (SPSS
version 22.0). All the normally distributed continuous
Data are shown as mean ± SD
variables were expressed as mean value ± SD, categorical
RIPC remote ischemic preconditioning, BMI body mass index, HR
heart rate, HDL-C high-density lipoprotein cholesterol, LDL-C low-
data as percentages. The normality of distribution for con-
density lipoprotein cholesterol, RHI reactive hyperemia index tinuous variables were determined via the Shapiro–Wilk
*P < 0.05 vs. before long-term RIPC therapy test. Comparison of continuous variables was analyzed
using the Paired t-Test. Categorical data were analyzed
from blood samples at 4 ℃, 10,000 × g for 10 min, and using the Chi-square test. In addition, Spearman’s rank
stored at −80 ℃. The concentrations of hSDF-1α examined correlation was conducted to identify any potential rela-
in plasma T0 and T1 (Fig. 1). tionship between ΔBP or ΔRHI and ΔhSDF-1α. In the
analyses, a value of P < 0.05 was considered significant.
Blood pressure

According to standardized recommended procedures, clinic Results

BP was measured with calibrated blood pressure monitor
(HEM-7071, Omron, Dalian, China) after resting at least 15
min. Clinic BP values were calculated as the average of 3
readings on the right upper arm at least 1 min interval. 24-h
ambulatory blood pressure was performed with the ABPM
(Oscar2, SunTech Medical, North Carolina, U.S) pro-
grammed to register BP at 30-min intervals during
the daytime (7:00 am to 9:59 pm) and every 1-h at night
Decreased clinic BP and 24-h ambulatory BP and
increased RHI after RIPC therapy in hypertensive
patients
As listed in Table 1, the baseline characteristics of the
hypertensives were described before and after long-term
RIPC therapy. Clinic blood pressure and 24-h ambulatory

012

Fig. 2 Effects of long-term RIPC treatment on clinic blood pres-
sure, 24-h ambulatory blood pressure and vascular endothelial func-
tion. a–b Clinic systolic and diastolic blood pressure showed a decline
after the long-term RIPC treatment that were statistically significant (P
< 0.05). c–d 24-h ambulatory blood pressure was obviously reduced
compared with the baseline (P < 0.05). e–f RHI was significantly
enhanced after the long-term RIPC (P < 0.05). Data are the mean ± SD.
SBP systol blood pressure, DBP diastolic blood pressure
blood pressure were significantly decreased after RIPC
(clinic SBP, 139.13 ± 6.68 versus 131.45 ± 7.45 mmHg,
P = 0.002; clinic DBP, 89.67 ± 4.98 versus 83.83 ± 6.65
mmHg, P = 0.004; 24-h SBP, 136.33 ± 9.10 versus
131.33 ± 7.12 mmHg, P = 0.019; 24-h DBP, 87.60 ± 6.22
versus 82.47 ± 4.47 mmHg, P = 0.002; Fig. 2a-d).
whereas endothelial function marker RHI was restored by
the long-term RIPC therapy (1.95 ± 0.34 versus 2.47 ±
0.44, P = 0.006; Fig. 2e, f). Furthermore, the directions
of trends of daytime was the same as those of 24-h BP
(daytime SBP, 138.33 ± 9.40 versus 132.33 ± 7.98
mmHg, P = 0.012; daytime DBP, 89.67 ± 4.98 versus
83.83 ± 6.65 mmHg, P = 0.002; Fig. S1a-b). The night-
time BP showed a decreased trend but the change was not
statistically significant (nighttime SBP, 128.47 ± 8.42 vs
125.40 ± 8.63 mmHg, P = 0.096; nighttime DBP,
79.53 ± 7.99 vs 78.67 ± 7.27 mmHg, P = 0.632; Fig. S1c-
d). This reason for above result might be due to small
sample size bias or other factors, such as less sleeping
time, poor sleeping quality, and so on. According to the
above-mentioned situation, we regarded 24 h overall
ABPM as the main evaluation index.
Increased plasma hSDF-1α levels after RIPC therapy
in hypertensive patients
Plasma hSDF-1α varied between different subjects (minimum
hSDF-1α: 1222.63 pg/ml, maximum hSDF-1α: 2172.34
pg/ml; Fig. 3a). Mean hSDF-1α levels were significantly
higher in protective RIPC-plasma T1 (1585.86 ± 167.17 pg/ml
versus 1719.54 ± 211.17 pg/ml, P = 0.011, Fig. 3b).
The increasing in hSDF-1α level was associated with
fall in clinic and 24-h ABPM and rise in RHI
Correlation analysis was performed between change in
hSDF-1α and changes in clinic BP, 24-h ABPM and
013
Xin-zhu Tong et al.
vascular endothelial function before and after the long-term
RIPC treatment. There was an inverse association between
change in hSDF-1α levels and changes in clinic SBP, clinic
DBP, 24-h SBP and 24-h DBP levels after long-term RIPC
treatment, respectively (clinic SBP: r = −0.546, P = 0.035,
Fig. 4a; clinic DBP: r = −0.537, P = 0.038, Fig. 4b; 24-h
SBP: r = −0.590, P = 0.021, Fig. 4c; 24-h DBP: r =
−0.602, P = 0.018; Fig. 4d). As shown in Fig. 4e, the
enhance in hSDF-1α levels was linearly correlated with
increase in RHI (r = 0.517, P = 0.049).
Discussion
Our present study demonstrated that RIPC significantly
reduces blood pressure and improves micro-vessel endo-
thelial function in patients with hypertension. Furthermore,
we firstly found that RIPC markedly enhances circulating
hSDF-1α levels of hypertensive patients. And the increase
in hSDF-1α level was associated with the fall in clinic and
24-h ABPM and rise in RHI. The present data suggests that
RIPC may be a novel alternative or complementary inter-
vention means to treat hypertension and protect endothelial
function.
RIPC has been regarded as a protective intervention that
could be conducted in a cheap, non-invasive procedure
since its inception in 1997 [16]. To date, numerous data
from clinical trials unveiled cardioprotective effect on dif-
ferent ASCVD, including chronic heart failure, myocardial
infarction and acute kidney injury [6, 8, 10]. These studies
mainly focused on protective effect upon ischemia-
reperfusion injury, indicating that repeated RIPC may
represent a potent, systemic stimulus for vascular adaptation
and protection.
Hypertension is a major risk factor for pathogenesis of
ASCVD in the world, posing a significant challenge in
prevention and treatment of hypertension. Despite numer-
ous pharmacological advances in the management of
patients with hypertension, a critical need exists for novel
effective and low-cost means that can reduce the BP levels,
which is the key target for the development of hypertension-
related ASCVD. Previous studies showed that serial ses-
sions of RIPC had a statistically significant blood pressure
lowing effect on a normotensive or prehypertensive subject
merely once and twice a day within a week, respectively
[4, 17]. Another study, which had enrolled in 21 patients
with both heart failure and hypertension and treated them
with RIPC once a day for a month, has the similar result as
the study we mentioned above [8]. These studies indicated
that RIPC may have a blood pressure lowing effect. How-
ever, from the point of view of clinical trial, the data and
efficacy of RIPC on patients with hypertension is somewhat
still insufficient. In this present study, we found that
Chronic remote ischemic preconditioning-induced increase of circulating hSDF-1α level and its relation. . .
Fig. 3 Quantification of hSDF-1α concentrations in human RIPC-
plasma. a hSDF-1α concentrations in RIPC-plasma before and after
long-term RIPC treatment from the various hypertensives (sample
1 month of daily RIPC leads to a significant fall in SBP and
DBP. To the best of our knowledge, the current investiga-
tion is the first time to demonstrate the impact of long-term
RIPC on reduction of BP in patients with hypertension by
using both clinic BP measurement and ABPM and
improvement of micro vessel endothelial function, which
may display a synergistic salutary effect to treat
hypertension.
It’s generally accepted that endothelial dysfunction
plays a key role in pathogenesis of ASCVD, and it is the
hallmark of hypertensive vascular injuries [18]. RHI is the
novel parameter of the clinical evaluation for microvascular
endothelial dysfunction [15]. One study provides an evi-
dence to show that RIPC improves microvascular endo-
thelial function in healthy volunteers [19]. However, there
is no any study to investigate the impact of RIPC on
microvascular endothelial function in patients with hyper-
tension. In our studies, we found that chronic RIPC
improves microvascular endothelial function. To the best of
our knowledge, the current study is the first time to
demonstrate that chronic RIPC can increase RHI value,
indicating that improvement of microvascular endothelial
function together with reduction in BP contributes to a
synergetic salutary effect in management of patients with
hypertension.
Notably, it has long been curious how RIPC provide a
vascular endothelial protection. Several possibilities have
been to propose to explain this phenomenon: (1) RIPC
release humoral factors into the systemic circulation to
reach the remote target organs; and (2) RIPC stimulus
induce a systemic anti-inflammatory and anti-apoptotic
014
1–15). b Recovery of hSDF-1α concentrations in RIPC-plasma after
1month therapy was improved in parallel with the baseline (P < 0.05).
Data display the mean ± SD
response; (3) the RIPC-protections were conferred by neu-
ronal pathways. Recently, research interest is focused on the
release of protective humoral factors. Such as SDF-1α [20],
nitrite [21], exosomes [22], or Interleukin [23]. However,
precious identification of the endogenous materials has been
suspicious in clinic. hSDF-1α seems to be the most con-
tributed one candidate according to the previous report.
SDF-1 as a member of the CXC group of chemokines is
mainly expressed in bone-marrow-derived stromal cells. It
is an important factor in physiological and pathological
processes, including hematopoiesis, angiogenesis, and
inflammation, because it activates and/or induces migration
of hematopoietic progenitor and stem cells, endothelial cells
and most leukocytes [14]. SDF-1 is expressed in the vas-
culature, and also involved in the stress-induced recruitment
of EPCs to the injury zone [24]. Our previous data and other
studies have demonstrated that SDF-1 as a ligand can serve
the adhesion and migration activities of EPCs in this pro-
cess by binding its receptors, CXCR4/CXCR7, which play
an important role to reflect the endothelial repair function
after vascular injury [12, 13, 25]. In an animal study, a 50%
plasma increment of the SDF-1α level was also detected in
rats subjected to a RIPC protocol compared to control [26].
Based on these data, SDF-1 could be regarded a potential
humoral factor of RIPC-induced endothelial protection.
The data reported here for the first time that RIPC is able
to significantly increase hSDF-1α level in hypertension.
Furthermore, there is a close association between the hSDF-
1α levels and RHI, indicating that endothelial function
improvement is, at least in part, related to upregulation of
circulating hSDF-1α concentration. The present data, taken

Fig. 4 The increasing in hSDF-1α level was associated with fall in
clinic and 24-h ABPM and rise in RHI. Δclinic SBP, Δclinic DBP,
Δ24-h SBP, Δ24-h DBP, ΔRHI and ΔhSDF-1α represent the values of
together with previous study [8, 26], suggest that RIPC-
stimulated hSDF-1α release contribute to amelioration of
endothelial function in patients with hypertension. On the
basis of the present study, hSDF-1α may be a surrogate
parameter for the clinical evaluation on the validation of
endothelial function improvement by using long-term RIPC
treatment in hypertension.
The present study has an important clinical implication.
Nowadays, efficacy of BP lowering and organ-targeted
protection are the two key aspects for the clinical evaluation
of a novel anti-hypertensive measure. The data reported
here showed that RIPC reach to these two requirements.
More importantly, RIPC intervention is costless, non-
pharmacological and safe in hypertension management.
Therefore, RIPC should be worthy of widely application in
clinical practice in patients with hypertension.
There are some limitations in the present study. First, this
is a single center study and sample size is small. In the
future, multicenter, double-blind and clinical trial and more
hypertensive patients should be required to further prove the
current data. Second, this protocol is lack of a control group
or the cross-over design. It could be better carried out by
taking conditional control group into consideration in the
next clinical trials, which is now under way in our labora-
tory. Finally, we found a correlation between hSDF-1α and
lower blood pressure, however, the potential mechanisms of
decline in RIPC-mediated BP in hypertension is largely
unclear and beyond the present study, which deserves fur-
ther study in the future.
In summary, our present study for the first time in
clinical practice identified that hSDF-1α is a protective
humoral factor induced by RIPC intervention in
hypertension, which is parallel to the decline in BP and
the improvement of microvascular endothelial
function. Our findings suggest that RIPC is a novel
costless and effective intervention means to m anage
patients with hypertension.
015
Xin-zhu Tong et al.
changes in clinic SBP, clinic DBP, 24-h SBP, 24-h DBP, RHI and
hSDF-1α of each individual after the 1-month treatment, respectively
Summary table
What is known about this topic
● Endothelial dysfunction plays a key role in the
pathogenesis of hypertension-related ASCVD.
● RIPC improves microvascular endothelial function in
healthy volunteers.
● SDF-1α is a novel protective humoral factor of
hypertensive vascular injury. The increment of SDF-
1α level was detected in rats subjected to a RIPC
protocol.
What this study adds
• RIPC markedly enhances circulating hSDF-1α levels of
hypertensive patients.
• Increase in hSDF-1α level was associated with the fall in
clinic and 24-h ABPM and rise in RHI in patients with
hypertension.
• RIPC is a novel alternative or complementary
intervention means to treat hypertension and protect
endothelial function. And hSDF-1α is a surrogate
parameter for the clinical evaluation on the validation of
endothelial function improvement by using long-term
RIPC treatment in hypertension.
Acknowledgements The work was supported financially by the
National Nature Science Foundation of China (No. 31530023) and
Science and Technology Plan Collaborative Innovation Key Projects
of Guangzhou (No. 201704020212).
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
 Remote Ischemic Preconditioning Acutely improves coronary
Microcirculatory Function

Background

Remote ischemic preconditioning (RIPC) attenuates myocardial damage during elective and primary
percutaneous coronary intervention. Recent studies suggest that coronary microcirculatory function is an
important determinant of clinical outcome.

Aim:

The aim of this study was to assess the effect of RIPC on markers of microcirculatory function.

Methods:

Patients referred for cardiac catheterization and fractional flow reserve measurement were randomized
to RIPC or sham. Operators and patients were blinded to treatment allocation.

Assessment:

Comprehensive physiological assessments were performed before and after RIPC/sham including the
index of microcirculatory resistance and coronary flow reserve after intracoronary glyceryl trinitrate and
during the infusion of intravenous adenosine. Thirty patients were included (87% male; mean age:
63.110.0 years). RIPC and sham groups were similar with respect to baseline characteristics.

Results:

RIPC decreased the calculated index of microcirculatory resistance (median, before RIPC: 22.6
[interquartile range [IQR]: 17.9–25.6]; after RIPC: 17.5 [IQR: 14.5–21.3]; P=0.007) and increased coronary
flow reserve (2.60.9 versus 3.81.7, P=0.001). These RIPC-mediated changes were associated with a
reduction in hyperemic transit time (median: 0.33 [IQR: 0.26–0.40] versus 0.25 [IQR: 0.20–0.30]; P=0.010).
RIPC resulted in a significant decrease in the calculated index of microcirculatory resistance compared
with sham (relative change with treatment [meanSD] was 18.124.8% versus +6.137.5; P=0.047) and a
significant increase in coronary flow reserve (+41.2% [IQR: 20.0–61.7] versus 7.8% [IQR: 19.1 to 10.3];
P<0.001).

Conclusion:

The index of microcirculatory resistance and coronary flow reserve are acutely improved by remote
ischemic preconditioning. This confirms that RIPC confers cardio protection during percutaneous coronary
intervention as a result of an improvement in coronary microcirculatory function.

016
 ORIGINAL RESEARCH

Remote Ischemic Preconditioning Acutely Improves Coronary

Microcirculatory Function
Jerrett K. Lau MBBS; Probal Roy, MBBS; Ashkan Javadzadegan, PhD; Abouzar Moshfegh, PhD; William F. Fearon, MD; Martin Ng, PhD;
Harry Lowe, PhD; David Brieger, PhD; Leonard Kritharides, PhD; Andy S. Yong, PhD

Background-—Remote ischemic preconditioning (RIPC) attenuates myocardial damage during elective and primary percutaneous
coronary intervention. Recent studies suggest that coronary microcirculatory function is an important determinant of clinical
outcome. The aim of this study was to assess the effect of RIPC on markers of microcirculatory function.
Methods and Results-—Patients referred for cardiac catheterization and fractional flow reserve measurement were randomized to
RIPC or sham. Operators and patients were blinded to treatment allocation. Comprehensive physiological assessments were
performed before and after RIPC/sham including the index of microcirculatory resistance and coronary flow reserve after
intracoronary glyceryl trinitrate and during the infusion of intravenous adenosine. Thirty patients were included (87% male; mean
age: 63.110.0 years). RIPC and sham groups were similar with respect to baseline characteristics. RIPC decreased the calculated
index of microcirculatory resistance (median, before RIPC: 22.6 [interquartile range [IQR]: 17.9–25.6]; after RIPC: 17.5 [IQR: 14.5–
21.3]; P=0.007) and increased coronary flow reserve (2.60.9 versus 3.81.7, P=0.001). These RIPC-mediated changes were
associated with a reduction in hyperemic transit time (median: 0.33 [IQR: 0.26–0.40] versus 0.25 [IQR: 0.20–0.30]; P=0.010). RIPC
resulted in a significant decrease in the calculated index of microcirculatory resistance compared with sham (relative change with
treatment [meanSD] was 18.124.8% versus +6.137.5; P=0.047) and a significant increase in coronary flow reserve (+41.2%
[IQR: 20.0–61.7] versus 7.8% [IQR: 19.1 to 10.3]; P<0.001).
Conclusions-—The index of microcirculatory resistance and coronary flow reserve are acutely improved by remote ischemic
preconditioning. This confirms that RIPC confers cardioprotection during percutaneous coronary intervention as a result of an
improvement in coronary microcirculatory function.
Clinical Trial Registration-—URL: www.anzctr.org.au/. Unique identifier: CTRN12616000486426. ( J Am Heart Assoc. 2018;7:
Downloaded from http://ahajournals.org by on April 18, 2024

e009058. DOI: 10.1161/JAHA.118.009058.)

Key Words: coronary flow reserve • coronary physiology • microcirculation • microcirculatory resistance • remote ischemic
preconditioning

I n remote ischemic preconditioning (RIPC), brief nonharmful

ischemia to a remote organ can protect the heart against
ischemia reperfusion injury.1,2 RIPC has been used before
elective and primary percutaneous coronary intervention (PCI),
resulting in reduced post-PCI troponin levels and reduced
myocardial infarct size.3–6 In addition, patients who receive RIPC
before elective and primary PCI have been found to have reduced
clinical events during long-term follow-up.7,8 However, large
multicenter randomized trials in the setting of cardiac surgery
have demonstrated no benefit of RIPC.9,10 These discrepant
results demonstrate a context-specific benefit of RIPC.11
The human coronary microcirculation is recognized as an
important determinant of patient prognosis. The coronary flow
reserve (CFR) and the index of microcirculatory resistance
(IMR), a pressure–temperature sensor wire–derived index,12
have been shown to predict outcome in patients with

From the Concord Repatriation General Hospital (J.K.L., P.R., H.L., D.B., L.K., A.S.Y.), ANZAC Research Institute (J.K.L., A.J., A.M., D.B., L.K., A.S.Y.), and Department of
Cardiology, Royal Prince Alfred Hospital (M.N.), University of Sydney, Australia; Faculty of Medicine and Health Sciences, Macquarie University, Sydney, Australia (A.J.,
A.M., A.S.Y.); Division of Cardiovascular Medicine, Stanford University School of Medicine, Stanford, CA (W.F.F.).
Accompanying Figures S1 and S2 are available at https://www.ahajournals.org/doi/suppl/10.1161/JAHA.118.009058
Correspondence to: Andy S. Yong, PhD, Department of Cardiology, Concord Hospital, Hospital Road, Concord, New South Wales 2139, Australia. E-mail:
andy.yong@sydney.edu.au
Received March 4, 2018; accepted August 20, 2018.
ª 2018 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley. This is an open access article under the terms of the Creative Commons
Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-
commercial and no modifications or adaptations are made.

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 Remote Preconditioning and Microcirculation Lau et al
Clinical Perspective
What Is New?
• Remote ischemic preconditioning causes an acute improve-
ment in coronary microcirculatory function, which is an
important determinant of prognosis during elective and
primary percutaneous coronary intervention.
What Are the Clinical Implications?
• An improvement in microcirculatory function may help
explain the benefit of remote ischemic preconditioning at
the time of coronary stenting and raises the possibility that
this treatment may be used to augment the microcirculation
in other clinical settings.
epicardial coronary artery disease (CAD), including those
undergoing primary and elective PCI.13–18 The combination of
high IMR and low CFR confers a substantially increased risk of
major cardiovascular events independent of the severity of
epicardial coronary stenosis.13,16–19 In addition, the coronary
microcirculation has been proposed as a target of RIPC-
mediated cardiac protection.20
Clarifying the mechanism by which RIPC may exert a
protective effect on the heart in the setting of PCI could help
guide future studies and clinical protocols by identifying
patient populations most likely to benefit. Studies demon-
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strating that RIPC protects against ischemia reperfusion
injury–associated forearm vascular endothelial dysfunction21
and increases CFR, as assessed by indirect echocardiographic
evaluation of the left anterior descending artery,22 suggest an
effect of RIPC on microcirculatory function. Given the
significant impact that the microcirculation has on the
prognosis of patients undergoing PCI17,18 and the demon-
strated benefits of RIPC in this population, we hypothesized
that RIPC may improve coronary microvascular function. To
explore the mechanism behind RIPC-mediated protection in
the setting of PCI, we undertook a randomized, blinded,
placebo-controlled proof-of-concept study to investigate the
effect of RIPC on invasively measured coronary physiological
parameters, IMR and CFR, in patients with CAD.
Methods
The data that support the findings of this study are available
from the corresponding author on reasonable request.
Patient Enrollment
Clinically stable patients referred for nonurgent coronary
angiography, with symptoms or noninvasive investigations
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ORIGINAL RESEARCH
suggestive of significant CAD, at a single tertiary referral
center were invited to participate in the study before their
planned procedure. Coronary angiography was performed,
and patients were included if they required a clinically
indicated fractional flow reserve (FFR) assessment of an
angiographically equivocal lesion in a major epicardial coro-
nary artery, as determined by the interventional cardiologist
performing the procedure. We included only patients who
required FFR measurement to justify the increased risk
associated with coronary artery wiring. Consecutive patients
who met these inclusion criteria were included in the study.
Exclusion criteria included the need for emergent coronary
angiography, contraindication to inflation of a sphygmo-
manometer on the left arm (eg, arteriovenous fistula for renal
dialysis, peripheral vascular disease involving the limb), and
existing neuropathy or myopathy that may predispose to
nerve or muscle damage from upper limb ischemia. In
addition, patients with prior myocardial infarction in the
target artery territory or coronary anatomy that would affect
accurate coronary physiology assessment, such as left or
right coronary ostial disease leading to guide pressure
“damping,” were also excluded, as were patients in atrial
fibrillation, because irregularity of the cardiac cycle could
affect thermodilution measurements. Patients with severe
asthma were not invited to participate because adenosine can
exacerbate airway disease.
Once participants met the inclusion criteria, they were
randomized to either RIPC or sham treatment by way of a
closed-envelope system during their procedure. Data regard-
ing patient demographics, comorbidities, medications, and
preprocedure investigations were collected.
Coronary Physiology Measurements
After informed consent, a 6F radial or femoral arterial sheath
was inserted, and coronary angiography was performed by
standard techniques under conscious sedation with at least
1 mg midazolam and 25 lg fentanyl administered intra-
venously. Quantitative coronary angiography (Artis; Siemens)
was performed off-line in 2 orthogonal views. Unfractionated
heparin was administered at a dose of 70 U/kg. Coronary
physiology measurements were performed immediately
before and immediately after the RIPC/sham treatment
protocol, as described previously.17,18,23 In brief, a 6F guiding
catheter without side holes was used to engage either the left
main coronary artery or the right coronary artery. A pressure–
temperature sensor guidewire (Certus; St Jude Medical) was
advanced to the tip of the guiding catheter for pressure
equalization before being advanced to the distal segment of
the target artery, ensuring that the sensor position was at
least 30 mm distal to the lesion in question. The wire position
was fluoroscopically stored and maintained throughout the
Journal of the American Heart Association
 Remote Preconditioning and Microcirculation Lau et al
study protocol. Intracoronary glyceryl trinitrate was adminis-
tered at a dose of 100 lg before each coronary physiology
study. Thermodilution curves were produced in triplicate to
determine the mean transit time at rest (shown as TmnR) by
briskly injecting 3 mL of room temperature saline down the
coronary artery. In addition, the mean proximal pressure and
mean distal pressure were recorded at rest.
Hyperemia was induced with an intravenous infusion of
adenosine (140 lg/kg per minute) via a 4F femoral venous
sheath. In a similar manner, thermodilution curves were
produced to determine the mean transit time during hyper-
emia (shown as TmnH). Mean proximal pressure (shown as Pa)
and mean distal pressure (shown as Pd) during hyperemia
were also recorded. As described previously, the FFR was
calculated by Pd/Pa during hyperemia and the CFR was
calculated by TmnR/TmnH.23 The IMR was calculated by 2
methods. In patients where there was no hemodynamically
significant epicardial stenosis, as determined by an FFR
>0.80, the IMR was calculated during hyperemia by the
formula Pd9TmnH.12 The IMR overestimates microcirculatory
resistance in the presence of significant epicardial stenoses
due to the presence of collateral flow. To avoid measuring the
coronary wedge pressure to account for collateral flow, which
requires coronary balloon inflation, the IMR was also calcu-
lated in all patients using the formula derived by Yong et al
(calculated IMR [IMRcalc]): Pa9TmnH9(1.349Pd/Pa 0.32).24
All measurements were recorded using the RadiAnalyzer
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console (St Jude Medical).
Patients then received either RIPC or sham treatment while
on the catheterization laboratory table. Physiology measure-
ments were repeated in an identical manner immediately after
RIPC/sham after ensuring that the pressure–temperature
sensor was in a position identical to that when pretreatment
measurements were performed. The procedure then pro-
ceeded as clinically indicated. All study measurements were
taken before coronary balloon inflation to avoid the effect of
local ischemic preconditioning and distal embolization con-
founding the results.
Remote Ischemic Preconditioning
In the RIPC group, a sphygmomanometer was inflated to
either 200 mm Hg or 50 mm Hg greater than systolic blood
pressure (whichever was greater) for 5 minutes on the left
arm, followed by deflation for 5 minutes, and this cycle was
repeated 3 times using an automated sphygmomanometer
(HeartGuard; Condicion).4,25 Sham treatment involved sphyg-
momanometer inflation to a pressure of 10 mm Hg but was
otherwise identical to the RIPC protocol. To confirm ischemia,
the radial pulse was examined in each patient, ensuring that it
was impalpable during RIPC and unaffected during sham
treatment. Figure 1 outlines patient flow and randomization.
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ORIGINAL RESEARCH
Patients were not informed of their treatment allocation
but were warned of possible discomfort from sphygmo-
manometer inflation. Patients were asked not to express
discomfort unless it was intolerable, with all patients
complying with this request. The operator performing phys-
iological measurements was also blinded to the treatment
allocation, with RIPC or sham treatment delivered by an
assistant who did not communicate with the operator. To
maintain blinding, the sphygmomanometer was obscured
from view, and music was played throughout the laboratory to
mask the sound of sphygmomanometer inflation.
To ensure that the RIPC protocol was effectively inducing
ischemia to the treated upper limb, in a cohort of patients
who did not undergo coronary physiology measurements,
venous blood was drawn into tubes containing lithium heparin
from the cubital fossa of the treated upper limb before and
immediately after the RIPC and sham protocols for measure-
ment of blood lactate.
Plasma Collection and Analysis
Nitric oxide is known to be a regulator of coronary microcir-
culatory function, with nitrite being its major metabolite.26 To
determine the effect of RIPC on circulating nitrite levels, blood
was collected from the femoral venous sheath after each
coronary physiology study, before and after the allocated
treatment, into tubes containing 7.2 mg (1.8 mg/mL)
K2EDTA. The blood was centrifuged at 2500g for 15 minutes,
and the plasma was stored immediately at 80°C. Because
circulating nitrite levels have been shown to be elevated and
important for cardiac protection induced by RIPC,27 plasma
that had not previously been thawed was analyzed for nitrite
concentration with a commercially available ELISA kit (R&D
Systems), as per the manufacturer’s instructions.
Statistical Analysis
The D’Agostino and Pearson normality test was used to
determine whether data were normally distributed. Categor-
ical variables are presented as frequency and percentage.
Continuous variables are expressed as meanSD for normally
distributed data and as median (interquartile range [IQR]) for
nonnormally distributed data. Categorical data were com-
pared using the v2 or Fisher exact test, as appropriate.
Comparisons between continuous variables were performed
using the paired or unpaired t test, as appropriate, for
normally distributed data and the Wilcoxon signed rank test or
Mann–Whitney U test, as appropriate, for nonnormally
distributed data. Correlations between continuous variables
were assessed with the Pearson correlation.
Sample size calculation was performed based on the
primary analysis, which was amount of change in the IMR with
Journal of the American Heart Association
 Remote Preconditioning and Microcirculation Lau et al
Figure 1. Patient recruitment and randomization. Patients were randomized to RIPC or sham treatment
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after coronary angiogram, and the need for FFR measurement was established. A coronary physiology study
was performed before and after the allocated treatment. FFR indicates fractional flow reserve; RIPC, remote
ischemic preconditioning.
RIPC. Based on preliminary data, an absolute decrease in
IMRcalc with RIPC was expected to be 5.57.0. With a power
of 80% and a 2-sided a value of 0.05, it was estimated that 15
patients would need to be studied for a paired difference
analysis to detect a change in IMRcalc with RIPC.
We aimed to perform a secondary analysis to determine
change in IMRcalc with sham treatment to ensure that there
was no artifactual change in coronary physiology indexes as
a result of a prolonged catheterization procedure; therefore,
we recruited 30 patients who were randomized to RIPC
or sham (15 RIPC and 15 sham). Other secondary
analyses included changes in IMR, CFR, mean transit time
at rest, mean transit time during hyperemia, and FFR with
RIPC.
In addition, the relative change in IMRcalc, IMR, and CFR
was calculated as [(post pre)/pre]9100%, where pre repre-
sents that value measured before RIPC/sham and post
represents the value measured after RIPC/sham. The relative
change in the markers of coronary microcirculatory function
in the RIPC cohort was compared with the relative change in
the sham cohort.
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ORIGINAL RESEARCH
All analyses were performed using SPSS v22 (IBM Corp) or
GraphPad Prism (GraphPad Software). A 2-tailed probability
value <0.05 was considered statistically significant.
Ethical Considerations
The study protocol conforms to the ethical guidelines of the
1975 Declaration of Helsinki. Ethics approval was granted by
the Sydney Local Health District Human Research Ethics
Committee of Sydney, Australia (CH62/6/2014-016). The
study was registered with the Australia and New Zealand
Clinical Trials Registry (ACTRN12616000486426). Each par-
ticipant gave written informed consent for participation and
for use of their health information. All patient data were
deidentified and analyzed anonymously.
Results
A total of 65 patients underwent coronary angiography and
were screened for participation in the study. As Figure 1
shows, 34 patients were excluded after coronary
Journal of the American Heart Association
 Remote Preconditioning and Microcirculation Lau et al

angiography because there was no clinical indication for FFR
measurement. One additional patient was excluded because
coronary wiring was associated with significant coronary
spasm. Thirty patients underwent randomization and the full
study protocol. An example of the coronary physiology data
obtained from 1 patient are presented in Figure 2. This
patient was randomized to receive RIPC on the catheteriza-
tion laboratory table during the procedure. Figure 2A
displays the coronary physiology indexes before RIPC, and
Figure 2B displays the same indexes after RIPC. In this case,
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ORIGINAL RESEARCH
RIPC was associated with a decrease in IMR and an increase
in CFR.
Of the 30 patients who underwent randomization to either
RIPC or sham treatment, the mean age was 63.110.0 years,
and 26 (87%) were male. Coronary physiology measurements
were performed in all patients before and after the allocated
treatment. Fifteen patients were randomized to RIPC, and 15
patients were randomized to sham treatment. The allocated
treatment was tolerated by all patients, and there were no
complications of treatment. The baseline characteristics,

Figure 2. Coronary physiology measurements obtained from 1 patient who was randomized to RIPC. Data
were obtained before (A) and after (B) RIPC. There was a reduction in IMR and TmnH with RIPC, whereas the
CFR increased. IMR=Pd9TmnH. CFR indicates coronary flow reserve; FFR, fractional flow reserve; IMR, index
of microcirculatory resistance; Pa, mean proximal pressure; Pd, mean distal pressure; RIPC, remote ischemic
preconditioning; TmnH, mean transit time during hyperemia.

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Table 1. Baseline Characteristics of Patients lesion characteristics, and medications of the RIPC and sham
groups did not differ significantly (Table 1).
Characteristic RIPC (n=15) Sham (n=15) P Value

Age, y 64.58.8 61.711.2 0.465

Male 13 (87) 13 (87) 1.000
Effect of RIPC on Blood Lactate Measurements
Prior myocardial infarction 1 (7) 4 (27) 0.142 Sixteen patients, who were not part of the main study cohort,
underwent blood lactate measurements before and after RIPC/
Prior PCI 2 (13) 6 (40) 0.099
sham. There was a significant rise in blood lactate in response to
Prior CABG 0 (0) 0 (0)



the RIPC protocol (before versus after RIPC: 1.360.61 versus
Heart failure 0 (0) 1 (7) 0.309
1.690.56 mmol/L, respectively; P=0.004, n=10), whereas
Prior stroke 1 (7) 2 (13) 0.543 there was no effect after sham treatment (before versus after
Peripheral vascular 0 (0) 2 (13) 0.143 sham: median: 1.4 [IQR: 1.1–1.6] versus 1.5 [IQR: 1.2–1.7];
disease P=0.500, n=6).
Hypertension 11 (73) 10 (67) 0.690
Diabetes mellitus 7 (47) 3 (20) 0.121
Effect of RIPC on Coronary Physiology
Dyslipidemia 13 (87) 9 (60) 0.099
Measurements
Current smoking 1 (7) 3 (20) 0.283
Twenty-two (73%) of the lesions assessed were in the left
Normal left ventricular 14 (93) 14 (93) 1.000
contractility anterior descending artery, 12 in the RIPC cohort and 10 in
Left ventricular hypertrophy 2 (13) 1 (7) 0.543 the sham cohort (P=0.409). The mean pretreatment (RIPC/
sham) FFRs in the RIPC and sham cohorts were not
Medications
significantly different (0.830.06 versus 0.820.08;
Aspirin (100 mg daily) 14 (93) 15 (100) 0.309
P=0.762). There were 3 (20%) and 6 (40%) pretreatment
P2Y12 antagonist 12 (80) 10 (67) 0.409 FFR measurements ≤0.80 in the RIPC and sham groups,
Clopidogrel 9 (60) 9 (60) respectively (P=0.232).
Ticagrelor 3 (20) 1 (7)
Primary analysis
Warfarin/NOAC 0 (0) 0 (0)



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Statin 14 (93) 14 (93) 1.000 The coronary physiology indexes recorded before and after
RIPC and before and after sham treatment are displayed in
b-Blocker 5 (33) 8 (53) 0.269
Table 2. Within the RIPC cohort, there was a significant
ACEI or ARB 11 (73) 7 (47) 0.136
reduction in IMRcalc when the pre- and post-RIPC measure-
Nitrate 1 (7) 1 (7) 1.000 ments were compared (Figure 3A).
Coronary assessment
LAD assessed 12 (80) 10 (67) 0.409 Secondary analyses
Lesion diameter 38.710.0 39.56.4 0.779 There was also a significant reduction in IMR in patients with
stenosis, %* pretreatment FFR >0.80 and a significant increase in CFR in
Vessel reference 2.90.5 2.60.5 0.051 patients who were randomized to receive RIPC (Figure 3B
diameter, mm* and 3C). There was no effect of RIPC on FFR. The
Lesion length, mm* 9.44.3 10.14.2 0.651 predominant driver of these physiological effects was a
Parameters during admission reduction in the hyperemic transit time (median: 0.33 [IQR:
Systolic blood pressure, 128.512.8 136.112.3 0.108 0.26–0.40] versus 0.25 [IQR: 0.20–0.30]; P=0.010;
mm Hg Figure 3D) with no change in mean distal pressure dur-
Heart rate, beats/min 68.411.0 67.910.5 0.893 ing hyperemia (68.515.4 versus 70.516.1 mm Hg;
Hemoglobin concentration, 138.119.9 130.915.2 0.276
P=0.495) or mean proximal pressure during hyperemia
g/L (81.514.5 versus 84.314.0 mm Hg; P=0.406). There
eGFR, mL/min/1.73 m2 83.111.1 84.913.4 0.682 was no change in the resting transit time (0.950.43 versus
1.030.55 seconds; P=0.293) with RIPC.
Data are shown as meanSD or n (%). ACEI indicates angiotensin-converting enzyme There was no change in IMRcalc, IMR, CFR, or mean transit
inhibitor; ARB, angiotensin receptor blocker; CABG, coronary artery bypass grafting;
eGFR, estimated glomerular filtration rate; LAD, left anterior descending artery; NOAC, time during hyperemia with sham treatment (Figure 4).
novel oral anticoagulant; PCI, percutaneous coronary intervention; RIPC, remote A comparison of the change in markers of coronary
ischemic preconditioning.
*Average value from quantitative coronary angiographic assessment of 2 views of each
microcirculatory function induced by RIPC and sham treat-
lesion per patient. ment is displayed in Table 3. Change in IMRcalc was

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 Remote Preconditioning and Microcirculation Lau et al

ORIGINAL RESEARCH
Table 2. The Effect of RIPC on Coronary Physiology Indexes

RIPC (n=15) Sham (n=15)

Mean Mean
Marker Pre Post Difference* P Value† Pre Post Difference* P Value†

IMRcalc 22.6 (17.9–25.6) 17.5 (14.5–21.3) 5.1 0.007 16.0 (10.8–20.5) 16.8 (10.8–21.2) 1.1 0.847
‡
IMR 24.3 (18.5–26.1) 17.7 (13.2–21.7) 5.1 0.005 16.1 (9.3–22.8) 11.4 (10.6–24.7) 1.0 0.820
CFR 2.60.9 3.81.7 1.2 0.001 3.11.5 3.11.6 0.0 0.971
FFR 0.830.06 0.830.07 0.0 0.999 0.820.08 0.810.09 0.0 0.052

Data are shown as meanSD or median (interquartile range). CFR indicates coronary flow reserve; FFR, fractional flow reserve; IMR, index of microcirculatory resistance; IMRcalc,
calculated index of microcirculatory resistance; RIPC, remote ischemic preconditioning.
*Absolute difference in mean between pre and post within each cohort.
†
Comparison between pre and post values within each group was performed with the paired t test or Wilcoxon signed rank test for normally or non-normally distributed data, respectively.
‡
Patients with FFR >0.80: 12 in the RIPC group and 9 in the sham group.

significantly greater when comparing the RIPC and sham

cohorts (Figure 5A). Change in CFR was also significantly
greater when comparing the RIPC and sham cohorts
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Figure 3. Remote ischemic preconditioning reduces the IMR and increases the CFR through an increase
in hyperemic coronary flow. There was a significant reduction in IMRcalc (A) and the IMR (B) with RIPC,
whereas the CFR (C) increased significantly. There was a significant reduction in TmnH (D) with RIPC,
suggesting an increase in hyperemic coronary flow. IMRcalc, CFR and TmnH: n=15; IMR: n=12; Individual
filled symbols represent measurements before or after RIPC in each patient joined with a line, and open
symbols and bars represent meanSD. Bas indicates baseline; CFR, coronary flow reserve; Hyp, hyperemic;
IMR, index of microcirculatory resistance; IMRcalc, calculated index of microcirculatory resistance; RIPC,
remote ischemic preconditioning; TmnH, mean transit time during hyperemia.

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ORIGINAL RESEARCH
Figure 4. Sham had no effect on IMR, CFR, or hyperemic coronary flow. There was no effect of sham on
IMRcalc (A), IMR (B), CFR (C), or TmnH (D). IMRcalc, CFR, and TmnH: n=15; IMR: n=9. Individual filled symbols
represent measurements before or after sham in each patient joined with a line, and open symbols and bars
Downloaded from http://ahajournals.org by on April 18, 2024

represent meanSD. CFR indicates coronary flow reserve; IMR, index of microcirculatory resistance;
IMRcalc, calculated index of microcirculatory resistance; RIPC, remote ischemic preconditioning; TmnH, mean
transit time during hyperemia.

No significant association or correlation was noted medications listed in Table 1. The P value was >0.1 for all
between the RIPC-induced change in IMRcalc and CFR and these factors on univariable regression analysis; therefore, a
any of the baseline demographics, comorbidities, and multivariable regression analysis was not performed.

Table 3. Comparison of the Effect of RIPC and Sham on

Plasma Nitrite Measurement
Coronary Physiology Indexes There was no change in plasma nitrite with RIPC (before
versus after RIPC: 1.60.2 versus l.60.3 lmol/L; P=0.997;
Marker RIPC (n=15) Sham (n=15) P Value* Figure S1).
IMRcalc 18.124.8% +6.137.5% 0.047
IMR† 22.525.2% +6.845.5% 0.074
CFR +41.2% (20.0–61.7) 7.8% ( 19.1 to 10.3) <0.001
Discussion
FFR 0.0% ( 2.4 to 1.4) 1.5% ( 3.4 to 1.2) 0.269 RIPC has been proposed to confer cardioprotection in patients
undergoing elective and primary PCI, with reductions in post-
Relative change in index with RIPC/sham is shown, with negative ( ) values indicating a PCI troponin and infarct size3–5 through multiple potential but
reduction after treatment compared with before treatment and positive (+) values
indicating an increase. Data are shown as meanSD or median (interquartile range). CFR as yet uncertain mechanisms.28 We demonstrated that RIPC
indicates coronary flow reserve; FFR, fractional flow reserve; IMR, index of acutely improved coronary microcirculatory function, as
microcirculatory resistance; IMRcalc, calculated index of microcirculatory resistance;
RIPC, remote ischemic preconditioning. assessed by validated coronary pressure–temperature sensor
*Comparison of relative change in RIPC cohort with relative change in sham cohort with wire–based techniques. To the best of our knowledge, this
the unpaired t test or Mann–Whitney U test for normally and nonnormally distributed
data, respectively.
study provides the first demonstration of these effects of RIPC
†
Patients with FFR >0.80: 12 in the RIPC group and the 9 in sham group. on coronary microcirculatory function.

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 Remote Preconditioning and Microcirculation Lau et al

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Figure 5. Comparison of change in markers of coronary microcirculatory function with remote ischemic
preconditioning and sham. The relative change in IMRcalc (A) and CFR (B) induced by RIPC was significantly
different to the change due to sham treatment. Individual filled symbols represent relative change in
measurement with RIPC/sham in each patient, with negative and positive values indicative of reductions
and increases with treatment, respectively. Open symbols and bars represent meanSD. CFR indicates
coronary flow reserve; IMRcalc, calculated index of microcirculatory resistance; RIPC, remote ischemic
preconditioning.

The coronary microcirculation is recognized as an impor-
tant determinant of prognosis in patients with CAD. The IMR
is an index that assesses resistance to flow in the coronary
microvasculature, whereas the FFR is an index that is used to
assess the hemodynamic significance of an epicardial coro-
nary lesion. The CFR is an index that provides assessment of
both the epicardial artery and the microcirculation.29
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failure who received RIPC twice per day for 1 week demon-
strated an increase in CFR, as assessed by echocardiographic
spectral Doppler analysis of flow in the distal left anterior
descending artery. In addition, RIPC has been shown to
improve endothelial function, reducing vasoconstriction after
acetylcholine administration during cardiac catheterization.34
Despite this, there is conflicting evidence regarding the

Multiple studies have shown the utility of the CFR to
predict cardiac events, and it has been shown to be of greater
importance than the degree of disease in the epicardial
coronary arteries in determining prognosis.14,16,19,30,31 The
IMR, which correlates with true microcirculatory resistance,12
has been shown to be predictive of post-PCI myocardial
infarction after elective PCI and the occurrence of death and
heart failure hospitalization after primary PCI.17,18 The IMR
has been shown to be reproducible and independent of
hemodynamic conditions and thus is a reliable method to
assess the microcirculatory resistance.23
We demonstrate that RIPC leads to a rapid reduction in the
IMR and an increase in CFR. This suggests that beneficial
effects on the coronary microcirculation may contribute to
RIPC-mediated cardioprotection during PCI. The baseline
clinical characteristics appeared to have little effect on the
change in IMRcalc and CFR with RIPC. The results of this study
raise the possibility that RIPC may be beneficial in other
clinical settings involving microcirculatory dysfunction such as
microvascular angina, congestive heart failure, and aortic
stenosis.32,33 Our results suggest that research into the use
of RIPC beyond CAD is warranted.
Data support the role of the coronary microcirculation as a
target of RIPC-mediated cardioprotection.20 In a study by
Kono et al,22 10 healthy volunteers and 10 patients with heart
effects of RIPC on the coronary microcirculation. Studies of
RIPC in the setting of primary PCI have reported no changes in
surrogate markers of microcirculatory function, such as the
Thrombolysis in Myocardial Infarction (TIMI) frame count and
the appearance of microvascular obstruction on magnetic
resonance imaging.3,5 Moreover, a study by Hoole et al found
that RIPC had no effect on coronary microvascular resistance
in 11 patients assessed by a Doppler/pressure wire–based
technique requiring coronary balloon inflation during cardiac
catheterization.35 The null result in this study may have been
due to local preconditioning, or distal embolization, induced
by the coronary balloon inflation. There was a small numerical
increase in the microcirculatory resistance after coronary
balloon inflation in patients who did not undergo RIPC in this
study, but the study may have been underpowered to detect a
statistically significant difference.
Our study demonstrated an effect on CFR, IMR, and transit
time and confirmed the absence of an effect of sham treatment.
It is notable that there was an outlier in both the RIPC and sham
cohorts, with each of these patients demonstrating a marked
reduction in IMRcalc and IMR with RIPC/sham. However, the
decrease in IMRcalc and IMR with RIPC remained significant
after removal of this outlier (Figure S2).
There was no change in mean transit time at rest,
suggesting that RIPC does not affect resting coronary flow.

DOI: 10.1161/JAHA.118.009058 Journal of the American Heart Association

025
 Remote Preconditioning and Microcirculation Lau et al

Conversely, there was an increase in hyperemic flow,
indicated by a reduction in mean transit time during
hyperemia, in the absence of change in distal or proximal
coronary pressures. This increase in hyperemic flow supports
the interpretation that RIPC reduces coronary microcircula-
tory resistance.
Adenosine and nitric oxide/nitrite contribute to the
coronary microcirculatory tone, and because they have been
implicated in RIPC-mediated cardiac protection,27,36,37 they
are candidate mediators for the effect of RIPC on the
microcirculation. However, given the supraphysiological doses
of adenosine and intracoronary glyceryl trinitrate that were
administered at the time of the coronary physiology study to
achieve hyperemia, it is unlikely that these mediators are
primarily responsible for the effects that we observed. The
lack of change in plasma nitrite levels with RIPC, the major
metabolite of nitric oxide, supports this conclusion with
respect to nitric oxide. Although nitrite has been shown
previously to be increased by RIPC in an animal model,27 the
administration of glyceryl trinitrate during the procedure may
have masked any effect of RIPC on nitrite in this study.
Other mediators and pathways that have been shown to be
involved in cardiac preconditioning, include bradykinin, potas-
sium ATP channels, and calcium-activated potassium chan-
nels of the BK type.21,38,39 Although angiotensin-converting
enzyme inhibitors are known to reduce degradation of
bradykinin, in the RIPC cohort, the relative reduction in
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ORIGINAL RESEARCH
Finally, because this was a mechanistic proof-of-concept
study with only small numbers of patients undergoing PCI,
clinical outcomes and the correlation of microcirculatory
change with changes in post-PCI troponin were not assessed.
Although the inclusion of patients who required FFR mea-
surement resulted in a cohort that predominantly did not
require PCI, the results of this study herald the need for a
study correlating change in coronary microcirculatory status
and clinical outcomes with RIPC.
Conclusion
The IMR and CFR are acutely improved by RIPC. This suggests
that RIPC confers cardioprotection during PCI as a result of
improvement in coronary microcirculatory function. The
application of RIPC to augment the coronary microcirculation
in other settings warrants investigation.
Acknowledgments
We would like to recognize the contribution of Ms Kitty Xu, Dr Rong
Bing, Dr Gabrielle J. Pennings, and Dr Caroline J. Reddel for their
assistance in conducting the trial and processing the plasma samples.
In addition, we would like to thank the Concord Hospital catheteriza-
tion laboratory staff for their assistance and patience with this study.
Sources of Funding

IMRcalc and the increase in CFR were numerically smaller, but

not statistically significant, in patients taking this class of This work was supported by a National Health and Medical
drug. Because potassium ATP channels are involved in RIPC- Research Council/National Heart Foundation of Australia
mediated protection against ischemia–reperfusion injury– Postgraduate Scholarship (1094384 to Lau) and a National
associated endothelial dysfunction,21 they may play a role in Health and Medical Research Council Program Grant
the improved microcirculatory function that we demonstrated (1037903 to Kritharides).
and warrant future investigation. Although a large body of
evidence supports a circulating humoral factor mediating the
physiological effects of RIPC, some studies suggest a Disclosures
contribution by a neural pathway. In animal models, the Dr Yong has received research support from Abbott Labora-
transection of a peripheral nerve supplying the limb under- tories and Philips, and minor honoraria from Abbott Labora-
going RIPC or the use of a nicotinic acetyl choline ganglion tories. Dr Fearon has received research support form Abbott
blocker attenuated the protective effects of RIPC.40,41 Laboratories. The remaining authors have no disclosures to
report.
Limitations
We are unable to comment on the durability of the RIPC effect
and cannot be certain that we identified the maximal effect on
the CFR and IMR. Despite this, we demonstrated an acute
enhancement of microcirculatory function with RIPC that may
contribute to the protective effects of RIPC demonstrated in the
literature when RIPC was delivered within 2 hours of PCI.
Because of the administration of glyceryl trinitrate, additional
assessments of endothelial function, such as response to
acetylcholine administration, were not performed in this study.

DOI: 10.1161/JAHA.118.009058 Journal of the American Heart Association

026
SUPPLEMENTAL MATERIAL

027
Downloaded from http://ahajournals.org by on April 18, 2024
 Figure S1. No change in plasma nitrite with remote ischemic preconditioning.
Downloaded from http://ahajournals.org by on April 18, 2024

There was no change in plasma nitrite concentration, the major metabolite of nitric oxide,

with RIPC or sham treatment. RIPC, n=13; sham, n=10. Open symbols and bars represent

mean ± SD.

RIPC, remote ischemic preconditioning;

028
 Figure S2. The effect of remote ischemic preconditioning on the index of microcirculatory
resistance with outliers removed.

a) b)

When the outlier in the RIPC arm is removed, the effect of RIPC in reducing IMRcalc (a)

and IMR (b) persists. IMRcalc, n=14; IMR, n=11. Open symbols and bars represent mean ±

SD.
Downloaded from http://ahajournals.org by on April 18, 2024

IMR, index of microcirculatory resistance; IMRcalc, calculated index of microcirculatory

resistance; RIPC, Remote ischemic preconditioning;

029
●

●
± ±

030
Intermittent cycles of remote ischemic preconditioning
augment diabetic foot ulcer healing
Gad Shaked, MD1; David Czeiger, MD PhD1; Anwar Abu Arar, MD1; Tiberiu Katz, MD2;
Ilana Harman-Boehm, MD3; Gilbert Sebbag, MD1
1. Department of General Surgery B,
2. Department of Orthopedics,
3. Department of Internal Medicine C and the Diabetes Unit, Soroka University Medical Center and Ben-Gurion University, Beer Sheva, Israel

Reprint requests: ABSTRACT

Gad Shaked, Head of Trauma Unit,
Department of General Surgery, Soroka
University Medical Center and Ben-Gurion
University, Beer Sheva 84101, Israel.
Tel: 972-54-2365600;
Fax: 972-8-6239930;
Email: shakedg@bgu.ac.il
Manuscript received: May 29, 2014
Accepted in final form: February 9, 2015
DOI:10.1111/wrr.12269
The morbidity and mortality caused by diabetic foot ulcer (DFU) are still
significant. Conservative treatment of DFU is often ineffective. Treatment
modalities using stem cells directly into the DFU or systematically have been
introduced recently. Ischemic preconditioning (IPC) has been proved to be a
cheap, simple, and safe method which can augment stem cells number in the
peripheral blood circulation. This study’s purpose was to test whether IPC can
improve DFU healing. Forty diabetic patients were enrolled and divided into study
and control groups. All patients received their regular treatment. The study group
patients received in addition brief, transient cycles of IPC while the control group
patients received a sham procedure only. The procedure was repeated every 2
weeks to complete a follow-up period of 6 weeks. The ulcers were photographed
to measure wound area, and the degree of granulation tissue was assessed. No
serious adverse events were noted. Twenty-two patients from the study group and
12 from the control group completed the entire follow-up. The ratio of patients
who reached complete healing of their ulcer was 9/22 (41%) in the study group
compared with 0/12 (0%) in the control group, p 5 0.01. Furthermore, the mean
remaining ulcer area at the end of the follow-up was significantly smaller in the
study group, 25 6 6% of the initial area vs. 61 6 10% in the control group,
p 5 0.007. The degree of granulation increased after one cycle of treatment in 8/24
(33%) study patients compared to 3/16 (19%) in the control group, p 5 0.47.
Remote, repeated IPC significantly improves the healing of DFU. This simple,
safe, inexpensive treatment method should be considered to be routinely applied to
diabetic patients with DFU in addition to other regular treatment modalities.

INTRODUCTION remote IPC in healthy volunteers demonstrating augmenta-

Despite much advancement in the management of diabetic
complications and in particular diabetic foot ulcer (DFU),
the morbidity and mortality caused by DFU are still signif-
icant. The incidence of DFU is 15% among all diabetic
patients and is even higher in those with long-standing dia-
betes and in those with poor glycemic control.1 It has been
estimated that up to 20% of the patients with DFU will
eventually require amputation.2 The high morbidity associ-
ated with DFU, primarily local and systemic infections,
explains the dismal 5-year survival rate of 27% following
amputations.1 The two combined etiologies of DFU are
neuropathia and ischemia. Conservative treatment of DFU
is often ineffective. Without proper treatment DFU might
increase in size and depth, become infected and involve
deeper soft tissues and even bone, thus, creating much
more complicated situations that are difficult to cure.
In contrast to ischemia-reperfusion (IR), ischemic pre-
conditioning (IPC) is considered a protective and benefi-
cial phenomenon. In a recent study, we performed a
tion potential of blood endothelial progenitor cells.3 Other
reports have showed that IPC has a role in the process of
stem cell mobilization which leads to a substantial eleva-
tion in the number of peripheral blood stem cells.4,5
Albeit multiple studies have been conducted in an
attempt to show beneficial effect of stem cell infusion on
the healing of DFU,6–10 there is currently no study—to the
best of our knowledge—that investigates the effects of
remote IPC. Along with the expected beneficial effects of
IPC, there is a concern regarding how the immune system
would function following the procedure. Recently pub-
lished findings suggest that remote IPC selectively modi-
fies functional responses of human neutrophils and
decreases their adhesion.11 This might adversely affect the
healing of infected wounds.
Our hypothesis was that remote IPC may improve DFU
healing, probably by increasing the number of mesenchymal
and endothelial stem cells in the area of the wound. The
aim of this clinical study is to evaluate the ability of IPC to
enhance the healing process of aseptic and infected DFU.

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031
Ischemic preconditioning and diabetic foot ulcer
Table 1. The inclusion and exclusion criteria that were used to enroll patients into this study
Inclusion Criteria
Age over 18
Having a current diagnosis of diabetes mellitus type 1
or 2
Having a DFU graded as Wagner II or III
Signed informed consent
METHODS
Setup
The study was conducted in the multidisciplinary diabetic
foot outpatient clinic at the Soroka University Medical
Center (SUMC), Beer Sheva, Israel. SUMC is a referral
tertiary center that serves a population of approximately 1
million people. The study received the approval of the
Institutional Review Board (IRB) and was carried out in
accordance with the Good Clinical Practice (GCP)
guidelines.
Study objectives
To evaluate the safety and efficacy of repeated IPC as an
adjunct to the standard therapy on DFU healing, as com-
pared to sham procedure performed with the standard ther-
apy. The treatment effect was determined during a period
of 6-week follow-up by measuring the wound area reduc-
tion and the degree of wound bed coverage by granulation
tissue.
Study endpoints
Primary endpoint: IPC efficacy to enhance DFU healing,
as measured by the percent reduction in wound area by
week 6, as compared to the baseline measurement. Sec-
ondary endpoints: a. IPC efficacy to enhance DFU healing,
as measured by the change in degree of wound bed granu-
lation tissue by week 2, as compared to baseline measure-
ments. b. IPC efficacy to enhance DFU healing, as
measured by the percentage of patients that have demon-
strated 75% or more reduction in wound area by week 6,
as compared to baseline measurements. c. The safety of
IPC in diabetic patients was evaluated by comparing
adverse events in both groups.
Study population
The study was designed to include 40 adult subjects ran-
domly divided between the study group and the control
group. Diabetic (type 1 or 2) male and female patients
aged over 18 years old who presented to the diabetic foot
032
Shaked et al.
Exclusion Criteria
Ulcers suspected to be of nondiabetic etiology
Diagnosed osteomyelitis
Known pregnancy
Current participation in any other study
Vascular procedures performed to the study foot within one
year prior to screening
ABI < 0.7
Use of sulfonylureas
Hba1c  12
Hypocalcemia
clinic with DFU were enrolled into the study after signing
an informed consent.
Table 1 presents the detailed inclusion and exclusion
criteria pertinent to this study.
Study design
This was a prospective, double-blind, randomized, shame
procedure-controlled study. A total of 40 patients who pre-
sented to the diabetic foot clinic at SUMC and fulfilled the
inclusion and exclusion criteria were enrolled. All subjects
received the standard wound care in use by the staff of the
diabetic foot clinic who were blinded as to the study allo-
cation of each patient. Subjects were divided randomly
into two equal groups. Pressure cuffs were applied to both
arms of all subjects. The cuffs were inflated and deflated
intermittently for three cycles of 5 minutes each. In the
study group, the cuffs were inflated to 200 mmHg while
the performing investigator examines the radial pulse to
ensure complete blood flow obstruction. In the control
group, inflation was up to 10 mmHg only. The patients
were followed for 6 weeks. During this period, they were
examined every 2 weeks; and in each of these visits the
above procedure was repeated. A “blinded” physician
measured the diameter of the wound and graded the granu-
lation tissue covering the wound on a scale from 0 to 5
[0 5 no granulation tissue, 1 5 <25% wound surface area
(WSA), 2 5 25–50% WSA, 3 5 50–75% WSA, 75–100%
WSA]. The examiner evaluated whether the wounds were
infected and cultured if required. The wounds were photo-
graphed and the wound area was measured digitally by a
blinded investigator using the ImageJ 1.47v software
(National Institutes of Health, Bethesda, MD).
Statistical analysis
A statistical analysis was performed with GraphPad Prism
software (version 5.01; San Diego, CA). Statistically sig-
nificant differences of means between two groups were
tested by using the nonparametric Mann–Whitney test for
continuous variables (two-tailed). Results are presented as
mean 6 SE. Categorical variables are presented as percen-
tages and analyzed by the Fisher’s exact test (two-tailed).
Significance was considered at p  0.05.
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Shaked et al. Ischemic preconditioning and diabetic foot ulcer

Table 2. The epidemiological and clinical characteristics of the patients who erolled into the study

Trial Control

Male/female (%) 17/7 (71/29) 8/8 (50/50)

Age range (mean 6 SE) 34–79 (55.6 6 2.1) 43–87 (61.5 6 3.7)
Diabetes mellitus type 1 or 2 (%) 4/20 (17/83) 2/14 (12.5/87.5)
Insulin treated yes/no (%) 16/8 (67/33) 10/6 (62.5/37.5)
Hg A1c (mean 6 SE) gr% 5.7–12 (9.7 6 0.4) 6.6–12 (8.9 6 0.4)
Ca (mean 6 SE) 8.1–10.2 (9.2 6 0.51) 8.8–10.1 (8.8 6 1.7)
Chronic obstructive lung disease 2 (8) 1 (6)
Nephropathy 6 (25) 1 (6)
Obesity 3 (12.5) 2 (12.5)
Chronic ischemic heart disease 6 (25) 5 (31)
Neuropathy 1 (4) 1 (6)
Smoking 1 (4) 2 (12.5)
Dyslipidemia 12 (50) 8 (50)
Retinopathy 1 (4) 1 (6)
Wagner 2A/2B/3A (%) 11/8/5 (46/33/21) 6/5/5 (37.5/31/31)
Ulcer size at recruitment (mean 6 SE) cm2 0.21–3.6 (1.3 6 1.1) 0.36–5.4 (1.8 6 1.4)
Ulcer local infection yes/no (%) 11/13 (46/54) 11/5 (69/31)
Wound culture (% of positive cultures)
Staphylococcus 2 (18) 3 (27)
Enterobacter 2 (18) 1 (9)
Staphylococcus aureus 3 (27) 3 (27)
Streptococcus 2 (18) 2 (18)
Proteus 1 (9) 0 (0)
Klebsiella 1 (9) 1 (9)
E-coli 0 (0) 1 (9)

RESULTS The ratio of patients who reached 75% reduction of

their ulcer area was higher in the study group, 14/22
Forty patients who enrolled into the study were randomly (64%) than in the control group, 3/12 (25%), p 5 0.07.
divided between a study group (n 5 24) and a control The ratio of patients who reached complete healing of
group (n 5 16). Table 2 summarizes patient and wound their ulcer was 9/22 (41%) in the study group compared
characteristics. There were no significant differences with 0/12 (0%) in the control group, p 5 0.01 (Figure 1).
between the two groups regarding mean age [56.6 6 2.1 Furthermore, the mean residual ulcer area at the end of the
(study group) vs. 61.5 6 3.7 (control group), p 5 0.28], follow-up was significantly smaller in the study group,
gender ratio [17:7 (71% male in the study group) vs. 8:8
(50% male in the control group), p 5 0.20], glyycemic
control reflected by mean HbA1c [9.7 6 0.4 gr% (study
group) vs. 8.9 6 0.4 gr% (control group), p 5 0.14], time
from wound manifestation to the first study visit [60 6 12
days (study group) vs. 120 6 59 days (control group),
p 5 0.21], the wound area on baseline documentation
[1.3 6 0.2 cm2 (study group) vs. 1.9 6 0.4 cm2 (control
group), p 5 0.20], and the ratio of of infected wounds
upon presentation [11/24 (46%) in the study group vs.
11/16 (69%) in the control group, p 5 0.20].
During the procedure several participants reported dis-
comfort or numbness of the hands but none experienced
severe pain. No neurovascular or neuromuscular deficit or
deep venous thrombosis were identified. Figure 1. The figure shows the differences between the
Two patients from the study group and four patients percentage of the study group patients that achieved at
from the controls dropped and did not complete the entire least 50%, 75%, and 100% healing at the end of the follow-
follow-up period. up compared to the control patients.

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033
Ischemic preconditioning and diabetic foot ulcer
Figure 2. The mean ratio between the residual ulcer area at
the end of the follow-up and the baseline area was lower in
the study group compared to the control group.
25 6 6% of the initial area vs. 61 6 10% in the control
group, p 5 0.007 (Figure 2).
In the subset of patients, who had infected ulcers when
enrolled into the study (n 5 11 in each group) 6 (55%)
patients in the study group reached complete healing vs.
none in the control group, p 5 0.01. However, within the
study group there was no statistical difference among the
patients who achieved complete healing between those
whose wounds were infected 6/11 (55%) compared to
those who had clean wounds 3/13 (23%), p 5 0.21.
The WSA covered with granulation increased by at least
one grade after one cycle of treatment (as recorded in the
2-week visit following the first treatment cycle) in 8/24
(33%) study patients compared to 3/16 (19%) in the con-
trol group, p 5 0.47.
DISCUSSION
Skin ulcers are a severe and frequent complication of dia-
betes and constitute a significant burden for healthcare sys-
tems all over the world.12 Diabetes affects the three phases
of wound healing; the inflammatory phase through com-
promising the immune system, the proliferative phase
through suppression of collagen deposition and formation
of new vessels (angiogenesis), and the remodeling phase in
which reorganization of collagen occurs to restore the tis-
sue structural integrity.13–15
In this study, an attempt was done to improve DFU
healing by using a remote IPC. The main results showed
that: the healing process as inspected at the granulation
phase was increased in the study group, though this trend
did not reach a statistical significance; a significant larger
percentage of the study group patients reached a complete
healing of the wound compared to the control group
patients; at the end of the follow-up the mean wound area
was significantly reduced in the study group.
Local tissue hypoxia is a major component of many
DFUs. Regional tissue ischemia resulting from peripheral
vascular disease complicates a large number of the
wounds. Patients exhibiting a significant peripheral vascu-
lar disease were excluded from this study. However, it has
been noted that even limbs free from overt arterial insuffi-
ciency may suffer from a number of abnormalities in the
microcirculation that contribute to local tissue
hypoxia.14,16–18 The peripheral neuropathy induces
034
Shaked et al.
increased pressure points on the diabetic foot, which in
turn lead to bouts of cutaneous IR injury. On a molecular
level, the injury process is initiated when adenosine tri-
phosphate production is reduced during ischemia, leading
to cellular events that result in leukocyte activation, oxida-
tive stress, and microvascular dysfunction on reperfusion.
Similar to other chronic wounds, both reperfusion and the
additive effects of repetitive IR cycles can escalate the
amount of injury in diabetic ulcers. With already altered
mediators of IR injury, diabetic patients appear to experi-
ence a further delay in wound healing.19
Treatment options are still very limited for patients in
whom revascularization by vascular surgery or interven-
tional angiology is not indicated or impossible to perform.
A large body of experimental evidence in mice, rats, and
larger animals has demonstrated the feasibility and efficacy
of stem cell therapies in restoring blood flow to the critical
ischemic limb. Bone marrow–derived mononuclear cells
(BMMNCs) have been locally applied to restore angiogen-
esis and promote wound healing in type 2 diabetic patients
with neuro-ischemic wounds.20
Formation and extension of small collateral vessels
appear to be the most important factors of the physiologi-
cal repair mechanism in peripheral artery disease, presum-
ably affected by the local production of growth factors by
BMMNCs.21 Nevertheless, the growth capacity of these
collateral vessels is reduced in atherosclerosis and espe-
cially in diabetic macroangiopathy.22 To optimize the ther-
apeutic procedure of intra-arterial and intramuscular
administration of BMMNCs, researchers have conducted
an IPC of the affected leg by means of exercise.23 Other
investigators reported that treatment with CD341-enriched
cells directly injected into diabetic wounds, decreased
wound size, accelerated epidermal healing, and dramati-
cally accelerated revascularization of the wounds.9 In a
previous study, we reported that transient, brief cycles of
extremities IPC are capable of increasing the availability
of CD34 1 angiogenic progenitor cells while maintaining
their ability to form vascular structures.3 Other beneficial
effects that have been attributed to IPC are improved stem
cell survival and effectiveness.24,25 It has also been shown
that IPC increases nitric oxide (NO) production and that
NO has a protective effect against IR injury. NO is an
angiogenic factor and a potent vasodilator. Furthermore, it
has been recently suggested that NO production increases
the recruitment of CD34 1 cells.26 IPC has been showed in
previous animal models and in clinical trials as capable of
preventing damage and improving healing of various tis-
sues and body systems such as the heart, kidney, flaps,
and gut. Some works suggested that the IPC resulted in
increased blood flow and oxygen saturation in the remote
examined tissue.27–32 Furthermore, there are reports that
applying cuffing on limbs after a stroke, or a myocardial
infarction has occurred, or after a flap became ischemic
(remote conditioning or perconditioning) had a beneficial
effect on the affected tissue.33–35 This study protocol of
IPC is based on the aforementioned clinical trials that used
the same regimen of upper extremities brief repeated
cycles of pressure cuffing to induce the required effect.
The anticipation that remote IPC can result in better heal-
ing of DFU is supported by previous reports that remote
intermittent IPC affects cutaneous tissue oxygen saturation,
arterial capillary blood flow, and postcapillary venous
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Shaked et al.

filling pressure at a remote cutaneous location of the lower

extremity.36,37
Little is known on the relationship between IPC and
infection, thus, the finding of this study that the effect of
IPC on wound healing was preserved in the presence of
infection is of importance.

Discussion
The study attendeds to serve as a pilot proof-of-concept
study to evaluate the possible effectiveness of IPC on the
healing of DFU. Levels of known blood borne healing fac-
tors or surrogates such as VEGF, IL-8, stem cells of the
angiogenic or mesenchymal lineages, and others were not
measured in this study. These factors should be investi-
gated in the future to understand the mechanism of the
current clinical observations. The current protocol of
inducing IPC every 2 weeks was empirically chosen. The
study does not relate to whether more frequent interven-
tions would have improved the healing process. The best
therapeutic protocol should be decided according to future
studies.
To summarize, the technique of remote (from the
affected leg) IPC, repeated every 2 weeks was proved in
this study to be safe, and yielded significant improvement
in wound size and healing process. The procedure is very
simple and can be done in any clinic and even at home. It
does not require any sophisticated expensive equipment, or
extensive physical exercise that might be problematic for
some of the pertinent patient population. Based on this
study, it seems that IPC may be a practical adjunct in
terms of effectiveness and cost to the current therapeutic
tool box of DFU patinets.
It was beyond the scope of this research to explore the
fundamental biologic basis of the clinical findings and this
should be studied in the future. Further investigations also
need to evaluate the response to IPC by patients sustaining
more severe grades of DFU and of diabetic patients suffer-
ing critical leg ischemia.

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035
036
 J D Maxwell, H H Carter and Ischaemic preconditioning and 181:6
Clinical Study others endothelial function in T2DM

Seven-day remote ischaemic preconditioning

improves endothelial function in patients
with type 2 diabetes mellitus: a randomised
pilot study
Joseph D Maxwell1,*, Howard H Carter1,2,*, Ylva Hellsten2, Gemma D Miller1, Victoria S Sprung1,3,
Daniel J Cuthbertson3, Dick H J Thijssen1,4 and Helen Jones1
1
Research Institute of Sport and Exercise Science, Liverpool John Moores University, Liverpool, UK, 2Department of
Nutrition, Exercise and Sports, Integrative Physiology Group, University of Copenhagen, Copenhagen, Denmark,
3
Obesity and Endocrinology Research Group, Clinical Sciences Centre, University Hospital Aintree, Liverpool, UK, and
Correspondence
4
Department of Physiology, Radboud Institute of Health Sciences, Radboud University Medical Center, Nijmegen,
should be addressed
The Netherlands
to H Jones
*(J D Maxwell and H H Carter contributed equally to this work) Email
H.Jones1@ljmu.ac.uk
European Journal of Endocrinology

Abstract
Background: Remote ischaemic preconditioning (rIPC) may improve cardiac/cerebrovascular outcomes of ischaemic
events. Ischaemic damage caused by cardiovascular/cerebrovascular disease are primary causes of mortality in type
2 diabetes mellitus (T2DM). Due to the positive effects from a bout of rIPC within the vasculature, we explored if daily
rIPC could improve endothelial and cerebrovascular function. The aim of this pilot study was to obtain estimates for
the change in conduit artery and cerebrovascular function following a 7-day rIPC intervention.
Methods: Twenty-one patients with T2DM were randomly allocated to either 7-day daily upper-arm rIPC (4 × 5 min
220 mmHg, interspaced by 5-min reperfusion) or control. We examined peripheral endothelial function using
flow mediated dilation (FMD) before and after ischemia-reperfusion injury (IRI, 20 min forearm ischaemic-20 min
reperfusion) and cerebrovascular function, assessed by dynamic cerebral autoregulation (dCA) at three time points;
pre, post and 8 days post intervention.
Results: For exploratory purposes, we performed statistical analysis on our primary comparison (pre-to-post) to
provide an estimate of the change in the primary and secondary outcome variables. Using pre-intervention data
as a covariate, the change from pre-post in FMD was 1.3% (95% CI: 0.69 to 3.80; P = 0.09) and 0.23 %cm/s %/mmHg
mmHg/% (−0.12, 0.59; P = 0.18) in dCA normalised gain with rIPC versus control. Based upon this, a sample size of
20 and 50 for FMD and normalised gain, respectively, in each group would provide 90% power to detect statistically
significant (P < 0.05) between-group difference in a randomised controlled trial.
Conclusion: We provide estimates of sample size for a randomised control trial exploring the impact of daily rIPC for
7 days on peripheral endothelial and cerebrovascular function. The directional changes outline from our pilot study
suggest peripheral endothelial function can be enhanced by daily rIPC in patients with T2DM.

European Journal of
Endocrinology
(2019) 181, 659–669

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Introduction failure patients (18). Some studies have also revealed

a potential clinical benefit of rIPC with a 6-week
Cardiovascular and cerebrovascular disease are leading
intervention reducing the size of diabetic foot ulcers (19)
causes of mortality in type 2 diabetes mellitus (T2DM)
and lower stroke recurrence following 1 year of rIPC (20,
(1). Importantly, the pathological consequences of
21). Whether an acute intensive rIPC intervention leads
T2DM predominately relate to vascular complications,
to improvements in cerebrovascular function assessed
encompassing both the macro- (e.g. cardio- and cerebro-
measuring dynamic cerebral autoregulation (dCA), a key
vascular disease) and microvasculature (e.g. retinopathy
mechanism protecting the brain from fluctuations in
and nephropathy) (2). Clinical studies show that diabetic
blood pressure, as well as peripheral endothelial function
individuals are more susceptible to ischemia-reperfusion
in T2DM patients is currently unknown, whilst such
injuries (IRI) compared to non-diabetics (3, 4), and reduced
benefits may have important clinical benefits, especially
tolerance to ischaemia has been considered responsible
those with functional limitations.
for the increase morbidity of ischaemic heart disease in
The primary aim of this pilot study was to obtain
T2DM (5). Conventionally, the main therapeutic target in
estimates of the change in conduit artery endothelial
T2DM has been glucose lowering but the importance of
function before and after endothelial IRI, a model that
targeting cardiovascular risk is increasingly recognised (6).
allows for the assessment of the efficacy of an intervention
Intensive glucose-lowering treatment has shown limited
to reduce the damage that is induced by reperfusion
benefits on all-cause morbidity and mortality from
following a period of ischaemia, succeeding a 7-day
cardiovascular causes (7). Lifestyle changes including
rIPC intervention. Acute intensive rIPC interventions
improved diet and physical activity are the mainstay
European Journal of Endocrinology

have improved conduit artery endothelial function (14)

of management with regular exercise promoted to
and attenuated the injury induced by an IRI in young
improve metabolic health and lower cardiovascular and
healthy individuals (16), yet it is not known whether rIPC
cerebrovascular risk in T2DM (8). Since a vast majority of
offers similar benefits to individuals with T2DM whereby
T2DM patients do not engage in regular physical activity
endothelial dysfunction is likely present (22).
(9, 10), perhaps because of disease complications (e.g. foot
The secondary aim was to obtain estimates of the
ulcers), alternative or adjunct interventions are required
change in cerebrovascular function after 7 days of daily
to improve cardiovascular and cerebrovascular disease
limb rIPC. Given the evidence rIPC has systemic beneficial
risk, similar to that of exercise, in this highly vulnerable
effects on vascular regulation and endothelial function
population.
(15, 17), improvements to blood vessel function may
Remote ischaemic preconditioning (rIPC) is a
translate to enhanced responsiveness to blood pressure
technique whereby short periods of cyclical tissue
within cerebral vessels (dynamic cerebral autoregulation).
ischaemia-reperfusion (of a limb) has been shown to
Additionally, application of rIPC can regulate several
have protective effects beyond the vascular bed directly
vasoactive biomarkers including, nitric oxide, adenosine
exposed to the IPC stimulus (11), potentially mediated
and bradykinin (12, 23), which may have the potential to
by neural and/or humoral signalling pathways (12, 13),
enhance dCA (24, 25, 26).
yet precise mechanisms remain elusive. When applied
prior to planned ischaemia (e.g. coronary artery bypass
surgery) or around spontaneous ischaemic events
(e.g. myocardial infarction), studies have reported the Methods
potential beneficial and protective effects of rIPC to
Participants
render remote (vascular) tissues and organs (e.g. heart)
resistant to ischaemic reperfusion injuries (12). More Twenty-one participants (13 males, 8 females, Table 1)
recently, studies have examined the impact of performing with clinically diagnosed T2DM who were managed with
multiple rIPC episodes and explored the potential of diet or metformin only were recruited for this randomised
rIPC as an intervention to improve vascular function controlled pilot study (Fig. 1). Participants were excluded
(13). Repeated rIPC interventions ranging from 1 to 8 if they had a history of stroke (including TIAs), diagnosis
weeks have been shown to improve vascular endothelial of chronic heart failure, were current smokers or were
function before and after ischemia reperfusion injuries being treated with sulphonylureas, DPPIV, GLP-1, SGLT2
(14, 15, 16), increase the levels of endothelial progenitor or insulin to control T2DM. Participants were informed of
cells (17), and increase coronary flow reserve in heart the study protocol verbally and in writing before providing

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Table 1 Descriptive characteristics of participants in rIPC and control groups (P values reported from independent samples
t-test). Values are presented as means ± s.d.

rIPC (n = 11, 5 females) Control (n = 10, 3 females) P value

Age (years) 58.8 ± 7.4 59.7 ± 9.6 0.72

Weight (kg) 92.7 ± 18.6 101.5 ± 32.5 0.62
BMI (kg/m2) 32.3 ± 6.6 33.9 ± 9.7 0.89
MAP (mmHg) 101 ± 14 107 ± 11 0.37
SBP (mmHg) 145 ± 16 151 ± 19 0.57
DBP (mmHg) 79 ± 9 84 ± 10 0.31
Metformin 9/11 4/10
Anti-hypertensive medication 4/11 0/10
Lipid-lowering medication 7/11 3/11

BMI, body mass index; DBP, diastolic blood pressure; MAP, mean arterial pressure; SBP, systolic blood pressure.

written informed consent. The study was approved
by the local NHS ethics committee and adhered to the
standards set out in the Declaration of Helsinki (2000).
All data collection took place at Liverpool John Moores
University. Registered clinical trial at ClinicalTrials.gov
NCT03598855. Trial is reported following CONSORT
European Journal of Endocrinology
of 3% for baseline artery diameter which is deemed good-
excellent based on previous analysis (31).
Analysis was performed using custom designed edge-
detection and wall-tracking software, which is largely
independent of investigator bias. Previous articles contain
detailed descriptions of our analytical approach (32,

recommendations (27). 33). Reproducibility of diameter measurements using

Research design
Participants attended the laboratory on three occasions,
separated by 7 days, having fasted overnight (12 h),
refraining from alcohol and exercise for 24 h and
caffeine for 12 h before each visit. Each visit consisted
of assessments of brachial artery function (before and
after ischemia reperfusion injury) and cerebrovascular
function. Assessments were performed at the same time
of day for each visit (28, 29) and occurred prior to group
randomisation (computer-generated-sequence) (Pre),
immediately following the cessation of the intervention
(Post) and 8 days following cessation of the intervention
(Post+8) (Fig. 2).
this semi-automated software is significantly better than
manual methods, significantly reduces observer error, and
possesses within-day coefficient of variation of 6.7% (33).
Allometric scaling for baseline diameter was performed
(34). FMD analysis was performed by a researcher blinded
to the group allocation using a single blinded coding-
randomised procedure.
Ischaemia reperfusion
Immediately following the baseline FMD, a temporary,
endothelial IRI was induced by inflating a cuff around

Measurements
Brachial artery endothelial function

Brachial artery endothelial function was assessed using

the flow-mediated dilation (FMD) technique following
20 min of supine rest (30). Images of the right brachial
artery were acquired using high-resolution ultrasound
(T3300; Terason, Burlington, MA, USA). Diameter, flow
and shear stress were measured prior to and following
5 min of forearm cuff inflation (D.E. Hokanson, Bellevue, Figure 1
WA, USA). All FMD measurements were performed by Screening, recruitment and completion of participants in the
the same sonographer with a day-to-day coefficient of study. A full colour version of this figure is available at https://
variation in FMD% of 11% and a coefficient of variation doi.org/10.1530/EJE-19-0378.

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 Clinical Study J D Maxwell, H H Carter and
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the upper arm to 220 mmHg for 20 min using a rapid
inflation pneumatic device. This was followed by a 20-min
reperfusion period before the FMD protocol was repeated.
A calculation of the relative % reduction in endothelial
function following endothelial IRI was performed.
The immediate decrease in FMD following temporary
endothelial dysfunction induced by the 20-min cuff
European Journal of Endocrinology
inflation is believed to reflect a reperfusion injury and
reduced nitric oxide (NO) bioavailability (35, 36, 37). The
relative % decrease in FMD following IRI was calculated
by dividing the absolute change between the two FMD’s
by the baseline FMD × 100.
Cerebrovascular function (baseline velocity and dynamic
cerebral autoregulation)
Following 20-min rest in the supine position, bilateral
middle cerebral artery velocity (MCAv) was continuously
measured through the temporal window using
transcranial Doppler ultrasonography (TCD). A 2-MHz
Doppler probe (Spencer Technologies, Seattle WA, USA)
was adjusted until an optimal signal was identified, as
described in detail previously (38), and held in place
using a Marc 600 head frame (Spencer Technologies,
Seattle, USA) to prevent subtle movement of the Doppler
probe and maintain insonation angle accuracy. Once the
optimal signals were attained in the temporal window,
the probe location and machine settings (depth, gain
and power) were recorded to identify the same imaging
site for all visits. Participants were instrumented with a
two-way valve-breathing (MLA1028, ADInstruments,
Colorado Springs, Colorado, USA) mouthpiece (MLA1026,
ADInstruments) from which partial pressure of end tidal
CO2 (PetCO2) was measured using a calibrated gas analyser
(ML206, ADInstruments). Continuous beat-by-beat
blood pressure (BP) was obtained from a digit (Finapres,
Amsterdam, Netherlands) and heart rate acquired from a
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Ischaemic preconditioning and 181:6
endothelial function in T2DM
Figure 2
Schematic of the study design. Each rIPC
consisted of four cycles of 5-min
ischaemia (220 mmHg) followed by 5-min
reperfusion applied unilaterally. At each
testing visit brachial artery flow mediated
dilation, ischaemic reperfusion injury and
cerebrovascular function were assessed.
rIPC, remote ischemic preconditioning. A
full colour version of this figure is
available at https://doi.org/10.1530/
EJE-19-0378.
three lead electrocardiogram (Powerlab, AD Instruments,
Oxford, UK). An index of cerebrovascular resistance
(CBVC) was calculated using the ratio of MCAv to BP. All
data was sampled at 50 Hz with a data acquisition system
(PowerLab, ADInstruments) and displayed on LabChart
(ADInstruments).
The relationship between BP and MCAv, referred to
as dynamic cerebral autoregulation (dCA), was assessed
using a squat to stand procedure in order to induce
transient changes in BP (39). Participants replicated the
experimenter whilst performing these manoeuvres in
order to achieve consistent movements. These manoeuvers
were performed at 0.10 Hz (5 s squat followed by 5 s stand)
for 5 min to create physiologically relevant changes in BP
via adjustments in posture that present challenges to the
autoregulatory system that are typically experienced in
daily life (40). The BP-MCAv relationship during these
manoeuvres were analysed in accordance with most
recent guidelines (41) using Transfer Function Analysis.
Resting measurements of MCAv, BP and PetCO2 were
extracted from LabChart averaged over a 5-min recording.
Data from 5-min recording of squat to stand manoeuvres
for dCA were extracted from LabChart beat-to-beat using
ECG tracing. Cerebrovascular conductance (CbVC) was
calculated using; MCAv/MAP. Transfer function analysis
was applied using MATLAB (2010a; MathWorks-Inc.,
Natick, MA, USA) in order to calculate associated power
(gain) and timing (phases) and linearity of MAP and MCAv
(coherence) using a Cerebral Autoregulation Network
(CARNet) provided script (41).
Interventions
rIPC
The participants randomised into the rIPC intervention
group (n = 11) each received a hand-held BP device (Welch
Allyn DuraShock™ DS45, NY, USA) to self-administer
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 Clinical Study J D Maxwell, H H Carter and
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rIPC. The cuff was placed around the upper arm and
inflated to 220 mmHg for 5 min, followed by 5 min
deflation, and this cycle was repeated a further three times.
This process was performed daily for 7 days. The arm to
which the participants applied the rIPC was randomised
between the same arm the FMD’s were performed (IPC
arm, n = 5) and the contra lateral arm (n = 6). Participants
were supervised for their first rIPC bout to ensure it was
correctly performed and were then free to perform the rIPC
at any time of day and noted this in a diary to monitor
compliance. Participants were instructed to follow their
normal routine and to abstain from any new physical
activity or changes in dietary habits
Control
Each participant (n = 10) was instructed to follow their
normal routine and to abstain from any new physical
activity or change in dietary habits.
European Journal of Endocrinology
Statistical analysis
Given that this is a pilot study to obtain estimates of
primary and secondary outcome variables, no a priori
sample size was calculated. The primary outcome in the
study is FMD and the primary comparison is between pre
to post intervention. Using the data collected (rIPC group
n = 11, control group n = 10) in the study we calculated post
hoc power of the present study, but also calculated the
sample size for a future, fully powered randomised control
trial for both primary and secondary outcome variables
(G*Power 3.1.5).
For exploratory purposes, we performed statistical
analysis on our primary comparison (i.e. pre-to-post) to
provide an estimate of the change in the primary and
secondary outcome variables. Delta changes (∆) from pre
to post were calculated for each group and entered as the
dependent variable in a linear mixed model (Statistical
Package for the Social Sciences, version 20: SPSS Inc.)
with pre-intervention data used as a covariate. Data are
presented in the text as mean and 95% confidence intervals
(95% CI). P values are presented, but not interpreted.
The changes in the data are described in relation to a
minimally clinical important difference (MCID) of 1%
for FMD, calculated based upon previous intervention
studies (14, 15, 42) and from a meta-analysis indicating
that 1% improvement in brachial FMD decreases the risk
of future cardiovascular events by 13% (43). The MCID
for LF gain was between 0.07 and 0.26%cm/s %/mmHg
mmHg/%. This was based on studies showing differences
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Ischaemic preconditioning and 181:6
endothelial function in T2DM
between healthy and diseased populations (44, 45) due to
the limited intervention studies to date.
Results
Participants allocated to each intervention were similar
in terms of age, BMI and BP status (Table 1). Participants
randomised into the rIPC intervention group (n = 11)
demonstrated 96% compliance to the rIPC intervention.
Brachial artery endothelial function
Baseline FMD
Brachial artery FMD improved by 1.3% (95% CI: 0.69 to
3.80; P = 0.09) with rIPC compared to control from pre to
post, which was greater than our MCID of 1%. Our data
provided 65% power to detect a between-group difference
in FMD from pre-post. Using this data, a sample size of
20 in each group would provide 90% power to detect a
statistically significant (P < 0.05) between groups in FMD
in a future randomised control trial.
In the current study, FMD was 0.9 (−3.9, 2.0%) lower
in the rIPC group compared to control at pre, but 0.9
(−2.3, 4.0%) higher than control at post, which remained
higher at post+8 (0.8 (−2.3, 3.9%), Fig. 3). The associated
changes in baseline diameter, peak diameter, shear rate or
time-to-peak diameter between interventions or over time
were negligible from pre to post and post 8 (Table 2).
Endothelial IRI
When examining the FMD after the endothelial IRI
(Table 2). FMD was 2.3 (−5.4, 0.8%) lower in the rIPC
group compared to control at pre, but only 0.1 (−2.8,
2.6%) lower at post and 0.5 (−2.9, 2.0%) at post+8. FMD
increased over the intervention period by 0.7% (−0.1,
1.6). These directional changes were similar when the
FMD data were expressed as a relative change. Prior
to the intervention, the relative % decrease in FMD in
response to IRI was 24.7% (−10.4, 49.7%) greater in the
rIPC group compared to control (Table 2). This difference
was attenuated to 4.5% (−23.9, 14.9%) at post and 1.4%
(−22.5, 19.6%) at post+8.
Cerebrovascular function
Low-frequency normalised gain changed by 0.23 %cm/s
%/mmHg mmHg/% (−0.12, 0.59; P = 0.18) following
rIPC compared to control from pre to post, which was
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 Clinical Study J D Maxwell, H H Carter and
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European Journal of Endocrinology
Figure 3
Baseline brachial artery FMD% (A), Post IR FMD% (B) and the
relative % decrease (C) before (Pre), immediately after (Post)
and 8 days following the intervention (Post+8) in the rIPC
group (closed circles) and control group (open circles).
*Denotes significant main effect from time (P < 0.05). FMD,
flow mediated dilation; rIPC, remote ischaemic
preconditioning; IR, ischaemia-reperfusion.
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Ischaemic preconditioning and 181:6
endothelial function in T2DM
greater than our lower level of the MCID of 0.07 and
0.26. Our data provided 29% power to detect a between-
group difference in LF normalised gain from pre-post.
Using these data a sample size of 50 in each group would
provide 90% power to detect a statistically significant
(P < 0.05) between group difference in LF normalised gain
in a future randomised control trial.
In the current study, the associated changes in
MCAv, PetCO2 or CbVC were negligible between both
conditions and over time from pre to post and post 8
(Table 3). MAP decreased by 4 mmHg (2, 6 mmHg) across
both interventions. Similarly, SBP decreased by 5 mmHg
(−9, −1 mmHg) and DBP by 3 mmHg (−5, −1 mmHg;
Table 3). The directional changes in all of the dCA
variables were negligible between conditions (Table 4).
Discussion
The aim of this study was to obtain estimates of changes in
peripheral conduit artery endothelial and cerebrovascular
function and the response to endothelial IRI to 7-days of
daily limb rIPC in T2DM. We provide preliminary evidence
that 7-days of daily rIPC in a representative sample of
patients can enhance conduit artery endothelial function
measured using FMD and provide protection against
a temporary decline in endothelial function following
ischaemia reperfusion. Although our observations suggest
that rIPC had little impact on cerebrovascular function,
our preliminary directional findings and sample size
estimations suggest the ability of a rIPC intervention to
improve peripheral vasculature in T2DM. These effects
should be explored further in a larger, fully powered trial.
We provide preliminary evidence that daily rIPC
can increase conduit artery endothelial function. This is
clinically important given that individuals with T2DM
exhibit endothelial dysfunction (46, 47) and are also at high
risk of microvascular disease of the small vessels. Chronic
hyperglycaemia limits the ability of the endothelial cells
to produce nitric oxide (NO) which has important anti-
atherogenic properties, contributing to the maintenance
of vascular homeostasis (48). This is relevant as vascular
dysfunction plays a major role in the development of
cardiovascular complications (49). Given that a meta-
analysis confirmed that a 1% improvement in brachial
FMD decreases the risk of future cardiovascular events
by 13% (43), strategies to improve vascular endothelial
function are crucial. Numerous clinical outcome studies
have demonstrated that brachial artery FMD is a good
predictor of cardiovascular risk (50). Improvements in
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Table 2 Brachial artery flow mediated dilation before (Pre), immediately following (Post) and 8 days (Post+8) after the end of the
intervention in both the intervention (rIPC) groups and control. Data in tables shows FMD characteristics in both before and after
ischemia-reperfusion injury. Values are presented as means ± s.d.

rIPC Group (n = 11) Control group (n = 10)

Pre Post Post+8 Pre Post Post+8
Baseline
Resting diameter (mm) 4.4 ± 0.6 4.3 ± 0.7 4.3 ± 0.6 4.5 ± 0.7 4.6 ± 0.7 4.6 ± 0.7
FMD% 5.5 ± 1.7 7.2 ± 2.4 7.2 ± 2.6 6.8 ± 2.9 7.0 ± 3.0 6.9 ± 2.5
Time to peak (s) 70 ± 30 65 ± 24 71 ± 25 68 ± 23 63 ± 22 69 ± 20
Shear AUC (103) 16.9 ± 12.5 19.3 ± 12.2 17.1 ± 11.9 18.3 ± 11.4 19.6 ± 15.4 18.2 ± 8.1
Post-ischaemia reperfusion
Resting diameter (mm) 4.5 ± 0.8 4.5 ± 0.8 4.5 ± 0.8 4.7 ± 0.7 4.8 ± 0.7 4.9 ± 0.7
FMD% 2.4 ± 3.7 4.5 ± 3.0 4.0 ± 2.8 4.7 ± 1.9 4.6 ± 2.5 4.5 ± 1.9
Time to peak (s) 72 ± 23 71 ± 27 74 ± 23 53 ± 24 53 ± 17 64 ± 21
Shear AUC (103) 14.9 ± 12.9 14.5 ± 11.6 12.4 ± 8.1 15.6 ± 12.0 15.7 ± 6.5 13.6 ± 6.6
Ischaemia-reperfusion injury
Relative % change following IRI 62.2 ± 44.3 38.0 ± 20.4 39.4 ± 25.0 37.6 ± 13.2 42.5 ± 15.4 40.8 ± 12.0

AUC, area under the curve; FMD, flow mediated dilation; IRI, ischemia-reperfusion injury; rIPC, remote ischemic preconditioning.

FMD are associated with enhanced NO production (51) leads to an increase in vascular endothelial growth factor
and NO pathways are impaired with diabetes (22, 52). and endothelial progenitor cells (17), which may improve
European Journal of Endocrinology

Our data suggest that vascular endothelial function
can be improved in 7 days and remain elevated 8 days
following the end of the intervention. Given that
rIPC was administered in the arm that received the
preconditioning stimulus as well as in the contralateral
arm our data supports the notion that rIPC has local
and systemic effects on the vascular system (14). As this
present study was not designed as a mechanistic study,
we can only speculate on potential mechanisms involved
in the change in FMD we observed. Episodic increases in
shear stress is likely to represent a major physiological
stimulus for the local improvements in FMD (13),
however, is unlikely to have effected contralateral arm
FMD. The mechanisms mediating the systemic effects
of rIPC remains elusive. Systemic stimuli or circulating
markers activated by rIPC more likely explain the remote
improvement in conduit artery FMD. For example, rIPC
endothelial function in remote areas (53). However, more
research studies are required to gain insight into exact
mediating mechanisms.
The present study provides evidence that daily rIPC
can provide protection against endothelial IRI in T2DM.
The endothelial IRI model performed in this study has
been used by previous studies (16, 54) and is acknowledged
as a surrogate model for myocardial reperfusion injuries.
A similar model using forearm IRI identified that the
decrease in FMD occurs as a result of a decrease in plasma
nitrite and plasma nitrate concentrations, indicating a
reduction in NO bioavailability which is still decreased
up to 50 min post reperfusion (37). Our findings agree
with previous rIPC studies showing (partial) prevention of
endothelial dysfunction after IRI when preceded by a bout
of rIPC (16). Reduced endothelial dysfunction against
IRI is of clinical significance given that patients with

Table 3 Baseline hemodynamics from 5-min recordings before (Pre), immediately following (Post) and 8 days (Post+8) after the
end of the intervention. Values are presented as means ± s.d.

rIPC group (n = 11) Control group (n = 10)

Pre Post Post+8 Pre Post Post+8
Resting data
MAP (mmHg) 101 ± 14 100 ± 10 96 ± 12 107 ± 12 104 ± 12 104 ± 9
MCAv (cm/s) 56.2 ± 8.0 55.5 ± 7.8 55.7 ± 10.9 53.6 ± 11.0 53.2 ± 10.1 53.5 ± 9.8
PetCO2 (mmHg) 38.4 ± 6.0 38.1 ± 5.8 37.7 ± 4.7 38.8 ± 6.4 41.5 ± 6.0 42.2 ± 6.2
CbVC (cm/s/mmHg) 0.56 ± 0.10 0.55 ± 0.10 0.58 ± 0.14 0.52 ± 0.12 0.53 ± 0.12 0.52 ± 0.12
SBP (mmHg) 145 ± 16 144 ± 13 139 ± 16 151 ± 19 151 ± 17 148 ± 17
DBP (mmHg) 78 ± 9 77 ± 9 75 ± 10 84 ± 10 81 ± 9 83 ± 10

CbVC, cerebral vascular conductance; DBP, diastolic blood pressure; MAP, mean arterial pressure; MCAv, middle cerebral artery velocity; PetCO2, partial
pressure of end tidal carbon dioxide; rIPC, remote ischemic preconditioning; SBP, systolic blood pressure.

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 Clinical Study J D Maxwell, H H Carter and
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Table 4 Transfer function parameters from dynamic cerebral autoregulation before (Pre), immediately following (Post) and 8
days (Post+8) after the end of the intervention using squat-stand manoeuvres (0.10 Hz). Values are presented as means ± s.d.
rIPC Group (n = 10)
Pre Post
PetCO2 (mmHg) 40.3 ± 3.7 39.2 ± 48
Coherence 0.65 ± 0.10 0.60 ± 0.12
Phase (radians) 0.44 ± 0.12 0.48 ± 0.28
Gain (cm/s/mmHg) 0.66 ± 0.16 0.69 ± 0.20
Normalised gain (%/mmHg) 1.12 ± 0.21 1.23 ± 0.20
PetCO2, partial pressure of end tidal carbon dioxide; rIPC, remote ischemic preconditioning.
T2DM demonstrate more extensive injury in response to
ischaemia reperfusion (55). Interestingly, a previous six-
week rIPC intervention performed on patients with T2DM
with foot ulcers identified an augmentation in the wound
size of the foot ulcers in the patients who received the
rIPC compared to a control (19), further demonstrating
the capability of a rIPC intervention to treat ischaemic
induced complications in a diabetic patient group.
We identified that a 7-day repeated rIPC intervention
European Journal of Endocrinology
had little impact on resting MCAv or dCA. Despite the
considerable literature on the effects of rIPC on cardiac
and peripheral vascular function in humans, there are
few studies on cerebrovascular function, even with stroke
and cerebrovascular disease being a leading cause of death
worldwide (56). We performed a post hoc analysis of power
in this study which revealed that more participants would
have been required for adequate statistical power; therefore,
the data should be interpreted with caution. It is likely
that control of cerebral autoregulation is multifactorial
encompassing neurogenic, metabolic, myogenic and
endothelial factors (57). The exact contribution of
each, including the endothelium is debated. Evidence
suggests that the endothelium carries mechanoreceptor
properties that allows it to actively contribute to cerebral
autoregulation following changes in arterial shear stress
and transmural pressure (58). Therefore, a healthier and
more active endothelium may have translated to improved
dCA; yet, this was not evident in the present study. Given
that dCA is controlled by highly sensitive and tight
regulatory factors, it is possible that 7 days of rIPC was
not a sufficient enough stimulus to result in any change/
adaption. This potential explanation is supported by the
fact that the only previous studies examining repeated
rIPC on human cerebrovascular markers employed daily
rIPC for 300 days (20), 180 days (21) and 365 days (59)
identifying increases in cerebral perfusion and reductions
in stroke reccurrence but did not assess functional
markers of the cerebral circulation. Whilst there is also
a strong association between T2DM and cerebrovascular
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044
Ischaemic preconditioning and 181:6
endothelial function in T2DM
Control group (n = 9)
Post+8 Pre Post Post+8
38.3 ± 3.4 38.8 ± 7.5 38.3 ± 6.6 39.3 ± 5.6
0.60 ± 0.21 0.61 ± 0.17 0.59 ± 0.18 0.60 ± 0.22
0.48 ± 020 0.61 ± 0.32 0.52 ± 0.25 0.52 ± 0.22
0.72 ± 0.27 0.71 ± 0.18 0.69 ± 0.26 0.71 ± 0.24
1.36 ± 0.56 1.40 ± 0.27 1.27 ± 0.50 1.37 ± 0.32
dysfunction (60), none of our participants had any
previous documented cerebrovascular complications
unlike the aforementioned studies and were of shorter
duration of T2DM.
Given our data was collected for the purposed of
generating estimates for a larger trial we acknowledge we
have a small sample and limited statistical power. We also
acknowledge a number of other study limitations. Pre-
intervention characteristics, primarily MAP, metformin
and statin use were different between the intervention
and control group and some evidence now suggests
that certain medication used to treat risk factors of
cardiovascular disease can alter the response to cardio
protective interventions (61). Additionally, HbA1c data
were not collected to examine clinical relevance to
glucose control nor biomarkers of NO bioavailability.
Stratification for medication and markers of glucose
control and NO bioavailability should be incorporated
into a larger fully powered future trial. Lastly, middle
cerebral artery blood velocity was measured using
transcranial Doppler, a technique that provides a reliable
surrogate for absolute cerebral blood flow providing the
insonated artery diameter remains constant across and
between the study conditions (62). Although we believe
it is unlikely, we cannot discount the possibility that rIPC
induced a change in middle cerebral artery diameter that
impacted our measures of cerebral blood flow. A future
trial may consider assessment of extra cranial vessels (e.g.
internal carotid artery) with ultrasound to assess changes
in artery diameter as an indicator of changes in diameter.
Clinical perspectives
Endothelial dysfunction represents a significant event in
the atherosclerotic cascade and predicts cardiovascular
and cerebrovascular events (43). Our findings suggest
that rIPC interventions have the potential to represent a
low-cost, simple and importantly, non-invasive strategy
to improve endothelial function in a patient group with
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via free access
 Clinical Study J D Maxwell, H H Carter and Ischaemic preconditioning and 181:6
others endothelial function in T2DM

likely endothelial dysfunction and at higher risk of vascular

complications and it may be especially useful in those
with functional limitations. Nevertheless, future trials
with adequate statistical power are required to identify if
rIPC has the ability to improve vascular outcomes in this
population.

Conclusion

The present study has provided estimates of sample

size for a randomised control trial exploring the impact
of daily rIPC for 7 days on peripheral endothelial and
cerebrovascular function. The directional changes
outlined from our study suggest peripheral
endothelial function and responses to endothelial IRI can
be enhanced by daily rIPC in patients with T2DM.
European Journal of Endocrinology

Declaration of interest
The authors declare that there is no conflict of interest that could be
perceived as prejudicing the impartiality of this study.

Funding
This study was funded by the Danish Diabetes Academy supported by the
Novo Nordisk Foundation and The Independent Research Fund Denmark-
Medical Science (DFF-6110-00021).

Author contribution statement

D H J Thijssen and H Jones: senior authors.

https://eje.bioscientifica.com
045 Downloaded from Bioscientifica.com at 10/21/2022 11:28:25PM
via free access
 JNHA

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±
± ±

046
 JNH, Vol. 3, No.2, Agustus 2015

THE EFFECT OF REMOTE ISCHEMIC PRECONDITIONING IN DIABETIC

PATIENTS AFTER ELECTIVE PERCUTANEUS CORONARY INTERVENTION

Novi Anggriyani*, Donna Paramita**, Sodiqur Rifqi**

Department of Cardiology and Vascular Medicine, Diponegoro University School of

Medicine, Dr Kariadi General Hospital Semarang

Background
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HGJHG VZRUG¶ DV UHSHUIXVLRQ ZRXOG UHVWRUH R[\JHQ DQG QXWULHQWV VXSSO\ WR WKH LVFKHPLF
myocardium to improve its functional recovery, but in the other hand reperfusion could
augment myocardial ischemic damage, known as myocardial ischemia-reperfusion (I/R)
injury. The brief and repeated cycles of I/R given at a distant organ before a sustained
ischemia and reperfusion, known as remote ischemic preconditioning (RIPC), would protect
the heart from lethal I/R injury.
Objective
The effect of ischemic preconditioning in a diabetic heart is a contradictory whether it could
improve or worsen the damage degree of myocardial I/R injury, as reported by some
previous studies. These inconsistent reports need further studies.
Methods
Twenty-four diabetic patients with stable CAD undergoing elective percutaneus coronary
intervention were randomly assigned to 2 groups: 14 patients submitted to RIPC and 10
patients were control group. We induced RIPC by inflating a blood pressure cuff placed on
the upper limb to 20 mmHg above systolic arterial pressure for 5 min and deflating the cuff
for 5 min; 4 cycles were performed. All patients had CK-MB level measured at baseline and
18-24 hours after the elective PCI. Myocardial injury was considered when post-PCI CK-MB
level rose up to 1-3 fold of the upper normal limit.
Results
A higher proportion in control group (40%) experienced myocardial injury, compared with
the group receiving RIPC (0%) (p = 0.02). The mean of baseline CK-MB was equal in both
control and RIPC groups (19.07 ± 2.84 and 17.5 ± 2.32, respectively; p = 0.165). While the
mean of post-PCI CK-MB level in two groups differed significantly (34.2 ± 10.43 and 24.42
± 4.03, respectively; p = 0.017).

Conclusions
RIPC lower the incidence of myocardial injury in diabetic patients after elective percutaneus
coronary intervention. These data suggest that diabetic patients still gain protection of RIPC.

Introduction

The strategy of reperfusion of ischemic myocardium in patients with coronary artery

disease has led to a significant improvement of outcomes. However, reperfusion after a
prolonged period of ischemia damages the myocardium, through a process known as

047
 JNH, Vol. 3, No.2, Agustus 2015

ischemia/reperfusion injury (I/R)1. Transient sublethal episodes of ischemia before a

prolonged I/R injury, known as ischemic preconditioning (IPC), have been shown to reduce
the extent of myocardial infarction (MI). This protection not only acts locally but also can
protect distant tissues, a phenomenon known as remote IPC (RIPC), and limits myocardial
infarction size in animal models2. In humans, RIPC protects against endothelial I/R injury3
and the extent of MI after adult coronary bypass surgery, pediatric surgery, and noncardiac
surgery4.

Chronic diabetes mellitus remains one of root causes of mortality in developed as well
as developing countries with towering prevalence. It has been stated that 23.5 million adults
of 20 years or older have had a diagnosis of both coronary artery disease and diabetes
mellitus, with an estimation that by 2025, an additional 9% of the total US population will
have a diagnosis of a combination of these disorders5. Studies have demonstrated
preconditioning-mediated cardioprotection in the diabetic myocardium. Moreover, Tatsumi et
al.5 suggested that the diabetic myocardium could rather benefit more from preconditioning
stimulus than the normal myocardium, possibly due to diabetes induced reduction in the
production of glycolytic metabolites during sustained ischemia and concomitant attenuation
of intracellular acidosis. However, it remains a question whether the diabetic myocardium
could be protected by the preconditioning stimulus, as considerable numbers of studies
paradoxically demonstrated no preconditioning mediated cardioprotection in the presence of
chronic diabetes mellitus.

Materials and Methods

Twenty-four diabetic patients with stable CAD undergoing elective percutaneus

coronary intervention, and informed written consent was obtained from each patient.
Exclusion criteria were patients with pre procedural CK-MB level above normal limit, acute
ST-elevation myocardial infarction undergoing primary PCI, acute coronary syndrome less
than 7 days before elective PCI, angina pectoris CCS III within the last 24 hours before
elective PCI, systolic blood pressure < 90 mmHg, pulmonary oedema or cardiogenic shock,
significant renal insufficiency (creatinine level > 2.5 mg/dL that may influence blood enzyme
kinetic), target lesion Chronic Total Occlusion (CTO) more than 3 months, with the presence
of collateral vessels, symptomatic peripheral arterial disease. The patients were randomly
assigned to 2 groups: 14 patients submitted to RIPC and 10 patients were control group.

048
 JNH, Vol. 3, No.2, Agustus 2015

Remote ischaemic preconditioning (RIPC)

We induced RIPC by inflating a blood pressure cuff placed on the upper limb to 20 mmHg
above systolic arterial pressure for 5 min and deflating the cuff for 5 min; 4 cycles were
performed, approximately 1 hour before PCI. RIPC was not performed in control group.
This protocol adapted the study conducted by Rentoukas1, which showed the useful role of
RIPC for the prevention of reperfusion injury in patients submitted to primary PCI.

Percutaneus Coronary Intervention

PCI was conducted with the monorail technique and 7F catheters. The selection of coronary
balloons and stents was left to the discretion of the interventional cardiologist. Coronary
stents were implanted in all patients of both groups after balloon pre-dilatation of the target
lesion according to the decision of the operators. All lesions treated were de novo lesions of
native coronary arteries.

Blood sampling and laboratory measurements

Venous blood samples for CK-MB level measurements were obtained at baseline and 12-48
hours after the elective PCI. Serum was obtained by centrifugation at 1000 g for 15 min. CK-
MB concentrations between 7 and 25 U/l were considered to be within the reference range.
Values are expressed as nanograms per millilitre. Myocardial injury was considered when
post-PCI CK-MB level rose up to 1-3 fold of the upper normal limit.

Statistical analysis
All data are expressed as mean. For comparisons EHWZHHQ WKH WZR PDLQ JURXSV 6WXGHQW¶V W
test (unpaired) was used. Significance was taken at a p value of 0.05.

RESULTS
1. Baseline characteristic
Table 1 shows the baseline epidemiological and clinical features of the groups. There
were no important differences between the groups. Except for the level of blood glucose,
which the mean of RIPC group and control group differed significantly (163.7 ± 41.8 and
124.8 ± 24.5, respectively ; p = 0.015).

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 JNH, Vol. 3, No.2, Agustus 2015

Table 1. Baseline Epidemiological and Clinical Features of the Study Population

RIPC Groups Control Groups p value
Age, mean ± SD 60.5 ± 5.5 58.1 ± 5.4 0.302
Male, n (%) 11 (78.6) 7 (70) 0.615
BMI, mean ± SD 23.6 ± 2.9 23.6 ± 3.1 0.959
Ejection fraction, mean ± SD 54.5 ± 8.8 53.9 ± 8.8 0.871
CAD Risk Factors, n (%)
Hypertension 11 (78.6) 9 (90) 0.437
Active smoker 2 (14.3) 1 (10) 0.643
Family history 2 (14.3) 0 (0) 0.332
Hypercholesterolemia 3 (21.4) 2 (20) 0.668
Laboratory findings, mean ± SD
Hb 12.29 ± 1.35 13.54 ± 1.88 0.071
Leukocyte 9142.14 ± 1341 9028 ± 1751 0.858
Platelet 270550 ± 69826 259210 ± 78867 0.714
Ureum 40.7 ± 16.3 30.6 ± 10.6 0.1
Creatinine 1.3 ± 0.2 1.3 ± 0.2 0.66
Blood glucose 163.7 ± 41.8 124.8 ± 24.5 0.015
Total cholesterol 209.7 ± 38.7 188.4 ± 42.5 0.215
HDL 31.1 ± 4.1 29.7 ± 3.3 0.372
Triglyceride 133 ± 55.6 122 ± 33.9 0.599
Medications at enrollment, n (%)
Beta blockers 6 (42.8) 3 (30) 0.418
Nitrates 8 (57.1) 7 (70) 0.418
Statin 13 (92.8) 10 (100) 0.583
ACE inhibitors 8 (57.1) 7 (70) 0.418
ARB 12 (85.7) 7 (70) 0.332
Trimetazidine 12 (85.7) 9 (90) 0.629

2. Angiographic and Interventional Procedure Characteristic

No differences were observed between the studied groups in angiographic and
interventional procedure characteristic in terms of lesion type (ACC/AHA), target vessels,
stenosis severity, predilatation type, stenting type, and total stent length, as shown in table 2.
In this study, all patients received DES.

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 JNH, Vol. 3, No.2, Agustus 2015

Table 2. Angiographic and Interventional Procedure Characteristic

RIPC Groups Control Groups p value
Lesion type (ACC/AHA), n
A 0 1 0.272
B 12 6
C 2 3
Target vessels, n
1 8 5 0.643
2 5 3
3 1 2
Stenosis severity, %, mean±SD 80.71 ± 6.75 85 ± 8.16 0.174
Predilatation time, s, mean±SD 71.5 ± 54.5 71.8 ± 53.3 0.989
Stenting time, s, mean±SD 76.7 ± 50.8 81.6 ± 37.4 0.802
Total stent length, mm, mean±SD 52.1 ± 26.4 61.5 ± 35.2 0.464

3. Baseline and Post-PCI CK-MB Level

Table 3 presents the mean of baseline and post-PCI CK-MB level of the two groups.
There were no significant differences between groups in baseline levels of CK-MB (19.07 ±
2.84 and 17.5 ± 2.32, respectively; p = 0.165). While the mean of post-PCI CK-MB level in
two groups differed significantly (34.2 ± 10.43 and 24.42 ± 4.03, respectively; p = 0.017). A
higher proportion in control group (40%) experienced myocardial injury, compared with the
group receiving RIPC (0%) (p = 0.02).

Table 3. Baseline and Post-PCI CK-MB Level

RIPC Groups Control Groups p value
Baseline CK-MB, U/l 19.0 ± 2.8 17.5 ± 2.3 0.165
Post-PCI CK-MB, U/l 24.4 ± 4.0 34.4 ± 10.4 0.017
Myocardial Injury, n (%) 0 4 0.02

DISCUSSION

This study shows that RIPC lower the incidence of myocardial injury in diabetic
patients after elective percutaneus coronary intervention, as expressed in the mean of baseline
and post-PCI CK-MB level. This finding was consistent with the study of Zhu et al.6

051
 JNH, Vol. 3, No.2, Agustus 2015

conducted in streptozotocin(STZ)-induced diabetic rats. In their study, Zhu et al.6

demonstrated that non-invasive limb IPC ameliorated ventricular arrhythmia, reduced
myocardial infarct size, increased activities of total superoxide dismutase (SOD), manganese-
SOD and glutathione peroxidase. It is concluded that non-invasive IPC reduces oxidative
stress and attenuates myocardium ischemia-reperfusion injury.

The protective action of ischemic post-conditioning has been known for some time.
One study by Tatsumi et al.5 demonstrated cardioprotective effects of IPC (2 cycles of
ischemia and reperfusion of 5 min each) in STZ-induced diabetic rats against myocardial I/R
involved preservation of mitochondrial oxidative phosphorylation, inhibition of glycolysis
during ischemia, and the simultaneous attenuation of intracellular acidosis in the diabetic
heart explaining the possible mechanisms involved. In addition, Ravingerova et al.7 showed
that STZ-induced chronic diabetic rat hearts could be more benefited from IPC (1 cycle of 5-
min ischemia and 10-min reperfusion) mediated cardioprotection. Interestingly, Tsang et al.8
observed cardioprotection by IPC with 3 cycles (5-min global ischemia followed by 10-min
reperfusion) in Goto-Kakizaki diabetic rat hearts, while IPC with 1 cycle did not have any
cardioprotective effect. This study suggested that induction of Akt phosphorylation could
have taken place in multiple cycles of IPC (but not in single cycle of IPC) that could have
afforded cardioprotection in diabetic rat hearts. In summary, myocardial Akt
phosphorylation, preservation of mitochondrial oxidative phosphorylation, inhibition of
glycolysis during ischemia, decreased production of glycolytic metabolites, reduced
intracellular acidosis and attenuated oxidative stress could be potential mechanisms involved
in IPC-mediated cardioprotection against I/R injury in the diabetic heart9, 10
.

CONCLUSION
RIPC lower the incidence of myocardial injury in diabetic patients after elective
percutaneus coronary intervention. These data suggest that diabetic patients still gain
protection of RIPC, possibly due to myocardial Akt phosphorylation, preservation of
mitochondrial oxidative phosphorylation, inhibition of glycolysis during ischemia,
decreased production of glycolytic metabolites, reduced intracellular acidosis and
attenuated oxidative stress.

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 Cardiovascular Diabetology

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Epps and Smart Cardiovasc Diabetol (2016) 15:130
DOI 10.1186/s12933-016-0444-z Cardiovascular Diabetology

REVIEW Open Access

Remote ischaemic conditioning in the

context of type 2 diabetes and neuropathy: the
case for repeat application as a novel therapy
for lower extremity ulceration
J. A. Epps and N. A. Smart*

Abstract
An emerging treatment modality for reducing damage caused by ischaemia–reperfusion injury is ischaemic condi-
tioning. This technique induces short periods of ischaemia that have been found to protect against a more significant
ischaemic insult. Remote ischaemic conditioning (RIC) can be administered more conveniently and safely, by inflation
of a pneumatic blood pressure cuff to a suprasystolic pressure on a limb. Protection is then transferred to a remote
organ via humoral and neural pathways. The diabetic state is particularly vulnerable to ischaemia–reperfusion injury,
and ischaemia is a significant cause of many diabetic complications, including the diabetic foot. Despite this, studies
utilising ischaemic conditioning and RIC in type 2 diabetes have often been disappointing. A newer strategy, repeat
RIC, involves the repeated application of short periods of limb ischaemia over days or weeks. It has been demon-
strated that this improves endothelial function, skin microcirculation, and modulates the systemic inflammatory
response. Repeat RIC was recently shown to be beneficial for healing in lower extremity diabetic ulcers. This article
summarises the mechanisms of RIC, and the impact that type 2 diabetes may have upon these, with the role of neural
mechanisms in the context of diabetic neuropathy a focus. Repeat RIC may show more promise than RIC in type 2
diabetes, and its potential mechanisms and applications will also be explored. Considering the high costs, rates of
chronicity and serious complications resulting from diabetic lower extremity ulceration, repeat RIC has the potential
to be an effective novel advanced therapy for this condition.
Keywords: Diabetes, Neuropathy, Diabetic foot, Ulcer, Remote ischaemic conditioning, Repeat remote ischaemic
conditioning, Endothelial function, Neovascularisation, Endothelial progenitor cells

Introduction between $9 and $13 billion per annum using conserva-

Diabetes-related foot disease is a frequent complication
of type 2 diabetes mellitus (T2DM), with up to 25 % of
diabetic patients eventually developing foot ulceration
[1–3]. The physical, emotional and financial costs for
patients, their carers and the community are substan-
tial. The utilisation of health care services and associated
expenses are high in diabetic patients with ulcers [4]. In
the US, it has been shown that the costs of foot ulceration
caused by diabetes and related complications amount to
*Correspondence: nsmart2@une.edu.au
School of Science and Technology, The University of New England,
Armidale, NSW 2351, Australia
tive measures [1, 5]. Responsible for a large proportion
of hospital admissions and health care costs in diabetes
[2], such figures are likely to increase. The prevalence of
T2DM is escalating worldwide [2, 6]. Although interna-
tional data are scarce, an improved survival rate of dia-
betic patients has been reported in wealthier nations in
particular [7, 8]. A potential consequence of this may be
a higher prevalence of diabetic ulcers, especially as dura-
tion of diabetes is a major risk factor for the development
of lower extremity ulceration [9].
Arguably the most feared complication of diabetic
foot ulceration is the need for amputation. A UK study
observed this occurred in 19 % of cases over a 5 year

© 2016 The Author(s). This article is distributed under the terms of the Creative Commons Attribution 4.0 International License
(http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium,
provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license,
and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/
publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

055
Epps and Smart Cardiovasc Diabetol (2016) 15:130
period in patients with a diabetic ulcer present for
2 weeks [10]. In a 2016 systematic review, amputation
rates associated with diabetic ulceration ranged from 5
to 35 % [11]. Rates have improved in some higher income
countries by 40 to 60 % [12], however data from countries
of lower income and poorer access to health care (yet a
higher prevalence of diabetes) are notably lacking [8, 12].
Further, some population groups even within wealthy
nations are clearly at much higher risk of amputation [13–
15]. In Australia, the Indigenous diabetic community is at
particularly high risk. A 2000–2008 analysis revealed in
some regions this population experienced up to 27 times
higher risk of minor amputation and 38 times higher risk
of major amputation [13]. The benefits of improved access
to primary health foot care, in addition to multidiscipli-
nary foot clinics, cannot be overemphasised. They are
highlighted by the impressive 72 % reduction, following
adjustment of variables, in lower extremity amputation
rates identified in a 15 year longitudinal observational
study of a West Australian city [16]. Despite this, from
2002 to 2012 there was a 30 % overall increase in diabetes-
related amputations in well-resourced Australia [17], with
one of the highest rates of amputation amongst devel-
oped nations [18]. The need to improve statistics world-
wide for diabetic ulcer prevalence, complication rate and
cost is undeniable, and new therapeutic options must be
considered. This article will assess the potential for repeat
remote ischaemic conditioning (RRIC) as a novel adjuvant
treatment in diabetic lower extremity ulceration.
Methods
Utilising PubMed and Scopus databases in January 2016,
publications relating to all forms of local and remote
ischaemic conditioning were searched. Articles with an
emphasis on neural mechanisms, diabetes or comorbid-
ity in ischaemic conditioning, RIC or RRIC were identi-
fied in particular. The papers retrieved were examined,
and further relevant references obtained by reviewing
cited articles and cross-referencing. Alerts utilising Sco-
pus and PubMed were established in these search areas
over a 7 month period ending August 2016, enabling
access to the most current research. Further searches
were performed as required for other material covered in
this article, including diabetic lower extremity ulceration,
diabetic neuropathy and diabetic mobilopathy.
Pathogenesis of lower extremity ulceration
in diabetes
Sustained hyperglycaemia and associated abnormal
metabolic pathways in T2DM lead to peripheral neu-
ropathy, micro- and macro- vascular peripheral arterial
disease, mechanical changes and subsequent foot trauma
(e.g. Charcot neuroarthropathy), high plantar pressure,
056
increased susceptibility to infection, skin changes result-
ing from autonomic neuropathy, increased pro-inflam-
matory cytokines, decreased neovascularisation and
tissue regeneration, decreased neuropeptides involved in
angiogenesis, and an altered extracellular matrix [2, 10,
19–22]. Diabetic neuropathy and bone marrow micro-
angiopathy have been shown to prevent mobilisation of
haematopoietic stem cells (stem cell “mobilopathy”), cru-
cial in the response to ischaemia [23, 24], and can result
in abnormal neurovascular regulation [25]. Decreased
production of stromal cell-derived factor-1α in diabetic
wounds impairs homing of endothelial progenitor cells
(EPCs) to the ulcer region [26]. Diabetes also impairs the
release of vascular endothelial growth factor (VEGF),
normally upregulated in response to hypoxia, with result-
ant abnormal neovascularisation [27, 28]. All of these
factors may contribute to the development of lower
extremity ulceration or prevent healing. Poor foot self-
care, age, and the presence of other microvascular com-
plications are also important risk factors [29].
Treatment of diabetic lower extremity ulcers
The standard principles of management of diabetic ulcers,
ideally co-ordinated by a multidisciplinary team, are
debridement (utilising various methods), infection con-
trol, pressure offloading, optimising glycaemic control,
application of dressings and surgical intervention (revas-
cularisation or orthopaedic), addressed in recent com-
prehensive reviews of these treatments [2, 22, 30–34].
Newer advanced or potential adjunctive therapies include
hyperbaric oxygen therapy [2, 22, 30–34], bioengineered
skin [2, 22, 30–32, 34], negative pressure wound therapy
[2, 22, 30–34], growth factors [2, 22, 30–34], electro-
physical therapy [2, 22, 30–32], and stem cells [22, 30, 33].
NorLeu3-angiotensin (1–7), substance P and extracellular
matrix proteins have also been studied [reviewed in 22].
Many of these newer therapies are either expensive, dif-
ficult to implement, lack evidence for substantial benefit,
or lack consensus on which method of administration is
most effective for each therapy [31]. Even though exten-
sive research has been undertaken investigating a wide
range of treatment options and optimal management,
chronic ulcers are still all too frequent. The average ulcer
takes approximately 3 months to heal, and at least a half of
diabetic ulcers recur within 3 years [3]. Further research
into treatment options is therefore needed.
Potential treatment candidate: ischaemic
conditioning
Background
It is clear there is a great need to identify novel treatments
to improve diabetic ulcer healing. One potential candi-
date is ischaemic conditioning. This phenomenon was
Epps and Smart Cardiovasc Diabetol (2016) 15:130
first discovered in 1986 by Murry, Jennings and Reimer,
when a study in dogs showed 4 cycles consisting of 5 min
of complete occlusion of a coronary artery, followed by
5 min of reperfusion, decreased the size of a subsequent
induced myocardial infarction by up to 75 % when com-
pared to control [35]. Termed ischaemic preconditioning,
this intervention protects against myocardial ischae-
mia–reperfusion injury [36]. Ischaemic postcondition-
ing was discovered in 2003, when it was identified that
the ischaemic stimulus remained cardioprotective even
when applied after myocardial reperfusion [37]. In 1993,
it was found that administering cycles of preconditioning
to a coronary artery supplying a different region of myo-
cardium to the site of a subsequent infarction remained
cardioprotective [38]. An extension of this concept lead
to a study in 2002, which demonstrated that applying the
ischaemic preconditioning more conveniently to skel-
etal muscle also provided cardioprotection [39]. Termed
remote ischaemic conditioning (RIC), this has been cat-
egorised into several forms: remote ischaemic precon-
ditioning (RIPC), remote ischaemic perconditioning
and remote ischaemic postconditioning [40]. Yet further
subcategories exist when the stimulus is applied up to
24 h before or greater than 15 min after the target organ
ischaemia: delayed remote ischaemic pre- and post-con-
ditioning respectively [40]. Although a variety of organs
have been utilised as the source of the ischaemic stimu-
lus in RIC studies [36], skeletal muscle in upper or lower
limbs have predominantly been used.
Ischaemic preconditioning has been shown to provide
both an early and late window of protection. The first
phase lasts for 2–3 h and commences immediately after
the significant ischaemic episode. The second phase com-
mences between 12 and 24 h after the episode and per-
sists for approximately 3 days of protection [41, 42].
RIC has recently been studied when applied repeat-
edly (RRIC). There has been considerable variation in the
timeframe of administration, ranging from one bout of 5
cycles of 5 min of ischaemia and reperfusion applied once
or twice daily [43], to three 5 min cycles applied once per
fortnight [44]. The period of intervention has ranged
from 1 week of application of RRIC [45] to 300 days of
RRIC [43]. In RIC and RRIC interventions, either single
or bilateral limbs have been studied, and upper or lower
limbs, as the effector organ(s). Different systolic pressures
have been used in RIC to induce limb ischaemia; typically
a pneumatic blood pressure cuff is inflated to 200 mmHg.
The optimal protocols for all forms of RIC and RRIC are
not yet known, and need to be standardised [46, 47].
Despite disappointing results in some translational
studies in humans [48], mounting evidence points
towards RIC as a potentially powerful new therapeutic
tool for an increasingly wider range of conditions. Early
057
studies focused upon cardiac [reviewed in 36] and renal
[reviewed in 49, 50] endpoints. More recently, the use
of RIC has been studied in subarachnoid haemorrhage
management, thromboembolic stroke, cardiovascular
complications following major vascular surgery, cutane-
ous blood flow, transplantation surgery [51, 52], skin and
lung applications [51], skeletal muscle [53], endothelial
function [54, 55] (particularly in RRIC) [reviewed in 56,
57], hypertension [58], and exercise performance [46],
some showing more promise than others.
Remote ischaemic conditioning mechanisms
Neural mechanisms
The mechanisms of RIC are complex and have been the
focus of intense study. The exact mechanisms remain
unknown, but it is evident that both neural and humoral
pathways are involved and that the two interact [36, 40].
It is likely that the ischaemic stimulus causes afferent
C fibre sensory nerves to be activated by local release
of autacoids, such as calcitonin gene related peptide
(CGRP), bradykinin and adenosine [40, 59–63]. Separate
animal model studies have demonstrated that the cardio-
protective effects of RIPC are lost if the sensory nerve to
the ischaemic limb [64, 65], spinal cord [66, 67], the dor-
sal motor nucleus of the vagus nerve [68], the vagus nerve
[67, 69], renal nerves [70, 71], or the posterior gastric
branch of the vagus nerve [72], are transected or silenced.
These key studies reveal how crucial neural pathways are,
from sensory afferent fibres to the efferent autonomic
reflexes, in transmitting the signal for cardioprotection
in RIPC. Importantly, it was found by Basalay et al. [73]
that vagotomy or sectioning nerves of the ischaemic limb
abolished RIPC cardioprotective effects, although not the
effects of remote ischaemic postconditioning. This study
demonstrated that both these forms of remote ischaemic
conditioning are cardioprotective, but utilise very differ-
ent pathways. The implications in the context of the dia-
betic patient will be discussed below, but it is apparent
that humoral pathways are more prominent in remote
ischaemic postconditioning.
Humoral mechanisms
Despite the importance of neural pathways, overwhelm-
ing evidence also exists for a humoral mechanism in
RIC. This was demonstrated effectively in a study by
Shimizu et al. where protection was transferred humor-
ally between animals and even between species, by trans-
fusion of dialysate of plasma, from a preconditioned
human to a rabbit that received no RIC stimulus. The
cardioprotection was also not reduced when transferred
to a denervated rabbit heart [74]. A substantial amount
of research has been undertaken to try and determine
the humoral effector(s) of RIC, of which many have been
Epps and Smart Cardiovasc Diabetol (2016) 15:130
studied, and is beyond the scope of this article. Readers
are directed to two very detailed recent review articles
[36, 75] for comprehensive analysis and discussion of the
latest evidence. To summarise, the most significant or
likely humoral effector candidates resulting in a reduced
myocardial infarct size (the most frequently used end-
point for RIC studies [48]) would appear to be adenosine,
CGRP, bradykinin, nitric oxide (NO) and plasma nitrite,
hypoxia inducible factor 1-α, erythropoietin, stromal-
derived factor-1α, and microRNAs [reviewed in 36, 40].
Apolipotrotein A1 [76, 77] and kynurenic acid [78] are
also potential effectors. Extracellular vesicles, which can
transport microRNAs, are likely to be important in con-
veying the cardioprotective signal [79]. Evidence exists
for the involvement of numerous substances, but none
appear to act as an effector in isolation and it is likely
that many pathways are involved [36]. The late phase
of RIC appears to include nitric oxide synthase, heat
shock proteins and also cyclooxygenase-2 [reviewed in
52, 80]. Most investigations of the late phase of ischae-
mic conditioning have assessed the mechanism of local
(not remote) ischaemic preconditioning [81]. It is felt the
mechanisms are similar, but that both the early and late
phase of RIPC are reliant on neural pathways to a greater
degree than local ischaemic preconditioning [42]. The
late phase of RIPC has also been shown to result in the
mobilisation of bone marrow-derived stem cells, includ-
ing CD34+ cells, into both the peripheral blood and
damaged myocardium, which have the capacity to induce
angiogenesis and endothelial repair [82, 83].
Neural and humoral mechanisms are closely linked
Shimizu et al.’s rabbit study proving cardioprotection
could be transferred to a denervated heart [74] is often
cited as evidence that neural pathways are less crucial to
the mechanism of RIC. This had appeared the case for
the target organ effects, but certainly does not exclude
neural pathways as being essential for the generation
of the protective signal, nor for the release of humoral
factor(s) either locally or via neural pathways to other
non-target organs [47]. In a very recent study by Pick-
ard et al.’s research team, an ex vivo rat heart received
dialysate from donor rats following RIC administra-
tion to the donor. Cardioprotection from a subsequent
induced myocardial infarction was abrogated by admin-
istration of either atropine or hexamethonium to the
recipient, immediately before dialysate treatment [69].
Vagotomy prior to the RIC protocol in donor rats also
abrogated the RIC cardioprotection in the naïve recipi-
ent rat heart [69]. These findings are evidence that both
neural and humoral pathways are integrally linked, at
least in the early phase of RIPC for cardioprotection, and
also confirm that neural pathways are important as far
058
downstream as the target organ itself. Pickard et al. sus-
pect the likely involvement of intrinsic cardiac ganglia in
the mechanism [69]. In addition to neural and humoral
pathways, there is evidence that an anti-inflammatory
effect is produced by RIC through alteration of inflam-
matory gene expression [84], neutrophil function [85],
and interleukin-10 upregulation [86].
End‑organ protection
The end result of cardioprotection occurs via activation
of the reperfusion injury salvage kinase (RISK) pathways,
protein kinase C pathways (via adenosine triphosphate-
sensitive potassium channels) and survival activating
factor enhancement (SAFE) during reperfusion. Mito-
chondrial influx of molecules and cell death during reper-
fusion is then prevented by closure of the mitochondrial
permeability transitions pore [36, 46, 48, 52, 75]. The
mechanism of neuroprotection, in contrast, is less well
studied as an end point of RIC than cardioprotection.
It is likely to involve similar mitochondrial pro-survival
pathways preventing apoptosis, the neuroprotective acti-
vation of synaptic N-methyl-d-aspartate receptors and
increased cerebral blood flow [87, 88]. The mechanism of
renoprotection in RIC is even less well understood and
researched, although cell cycle arrest is thought to be
involved [89].
Nociceptive‑induced conditioning
In a study by Jones et al. [90] using a mouse model, and
in a study by Gross et al. [91] in dogs, the intriguing
phenomenon of remote preconditioning of trauma was
identified. The animals in both studies were subjected to
a shallow wound being administered prior to inducing
myocardial infarction, and this procedure was found to
be cardioprotective, reducing infarct sizes by 80 and 59 %
respectively. This protection could be blocked by admin-
istration of local anaesthesia at the wound site [90]. The
application of topical capsaicin also had a similar effect
in decreasing myocardial infarction size [90, 92]. These
findings have been termed nociceptive-induced remote
conditioning [66]. Redington et al. took the further step
to demonstrate that electroacupuncture also was cardio-
protective, with many similarities to RIPC and previous
nociceptive conditioning studies [93].
Considering the impressive findings of the nocice-
ptive-inducing cardioprotection studies, we propose
that this may also be a substantial confounding factor
in many translational RIPC studies, resulting in mis-
leading conclusions. An example would be human stud-
ies in RIPC used prior to coronary artery bypass graft
(CABG) surgery. The control groups do not receive the
RIPC protocol in these trials, but both groups necessarily
require a large central sternotomy wound. Applying the
Epps and Smart Cardiovasc Diabetol (2016) 15:130
conclusions from the nociceptive-induced remote myo-
cardial conditioning studies described above, one could
argue that the central sternotomy wound in itself could
provide conditioning, to which RIPC may not contribute
significant additional benefit. The impact, location and
timing of any co-administered local, regional and/or gen-
eral anaesthesia could also potentially alter nociceptive-
induced conditioning, varying with the protocol used.
The same concepts may apply to any medical or surgical
procedure or treatment involving trauma or nociception.
Logically, it could therefore be anticipated that study
groups receiving non-invasive treatment for relatively
painless conditions would benefit most from RIC, with
no co-conditioning from pain or trauma. The promising
early use of RIC for ischaemic stroke is one such example
[87].
To ignore the potential for nociceptive co-conditioning
in a study may lead to incorrect conclusions regarding
the response to RIPC. We suspect it would be important
to evaluate previous and future study designs carefully, to
establish whether pain or trauma could be a confound-
ing factor due to co-conditioning. To the authors’ knowl-
edge, the lowest threshold or dose necessary to induce
nociceptive conditioning has not yet been established.
If superficial skin wounds [91] and electroacupuncture
[93] are sufficient, it remains to be seen whether other
very minor procedures, such as intramuscular injections,
cannulations and capillary blood sampling, can trigger
nociceptive conditioning. Should small, minor stimuli be
sufficient, it is plausible that the earliest recorded thera-
peutic use of nociceptive conditioning in humans was in
fact 100BC (or earlier) in China, when acupuncture prac-
tice was first clearly documented [94].
Difficulties in clinical translation of remote
ischaemic conditioning
Clinical studies that show benefits, such as the applica-
tion of RIC in CABG surgery and elective and primary
percutaneous coronary intervention (PCI), have not
always been reproducible [36, 48, 95–99]. The most fre-
quent positive findings have been seen in the use of RIC
intervention in primary PCI [47]. Results from animal
RIC studies are typically more promising than those in
humans [98, 100]. Conflicting findings may be due to dif-
ferences between the animal models studied and humans
[48, 98, 100], or to limitations in study designs [48]. In
human studies, it is also very likely to be due to co-mor-
bidity [48, 98, 101], co-medication and differing anaes-
thetic protocols [98, 101–105], heterogeneity in study
designs [48, 62], and other factors that cannot be pre-
dicted due to an incomplete understanding of the mecha-
nisms and interaction of neural and humoral pathways in
RIC. As a consequence, confounding factors are likely to
059
be unwittingly present in studies, which could result in
misleading conclusions.
Difficulties in clinical translation of ischaemic
conditioning in diabetes
As ischaemia plays an important role in the pathophysi-
ology of many complications of T2DM, and the diabetic
state appears particularly vulnerable to ischaemia–reper-
fusion injury [106], it seems intuitive that patients with
T2DM would benefit from ischaemic conditioning. How-
ever, mixed and neutral findings have been reported in
preclinical and clinical studies in diabetes [48, 107–110],
with particularly poor results in preclinical postcondi-
tioning studies [48]. The majority of human studies in
diabetes have assessed cardioprotection in acute myocar-
dial infarction (AMI) following ischaemic conditioning,
with disappointing results [98]. Conversely, evidence for
the preservation of RIPC cardioprotection in human dia-
betics undergoing percutaneous coronary intervention
was found in a 2014 meta-analysis [111].
The number of human ischaemic conditioning studies
specifically in T2DM is, however, very small, particularly
with the gold-standard endpoint of myocardial infarction
size, and most of these do not assess remote ischaemic
conditioning efficacy [98, 100, 107]. The first early win-
dow of protection in diabetes has also received consid-
erably more study than the second window. In studies
performed with favourable cardioprotective outcomes
for ischaemic (pre- and post-) conditioning in diabetes, a
common finding is that an increased ischaemic stimulus
is often required [112–115].
Potential factors in type 2 diabetes limiting efficacy
Cardiomyocyte changes
Complex changes have been detected in diabetic hearts,
in human and animal model studies (particularly assess-
ing postconditioning [48]), that are likely to result in the
myocardium being less responsive to ischaemic con-
ditioning and RIC. These include impaired kinase and
signalling pathways. Specifically, these are altered extra-
cellular signal-regulated kinase (ERK) 1/2 [116, 117],
decreased phosphorylation of glycogen synthase kinase-3
beta [117, 118], impaired [112, 113, 117] or chronic [114]
Akt phosphorylation and Akt activity [119], effects on
mitochondrial adenosine triphosphate (ATP)-dependent
potassium channels [106, 120–124], decreased CGRP
[125], reduced adenosine [108], altered bioavailability
of NO [126] and abnormalities of other apoptotic path-
ways [127, 128]. In a human RIPC diabetes study, raised
O-linked β-N-acetylglucosamine was found to be associ-
ated with a state of chronic cardioprotection, with sub-
sequent RIPC providing no additional benefit [129].
Perplexingly, the diabetic state has also been found to
Epps and Smart Cardiovasc Diabetol (2016) 15:130
limit ischaemia–reperfusion injury [110, 130]. This was
highlighted in a study by Ma et al. using streptozotocin-
induced diabetic rat models, although protection was
limited to 2 weeks following induction of diabetes. Akt
activation, increased NO and VEGF formation leading to
angiogenesis were identified as likely mechanisms [131].
A study by Ravingerová et al. using a similar diabetic
rat model also concluded that early diabetes conferred
resistance to myocardial necrosis [132]. In this instance,
RIC may offer little additional benefit.
Hyperglycaemia
Hyperglycaemia and resultant oxidative stress are likely
to be involved in the changes of the abovementioned
pathways in diabetes [108, 133]. Whilst many studies
have demonstrated the loss of cardioprotection from
ischaemic conditioning in hyperglycaemia [116, 121,
134–137], this loss of cardioprotection was identified in
a Zucker obese rat study before the onset of hypergly-
caemia [123]. This demonstrated that hyperglycaemia is
not the only underlying mechanism in the loss of cardio-
protection in type 2 diabetes. To further complicate the
picture, studies of hyperglycaemia in animal models of
type 1 diabetes demonstrated that acute diabetes is car-
dioprotective, but these benefits were lost after 6 weeks
of onset of diabetes [reviewed in 109]. In addition to
hyperglycaemia, insulin is also an important factor in
cardioprotection. Activation of Akt by administration of
insulin has an inhibitory effect on ischaemic precondi-
tioning efficacy [138]. The restoration of cardioprotection
from ischaemic postconditioning with return to normo-
glycaemia, through transplantation of islet cells, has been
identified in a type 1 diabetic mouse model study [116].
In a separate rat study, however, insulin pretreatment had
very different findings, with no restoration of cardiopro-
tective effects from ischaemic postconditioning [139].
Most research assessing the sensitivity to ischaemia–
reperfusion injury has focused upon type 1 rather than
type 2 diabetes [109]. Findings have often been conflict-
ing, perhaps due to the differing methods of inducing
diabetes, duration and severity of hyperglycaemia, animal
models used, study protocols, and difficulties in compar-
ing ex vivo, in vivo and in vitro scenarios [109].
Impaired endothelial progenitor cell mobilisation
Diabetes has a negative impact on EPC mobilisation in
response to ischaemia [140], particularly in the context
of diabetic neuropathy [141, 142]. It has been demon-
strated that EPC mobilisation occurs following RIPC in
both mice and humans [82, 83], and this appears likely to
contribute significantly to the late phase cardioprotec-
tive effects of RIPC [82]. We therefore hypothesise that
this may be another important and under-recognised
060
mechanism behind the poorer response to RIPC in
T2DM, particularly in the rarely-studied late phase of
protection. Poor EPC mobilisation following AMI is
known to be associated with adverse outcomes, espe-
cially in diabetes [143], and results in impaired neovas-
cularisation [142]. Interestingly, diabetic mobilopathy has
improved following administration of dipeptidyl pepti-
dase-4 (DPP-4) inhibition [144].
Comorbidity and co‑medication
Evidence exists that many conditions frequently occur-
ring in T2DM also have a negative effect on cardio-
protection in ischaemic conditioning. These include
hyperlipidaemia, particularly in ischaemic postcondi-
tioning [98, 100, 145, 146], obesity [123, 147], and hyper-
tension [100, 148]. Ventricular hypertrophy abolished
cardioprotection in some animal model studies and not
others [100, 149]. Chronic kidney disease surprisingly
does not appear to abrogate the protective effects of
ischaemic conditioning [98] and RIPC shows promise in
providing renoprotection from acute injury [89], particu-
larly contrast-induced acute kidney injury [50]. Diabetic
neuropathy is known to abrogate the effect of RIPC [64],
and is outlined in the section below.
Many medications used in the management of diabetes
and its complications have been shown to have an effect
upon ischaemic conditioning. Some are inherently car-
dioprotective, providing pharmacological conditioning.
Examples include insulin [102, 150], metformin [151–
153], sitagliptin [151], exenatide [154], sildenafil [102,
155], beta blockers [156] and potentially ACE inhibitors
[102]. It is suspected that ischaemic conditioning may
offer limited additional benefit when applied concur-
rently with pharmacological conditioning [95]. Other
medication, such as statins [157], glimepiride [158, 159]
and losartan or valsartan (angiotensin II receptor type
1 blockers) [160] have demonstrated augmentation or
restoration of cardioprotection from ischaemic condi-
tioning in diabetes and/or hyperglycaemia. Yet other
classes abrogate the effects of ischaemic conditioning
altogether, including older sulfonylureas such as gliben-
clamide [reviewed in 151]. Despite the knowledge about
the effects of these medications, study findings of these
effects are not always consistent, limiting further under-
standing. For example, in a retrospective analysis assess-
ing the impact of likely confounders of RIC in CABG
surgery outcomes, statins and cardioprotective antihy-
pertensives were reported as not altering RIC cardiopro-
tection [161].
It is essential to note that the majority of studies assess-
ing the impact of comorbidity and co-medication upon
cardioprotection in T2DM have used a local (not remote)
ischaemic conditioning protocol [100], and small animal
Epps and Smart Cardiovasc Diabetol (2016) 15:130
models [48, 151]. Many of the preclinical studies assess-
ing the impact of medication used in human T2DM have
been conducted utilising non-diabetic animals [151].
Most of the diabetic animal models and protocols used
would not reflect the duration of T2DM and extent of
diabetic complications seen in humans (including neu-
ropathy), nor the true impact of polypharmacy, drug
doses used in clinical practice, lifestyle choices and con-
siderable comorbidity frequently encountered in T2DM
[48, 100, 109]. Although such preclinical studies are
much needed in T2DM, it is difficult to be certain of the
degree of translatability and relevance through extrapola-
tion of their key findings to human T2DM, particularly in
the context of RIC. Thus, it is more than likely that these
issues contribute to conflicting results from many ischae-
mic conditioning and RIC clinical studies in T2DM.
Readers are referred to comprehensive reviews of the
impact of diabetes in ischaemic conditioning of the myo-
cardium [98, 107, 108, 145], and medications commonly
prescribed in T2DM [98, 151], for a more in-depth dis-
cussion of the difficulties in clinical translation of RIC in
T2DM.
Neural pathways in remote ischaemic conditioning
and neuropathy of type 2 diabetes
Both the early and late phases of RIPC are dependent
upon neural pathways, identified in a key human study
by Loukogeorgakis et al. assessing the effects of RIPC on
endothelial function as an endpoint [42]. More evidence
for the requirement of a neural pathway in RIPC, spe-
cifically in the diabetic population, comes from another
crucial study by Jensen and colleagues, who successfully
demonstrated that intact peripheral nerves in human
T2DM subjects were critical for generating the dialys-
able, transferrable cardioprotective factors that reduced
myocardial infarct size in rabbits [64]. Jensen et al. found
that the protective effect was the same in both non-dia-
betic subjects and diabetics without peripheral neuropa-
thy, but that there was no protective effect in the dialysate
obtained from diabetic patients with peripheral neuropa-
thy, even with an intensified RIPC stimulus [64].
Peripheral neuropathy
Of interest is that Jensen et al.’s study protocol utilised
the upper arm for the RIPC administration. The exact
location of the peripheral neuropathy in the T2DM
neuropathy group is not documented in the article
[64]. Unless in advanced cases of peripheral sensorimo-
tor neuropathy, sensory deficits occur predominantly
in the lower limb neural pathways, as the neuropathy is
nerve length-dependent in its pathophysiology [162]. It
would be beneficial to establish the site(s) of the bioth-
esiometer readings obtained from those in the peripheral
061
neuropathy group of Jensen et al.’s study; unfortunately,
the author was not contactable. If lower limb sensory
neuropathy in one or both limbs abrogated the benefit
of RIPC applied to an upper limb without any detectable
sensory deficits, this would raise yet further questions
about the complex mechanisms of RIPC. It could also
raise the possibility that the cardioprotective effects of
RIPC may have been reduced by coexisting neuropathy at
other sites. Tentolouris et al. found that peripheral neu-
ropathy and autonomic neuropathy, specifically cardio-
vascular autonomic neuropathy (CAN), coexisted in 45 %
of cases of T2DM [163].
Peripheral neuropathy is prevalent in the T2DM popula-
tion, with distal symmetric polyneuropathy being the most
common [162]. Diabetic sensorimotor polyneuropathy
(DSPN) is initially subclinical, and progresses to overt clini-
cal symptoms and increased severity [164]. Research has
shown that diabetic neuropathy may often be present when
T2DM is first diagnosed. A study of 39 patients at diagnosis
of T2DM found 82 % of the patients had electrophysiologi-
cal abnormalities in peripheral nerve conduction studies,
62 % of these in more than one parameter [165].
Diabetic sensory neuropathy, particularly in relation to
nociceptive fibres, decreases the release and homing of
bone marrow-derived haematopoietic stem cells [141].
Evidence suggests that this stem cell response is impor-
tant for angiogenesis and vascular regeneration [140,
166], including wound healing in diabetic model mice
[167], and could be part of the mechanism for the late
window of protection in RIPC [80, 82]. The pathogenesis
of diabetic stem cell mobilopathy is also likely to involve
diabetic autonomic neuropathy [168–170]. Bone mar-
row receives sympathetic nervous system innervation,
involved in the mobilisation of stem cells [170, 171], and
diabetes can lead to autonomic neuropathy of the bone
marrow [168, 170].
Autonomic neuropathy
The autonomic nervous system (ANS) is commonly
abnormal in patients with longstanding diabetes. Human
and animal studies referred to above have demonstrated
the integral role of the ANS in RIPC. Despite the fact
that the ANS and the vagus nerve have been identified
as being critical for the cardioprotection of RIPC, we are
not aware of any studies published exploring the impact
of autonomic neuropathy upon the effectiveness of RIPC
in a clinical or human context. Decreased parasympa-
thetic tone and dysfunction is mentioned, however, by
Mastitskaya et al. as being a possible explanation for dis-
appointing results in some studies, when their research
team discovered that sectioning the posterior gastric
branch of the vagus nerve in rats abrogated the effects of
RIPC [72].
Epps and Smart Cardiovasc Diabetol (2016) 15:130
Diabetic autonomic neuropathy (DAN) can occur
subclinically in T2DM within a year of onset, although
symptoms typically develop after years [172]. The lengthy
vagus nerve can be one of the first affected by neuropa-
thy [172, 173]. DAN may occur before diabetic neuropa-
thy involves the peripheral sensory nervous system. The
commonest form of DAN is CAN, defined as impaired
autonomic control of the cardiovascular system [164].
The prevalence of DAN, using various criteria, has been
reported to be between 15 and 35 % in T2DM, and is
commonly undiagnosed [172]. CAN, like DAN, can
develop subclinically within a year of onset of diabe-
tes [173]. The reported prevalence of CAN in T2DM
also varies widely, listed as between 20 and 70 % in one
review, depending on differing diagnostic criteria and
duration of diabetes [174]. DePace et al. report one-third
of T2DM patients at diagnosis have CAN [175].
Given knowledge about the essential role of the vagus
nerve in RIPC and the high prevalence of (often undiag-
nosed) CAN, we suspect that the presence of autonomic
neuropathy may be an unrealised but significant contrib-
uting factor in the poorer or variable response to RIPC in
diabetic (and non-diabetic) patients identified in studies,
and thus a confounding variable. CAN is associated with
a significantly increased risk of myocardial infarction
and mortality, and an independent risk factor even in
those at high cardiovascular risk [174–176]. We propose
a decreased response to RIPC, and by extension, poten-
tially even to physiological conditioning such as regular
physical exercise, in patients with CAN.
Another common form of DAN in T2DM is gastro-
intestinal autonomic neuropathy (GAN). The most fre-
quent form of GAN is gastroparesis, which occurs in
30–50 % of patients with longstanding T2DM [177, 178].
Mastitskaya et al.’s 2016 study indicated the importance
of the posterior gastric branch of the vagus nerve in the
RIPC mechanism for cardioprotection in a rat model
[72]. It remains to be seen whether GAN affects this spe-
cific branch of the vagus nerve, and thus could inhibit
RIPC cardioprotective pathways.
Bilateral renal denervation has been shown to abrogate
RIPC cardioprotective effects, concomitantly inhibiting
erythropoietin production, in a murine model [70]. Eryth-
ropoietin-neutralising antibodies also abrogated the effect
of RIPC in the absence of renal denervation [70]. The renal
nerve consists of efferent and afferent sympathetic fibres
[179], which have been hypothesised to be abnormal in
severe DAN, resulting in erythropoietin deficiency (inde-
pendent of nephropathy) in these patients [180].
Calcitonin gene related peptide
Decreased secretion of the neurotransmitters CGRP
and substance P from neurons following noxious stimuli
062
is a consequence of diabetic neuropathy [23]. The man-
agement of painful diabetic peripheral neuropathy, and
neuropathic pain in non-diabetics, has been substan-
tially improved by the use of pregabalin and gabapentin
(gabapentinoids) [162, 181, 182]. Gabapentinoids are
believed to inhibit the modulation of neuronal excit-
ability, and also decrease neurotransmitters release in
the presence of inflammation, including CGRP and sub-
stance P [183, 184]. This prevents nociceptive signal
transmission [184].
Calcitonin gene related peptide has been identified
as important in the cardioprotective neurohumoral
pathways of RIPC [63], and in local pre- and post-con-
ditioning [185]. As discussed earlier, activation of nocice-
ptive-inducing pathways (C fibres, or capsaicin-sensitive)
is crucial in the neural mechanisms of RIPC [59] as well
as for the release of haematopoietic stem cells [141]. If
gabapentinoids block these pathways, they potentially
could reduce the effectiveness of RIPC and local ischae-
mic conditioning. Further well-designed studies are
therefore required to clarify if anti-nociceptive phar-
macotherapy may have an impact on different forms of
ischaemic conditioning.
Implications of abnormal neural pathways
We were unable to identify any articles or studies under-
taken to date primarily addressing DAN, CAN, or DSPN
in RIC (other than the study by Jensen et al. [64]) and
their potential impact upon RIC efficacy. Indeed, despite
Jensen et al.’s study findings, the presence, absence or
degree of peripheral sensory neuropathy is rarely men-
tioned when comparing groups within a RIPC study,
even when such a study is specifically investigating diabe-
tes, or diabetes as a co-variate. As both DSPN and CAN
are commonly undiagnosed and may be subclinical, yet
prevalent in T2DM and even early T2DM (for CAN in
particular), they have the potential to be significant but
overlooked confounding variables in many studies of
RIPC in diabetic participants to date, and further stud-
ies are warranted. Examination is not difficult to perform
for CAN or DSPN; the screening is straightforward, non-
invasive, safe, inexpensive and can be undertaken in an
office setting [173, 186, 187].
Repeat remote ischaemic conditioning
A more recent method of ischaemic conditioning is
repeat ischaemic conditioning, typically administered
remotely. Although often termed repeat, regular or inter-
mittent remote ischaemic preconditioning, intervention
studies utilising this method are generally applied to a
chronic condition (such as heart failure [188], endothe-
lial dysfunction [189], stroke recurrence [43] or chronic
wounds [44]) or following an acute ischaemic episode
Epps and Smart Cardiovasc Diabetol (2016) 15:130
(such as in a lower limb [190], post-myocardial infarc-
tion [191] or cerebrovascular accident [43]). Depending
on the context, the chronicity of pathological ischaemia
in the cohort, and endpoints studied, this would more
accurately represent combinations of pre, per and post-
conditioning, reflected in the term repeat remote ischae-
mic conditioning (RRIC). Neural plasticity effects of RRIC
have also been examined in an intriguing small study
of healthy adult participants, demonstrating that RRIC
aided motor learning and retention [192]. Although
few in number and with a broad variety of endpoints,
it would appear that both preclinical and clinical RRIC
studies cited above have more consistently yielded sig-
nificant beneficial results, particularly in comparison to
clinical single-dose RIC and local ischaemic condition-
ing studies. Importantly, early evidence shows efficacy of
RRIC in humans is not lost with advanced age. A study
of 58 patients aged 80–95 years found twice daily RRIC
for 180 days was effective in preventing stroke recurrence
compared to sham control, without any safety concerns
[193]. In contrast, RIC studies examining the impact
of ageing, most assessing cardioprotection in animals,
have revealed that efficacy predominantly declines with
increasing age [48].
Mechanisms
The mechanisms of RRIC are less well studied than in
RIC [57]. Although it would seem logical that they may
be similar to RIC, gene expression studies by Depre et al.
and Shen et al. demonstrated this is not correct [194,
195]. Further, unlike the late phase of protection in RIPC,
RRIC pathways do not appear to involve cyclooxyge-
nase-2 [196]. A 4 week RRIC study by Yamaguchi et al.
assessing cardiac remodelling in rats following myocar-
dial infarction, revealed beneficial effects were associated
with higher levels of miR-29a expression [188]. Kimura
et al. also conducted a 4 week study, assessing endothelial
function in healthy humans, and found RRIC was associ-
ated with improved endothelial function, and raised EPC,
VEGF and NO levels [197]. Neutrophil function changes
(including decreased adhesion and suppressed phagocy-
tosis) and cytokine changes, associated with modulation
of the systemic inflammatory response, have been identi-
fied in a 10 day human RRIC study by Shimizu et al. [85].
These changes were significant, and felt to be beneficial
by the research team since tissue damage in ischaemia–
reperfusion injury is facilitated by neutrophil adhe-
sion [198]. RRIC improves skin perfusion in addition to
endothelial function, even 8 days beyond RRIC interven-
tion cessation, as demonstrated by Jones et al. [45]. It is
also suspected that the effects from repeated ischaemia
may be due in part to shear stress [57], leading to the
063
mobilisation of EPCs recorded by Kimura et al. and thus
neovascularisation and improved vascular function [197].
Effectiveness of repeat remote ischaemic
conditioning in type 2 diabetes
As we have demonstrated above, many factors poten-
tially limit the benefits from ischaemic conditioning and
RIC in diabetic patients. It is therefore unexpected that
a recent study showed diabetic patients with refractory
lower leg ulcers had significant improvement following
repeat RIC [44]. In Shaked et al.’s double-blinded ran-
domised study with sham control, 22 patients received
3 fortnightly cycles of RRIC to both arms, in addition to
standard wound care. At the completion of the 6 week
study, 9/22 (41 %) of the study group had complete heal-
ing of their ulcer compared to 0/12 of the control group
[44]. Questionably, only one participant in each group
was reported as having “neuropathy”, even though this
was not listed in the exclusion criteria for the study. 46 %
of the study group at commencement were documented
as having local infection in the ulcer and 69 % in the con-
trol group. There were some further limitations with the
study due to other important variables being unmatched
between groups.
Despite the weaknesses and limitations of Shaked et al.’s
small study, the wound healing rate of 41 % of refractory
ulcers in just 6 weeks in a cohort of patients with very
poorly controlled diabetes, after a mere 3 fortnightly
remote ischaemic conditioning cycles, is not to be dis-
missed. Further, 14/22 patients in the intervention group
reached 75 % healing at 6 weeks compared with 3/12 in
the control group. 55 % of patients in the study group
with ulcer infection at enrolment had completely healed
wounds, compared to none in the control group with
infection [44]. The findings could indicate the potential
for RRIC providing significant benefit in this group, which
is easily and cost-effectively administered. Should these
results be reproducible in larger trials with a more satis-
factorily matched control group, RRIC could revolution-
ize treatment of chronic diabetic foot ulcers. If this were
the case, it may also point to the potential for RRIC to be
effective in other chronic diabetic complications or causes
of ulceration. Given the findings of improved endothe-
lial function and increased NO, EPCs and VEGF levels in
RRIC studies (albeit with predominantly healthy cohorts)
discussed above, the effects of RRIC in diabetic nephropa-
thy, neuropathy, coronary artery disease, peripheral arte-
rial disease (PAD) and erectile dysfunction would be
interesting to study. Endothelial progenitor cell therapy is
being researched with some success as a potential treat-
ment for stroke [199], another frequent complication of
diabetes where RRIC may prove effective.
Epps and Smart Cardiovasc Diabetol (2016) 15:130
Shaked et al.’s study involved administration of remote
conditioning. As local ischaemic preconditioning (IPC)
appears to rely upon humoral and not neural mecha-
nisms, this could mean local IPC may be relatively unaf-
fected by the presence of DAN or DPSN. We propose
repeat local IPC in the limb affected by the ulcer could
possibly prove even more effective than remote ischae-
mic conditioning on this basis, assuming it is as safe and
well tolerated as remote conditioning. Comparative stud-
ies between repeat remote and local conditioning inter-
ventions in diabetic patients with foot ulcers could be
worth performing to test this theory. It is also interesting
to consider Shaked et al.’s diabetic ulcer study had a con-
trol group where standard care did not involve significant
pain or trauma, thus making co-conditioning from nocic-
eption less likely to confound the outcome.
Shaked et al.’s study excluded those with moderate-
to-severe PAD, where ankle brachial index was <0.7.
It is important to note that even in the absence of large
vessel peripheral arterial disease, diabetic patients fre-
quently have small vessel disease and tissue hypoxia in
the lower limbs. Shaked et al. write that “peripheral neu-
ropathy induces increased pressure points on the diabetic
foot, which in turn lead to bouts of cutaneous IR injury”
[44, p 194], from which RRIC appears likely to provide
protection.
Diabetes suppresses angiogenesis in wound healing
[200]. Injection of CD34+ cells into diabetic wounds
has been found to improve diabetic ulcer wound heal-
ing and significantly enhanced wound revascularisation
within 7 days [167]. In addition, serum CD34+/CD45-
dim levels measured soon after the development of neu-
ropathic diabetic ulcers have shown to be predictive of
subsequent wound healing [201]. It is unfortunate EPCs/
CD34+ cells and other factors such as VEGF were not
measured in Shaked et al.’s study, but it is plausible that
RRIC may have increased VEGF and/or NO, and thus
EPCs (potentially overcoming mobilopathy), in the inter-
vention group, consequently improving the impaired
neovascularisation and wound healing. Both study
cohorts were likely to have a high prevalence of CAN
and (potentially undetected) DPSN. Neural mechanisms
have not been identified nor studied in RRIC. Should the
findings of Shaked et al. be replicated with more appro-
priately matched control and intervention groups, this
may suggest that RRIC mechanisms are not as inhibited
by DPSN as RIPC in diabetes. The issues regarding the
beneficial effects of local ischaemic conditioning and
RIC being decreased in the diabetic state, particularly
relevant to the cardiomyocyte, may be less problematic
with a different endpoint or target organ, such as wound
healing. Similarly, it appears likely that the mechanisms
in RRIC are significantly different to RIC, and coexisting
064
neuropathy or co-medication may not result in such neg-
ative effects. Considering the evidence suggesting that
RRIC leads to a state favouring neovascularisation, raised
NO and EPCs, and improved microcirculatory function,
one is tempted to postulate that RRIC may even slightly
reverse the effects of diabetic neuropathy [202], or micro-
angiopathy- and neuropathy-linked mobilopathy [23],
hence simultaneously improving the response to each
subsequently applied dose of RIC. Intermittent hypoxia is
known to induce neuroplasticity and improve recovery of
neurological function, including in motoneurons [192].
Raising levels of EPCs in diabetic patients is highly
desirable; lower levels being associated with adverse car-
diovascular outcomes [143, 203]. Current methods to
increase EPCs include administration of granulocyte-col-
ony stimulating factor, which has serious cardiovascular
side effects and is often ineffective in diabetics [23, 169],
or by culturing and re-administering EPCs [83]. RRIC
would offer a safer, more cost-effective and easily-admin-
istered alternative, with automated RRIC devices already
designed and manufactured for self-administration at
home.
It is interesting to note that smoking [204] and hyper-
lipidaemia [205] are associated with poor bone marrow
release of EPCs, in addition to a poorer response to RIPC
[98, 206]. Statins [207], independent of effects on serum
lipid levels, and sitagliptin [151, 159, 208] have cardio-
protective properties in diabetes, and statins enhance
cardioprotection in RIC [157, 206]. Studies have demon-
strated that statins [209, 210] and sitagliptin [211] aug-
ment mobilisation of EPCs from bone marrow. Many of
these facts may be interlinked, particularly in relation to
the late phase of RIC and possibly RRIC. The effects of
statins, smoking and anti-diabetic medication on RRIC
are yet to be determined.
The potential use of ischaemic conditioning in PAD has
received some attention. A crossover study by Delagarde
et al. examined claudication distance in 20 patients with
intermittent claudication after administering 3 intermit-
tent cuff inflations and deflations [212]. A treadmill test
was undertaken 5 min after completing both the RIPC
and the control sham procedure, which were 7 days
apart. Results did not show any alteration in claudication
distance. It was surmised by Delagarde et al. that patients
with PAD suffer from chronic ischaemia and reperfu-
sion, and thus are already maximally conditioned. Conse-
quently, they proposed that this may be why RIPC offered
no additional benefit [212]. It is important to consider
that performing a treadmill test 5 min after one cycle of
RIPC would not allow for second window of protection
(nor RRIC) effects to be assessed other than, to some
extent, in those participants who performed the RIPC
protocol first and then control 7 days later. In addition,
Epps and Smart Cardiovasc Diabetol (2016) 15:130

only 25 % of the participants in the study were recorded

as being diabetic, and presence of neuropathy was not
documented. Karakoyun et al. recently undertook a key
study of leg ischaemia, with a comparison between repeat
local ischaemic conditioning, repeat remote ischaemic
conditioning, no conditioning (ischaemic group) and
sham control groups in non-diabetic rats [190]. The right
iliac artery and vein were ligated to create critical limb
ischaemia in all except the sham group. In the repeat
local ischaemic conditioning group, the ischaemic stimu-
lus was applied to the right leg and in the RRIC group,
to the left leg. Both the RRIC and repeat local ischaemic
conditioning group received 3 cycles of 10 min of ischae-
mia followed by 10 minutes of reperfusion daily until
sacrificed at days 1,7,14 and 30. The ischaemic group and
sham group received no further intervention. The find-
ings of Karakoyun et al.’s study were that RRIC resulted
in increased angiogenesis in the ischaemic right limb,
although with no improvement in blood flow. The repeat
local ischaemic conditioning group demonstrated both
improved angiogenesis and blood flow. These positive
findings were significant when compared to the ischae-
mic and sham control groups. EPCs were also measured,
and were particularly higher in the RRIC group [190].

Conclusions
Repeat RIC is an effective new method to consider in the
treatment of diabetic lower extremity ulceration and
peripheral arterial disease that is inexpensive, easy to
administer in any location, and well tolerated.
Abbreviations
T2DM: type 2 diabetes mellitus; RIC: remote ischaemic conditioning; RIPC:
remote ischaemic preconditioning; RRIC: repeat remote ischaemic condition-
ing; ANS: autonomic nervous system; DSPN: diabetic sensorimotor poly-
neuropathy; DAN: diabetic autonomic neuropathy; CAN: cardiac autonomic
neuropathy; GAN: gastrointestinal autonomic neuropathy; EPCs: endothelial
progenitor cells; VEGF: vascular endothelial growth factor; CGRP: calcitonin
gene related peptide; NO: nitric oxide; CABG: coronary artery bypass graft; PCI:
percutaneous coronary intervention; AMI: acute myocardial infarction; PAD:
peripheral arterial disease; DPP-4: Dipeptidyl peptidase-4.

Authors’ contributions
JE and NS conceived the idea for the article. JE conducted the literature search
and identified suitable studies and articles to include. JE and NS planned the
structure of the article. JE reviewed the literature and was the major contribu-
tor in preparing the manuscript. NS assisted with reviewing the literature,
revised and edited the intellectual content of all drafts. Both authors read and
approved the final manuscript.

065
Remote Ischemic Preconditioning Protects Against Endothelial Dysfunction in a
Human Model of Systemic Inflammation: A Randomized Clinical Trial

OBJECTIVE:
Inflammation, oxidative stress, and endothelial dysfunction are known to contribute to ischemia-
reperfusion injury. Remote ischemic preconditioning (RIPC) protects from endothelial
dysfunction and the damage induced by ischemia-reperfusion. Using intensive periodontal
treatment (IPT), an established human model of acute systemic inflammation, we investigated
whether RIPC prevents endothelial dysfunction and modulates systemic levels of inflammation
and oxidative stress.
APPROACH:
Forty-nine participants with periodontitis were randomly allocated to receive either 3 cycles of
ischemia-reperfusion on the upper limb (N=24, RIPC) or a sham procedure (N=25, control) before
IPT.
ASSESSMENT:
Endothelial function assessed by flow-mediated dilatation of the brachial artery, inflammatory
cytokines, markers of vascular injury, and oxidative stress were evaluated at baseline, day 1, and
day 7 after IPT.
RESULTS:
Twenty-four hours post-IPT, the RIPC group had lower levels of IL-10 (interleukin-10) and IL-12
(interleukin-12) compared with the control group (P<0.05). RIPC attenuated the IPT-induced
increase in IL-1β (interleukin-1β), E-selectin, sICAM-3 (soluble intercellular adhesion molecule 3),
and sTM (soluble thrombomodulin) levels between the baseline and day 1 (P for interaction
<0.1). Conversely, oxidative stress was differentially increased at day1 in the RIPC group
compared with the control group (P for interaction <0.1). This was accompanied by a better flow-
mediated dilatation (mean difference 1.75% [95% CI, 0.428–3.07], P=0.011).
CONCLUSIONS:
RIPC prevented acute endothelial dysfunction by modulation of inflammation and oxidation
processes in patients with periodontitis following exposure to an acute inflammatory stimulus.

066
 Arteriosclerosis, Thrombosis, and Vascular Biology

CLINICAL AND POPULATION STUDIES

Remote Ischemic Preconditioning Protects

Against Endothelial Dysfunction in a Human
Model of Systemic Inflammation: A Randomized
Clinical Trial
Marco Orlandi , Stefano Masi, Devina Bhowruth, Yago Leira , Georgios Georgiopoulos, Derek Yellon, Aroon Hingorani,
Scott T. Chiesa , Derek J. Hausenloy, John Deanfield, Francesco D’Aiuto

OBJECTIVE: Inflammation, oxidative stress, and endothelial dysfunction are known to contribute to ischemia-reperfusion injury.
Remote ischemic preconditioning (RIPC) protects from endothelial dysfunction and the damage induced by ischemia-
reperfusion. Using intensive periodontal treatment (IPT), an established human model of acute systemic inflammation, we
investigated whether RIPC prevents endothelial dysfunction and modulates systemic levels of inflammation and oxidative stress.

APPROACH AND RESULTS: Forty-nine participants with periodontitis were randomly allocated to receive either 3 cycles of
ischemia-reperfusion on the upper limb (N=24, RIPC) or a sham procedure (N=25, control) before IPT. Endothelial function
assessed by flow-mediated dilatation of the brachial artery, inflammatory cytokines, markers of vascular injury, and oxidative
stress were evaluated at baseline, day 1, and day 7 after IPT. Twenty-four hours post-IPT, the RIPC group had lower levels
of IL-10 (interleukin-10) and IL-12 (interleukin-12) compared with the control group (P<0.05). RIPC attenuated the IPT-
induced increase in IL-1β (interleukin-1β), E-selectin, sICAM-3 (soluble intercellular adhesion molecule 3), and sTM (soluble
Downloaded from http://ahajournals.org by on April 17, 2024

thrombomodulin) levels between the baseline and day 1 (P for interaction <0.1). Conversely, oxidative stress was differentially
increased at day1 in the RIPC group compared with the control group (P for interaction <0.1). This was accompanied by a
better flow-mediated dilatation (mean difference 1.75% [95% CI, 0.428–3.07], P=0.011). After 7 days from IPT, most of the
inflammatory markers, endothelial-dependent and -independent vasodilation, were similar between groups.

CONCLUSIONS: RIPC prevented acute endothelial dysfunction by modulation of inflammation and oxidation processes in
patients with periodontitis following exposure to an acute inflammatory stimulus.

REGISTRATION: URL: https://clinicaltrials.gov/ct2/show/NCT03072342; Unique identifier: NCT03072342.

GRAPHIC ABSTRACT: A graphic abstract is available for this article

Key Words: endothelium ◼ inflammation ◼ oxidative stress ◼ periodontitis ◼ reperfusion

C
ardiovascular diseases are the leading cause of
death and disability worldwide.1 Central to the
pathophysiology of many cardiovascular diseases
is endothelial dysfunction,2 and as such new treatment
modalities are needed to prevent endothelial dysfunc-
tion and improve clinical outcomes in patients with
cardiovascular disease. In this regard, cycles of brief
nonlethal ischemia and reperfusion to the arm or leg
have been reported to prevent endothelial dysfunc-
tion induced by sustained ischemia and reperfusion, a
phenomenon which has been termed remote ischemic
preconditioning (RIPC).3,4
The exact mechanisms by which RIPC protects
the endothelium remain to be elucidated. However,

Correspondence to: Marco Orlandi, DDS, PhD, Eastman Dental Institute, Rockefeller Bldg, 21 University St, WC1E 6DE, United Kingdom. Email m.orlandi@ucl.ac.uk
The Data Supplement is available at https://www.ahajournals.org/doi/suppl/10.1161/ATVBAHA.121.316388.
For Sources of Funding and Disclosures, see page e426.
© 2021 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol is available at www.ahajournals.org/journal/atvb

067
 Orlandi et al
CLINICAL AND POPULATION
Nonstandard Abbreviations and Acronyms
STUDIES - VB
d-ROMs reactive oxygen metabolites
FMD flow-mediated dilatation
GTN glyceryl trinitrate
GTNMD glyceryl trinitrate-mediated dilatation
hs-CRP high sensitivity C-reactive protein
IFN Interferon
IL interleukin
IPT intensive periodontal treatment
IR ischemia-reperfusion
mtROS mitochondrial reactive oxygen species
PBMC peripheral blood mononuclear cells
RIPC remote ischemic preconditioning
sICAM-3 soluble intercellular adhesion molecule 3
TNF tumor necrosis factor
modulation of systemic inflammation and oxidative stress
has been implicated as possible contributing factors of
the observed vasculoprotective effects.5–7 Limited evi-
dence, however, is available on the interplay between
RIPC, acute systemic inflammation, oxidative stress, and
endothelial function in humans.
Our group has developed and extensively character-
ized a novel model to study human inflammation: the
intensive periodontal treatment (IPT) model. Periodontitis
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is a common chronic inflammatory and infectious disease
caused by a dysbiotic dental biofilm in susceptible individ-
uals and affecting the tissues supporting the dentition.8
Management of the disease relies upon professional
cleaning of the teeth affected (mechanical disruption of
the dental biofilm), resulting in a dramatic reduction of
local gingival inflammation.9 A single session of IPT not
only improves gums’ health but also results in a 1-week
acute elevation of the systemic inflammation and oxida-
tive stress.10 This is thought to be related to the systemic
dissemination of molecules of bacterial origin and a sys-
temic inflammatory response associated with a profound
but transient alterations of endothelial function.11
Acute inflammation and its detrimental effects on
the endothelium have been implicated as a plausible
biological mechanism behind the link of common acute
infections and increased vascular risk.12 The exposure
of endothelial cells to proinflammatory cytokines could
stimulate the expression of tissue-factor, cell-surface
adhesion molecules, and induction of procoagulant
activity.
Building on our previous experience, we designed a
single-blind, parallel-group, randomized controlled trial to
evaluate whether RIPC can modulate the inflammatory
and oxidative response and prevent endothelial dysfunc-
tion following an acute inflammatory stimulus induced by
IPT in patients with periodontitis.
068
RIPC and Endothelial Function
Highlights
• Remote ischemic preconditioning has been investi-
gated as method to attenuate the ischemia-reperfu-
sion damage that follows an acute ischemic injury.
• This mechanistic trial tested the effect of remote
ischemic preconditioning on endothelial function in
a human model of systemic inflammation.
• These results suggest a protective effect of remote
ischemic preconditioning on endothelial function via
the modulation of the inflammatory response and
the redox activity following exposure to a validated
systemic acute inflammatory stimulus in humans.
METHODS
The data that support the findings of this study are available
from the corresponding author upon reasonable request.
Population
Patients referred to the Eastman Dental Hospital, University
College London (United Kingdom) for periodontal screening
and therapy were invited to participate in this study if they had
active generalized periodontitis defined as at least 30 peri-
odontal pockets with probing pocket depth >4 mm and con-
firmed radiographic alveolar bone loss. Patients were excluded
if they were (1) pregnant, breastfeeding or of childbearing
potential, (2) on chronic treatment (ie, 2 weeks or more) with
specific medications known to affect periodontal status (phe-
nytoin or cyclosporine) within 1 month of the start of the study,
(3) suffering from any systemic disease (assessed by a medical
history questionnaire), (4) with limited mental capacity or lan-
guage skills such that simple instructions cannot be followed or
information regarding adverse events could not be provided, (5)
on any chronic medications or requiring antibiotic coverage for
dental/periodontal procedures, and (6) had received a course
of periodontal therapy in the preceding 6 months. All patients
gave written informed consent. The study was approved by the
London Queen Square Ethics Committee (06/Q0512/107).
Study Design
This was a randomized controlled clinical trial with a 7 day follow-
up with 2 parallel groups. At baseline, full medical and dental
histories were collected by a single trained examiner (M. Orlandi).
Anthropometric measures, including height, weight, waist, and
hip circumferences were recorded using standard protocols.
Arterial blood pressure was measured in triplicate, and the aver-
age of the readings was recorded. Patients underwent full dental
examinations, fasting blood samples collection, and endothelial
function assessment at baseline, as well as at 1 and 7 days fol-
lowing periodontal treatment (Figure 1). This trial was reported
following the CONSORT guidelines (Consolidated Standards of
Reporting Trials)13 (Appendix I in the Data Supplement).
Randomization
Following the baseline visit, study participants undergoing IPT
were randomly assigned with the use of a computer-generated
 Orlandi et al
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Figure 1. CONSORT study flowchart.
IPT indicates intensive periodontal treatment; and RIPC, remote ischemic preconditioning.
table and in a 1:1 ratio to receive RIPC (test group) or a sham
procedure (control group) 30 minutes before treatment. Smoking
status, sex, age, and severity of periodontitis differences were
accounted for in the randomization by minimization.14 Treatment
assignments were concealed in opaque envelopes and revealed
to the research staff performing the RIPC on the day the treat-
ment was administered by the trial coordinator. The clinician per-
forming IPT was unaware of the group allocation. The data were
collected and analyzed in masked fashion.
Periodontal Examination and Therapy
A single, trained dental examiner (M. Orlandi) performed all
dental assessments/treatment. The number of teeth, probing
pocket depth, gingival recession, and clinical attachment lev-
els were recorded. The presence or absence of supragingival
069
RIPC and Endothelial Function
CLINICAL AND POPULATION
STUDIES - VB
dental plaque and gingival bleeding on probing on whole mouth
was also recorded. All study participants underwent IPT within
1 month from the baseline visit. This consisted in a single-sit-
ting full-mouth session of scaling and root planning, which was
performed under local anesthesia using hand and ultrasonic
instruments as previously described.11
RIPC and Sham Control Procedure
RIPC was induced using a 9 cm blood pressure cuff placed on
the upper arm and inflated to a pressure of 200 mm Hg for 5
minutes followed by completed deflation for 5 minutes, a cycle
which was repeated 3 times in total.15 For the sham procedure
(control group), a 9 cm blood pressure cuff placed on the upper
arm and inflated to a pressure of 10 mm Hg for 5 minutes fol-
lowed by completed deflation for 5 minutes, a cycle which was
 Orlandi et al
repeated 3 times in total. IPT commenced after the completion
CLINICAL AND POPULATION
of the RIPC protocol.
STUDIES - VB
Vascular Function
All the participants were instructed to fast for at least 8 hours,
refrain from drinking beverages containing caffeine, and to not
smoke on the day of the examination. A temperature-controlled
room (22 °C degrees) was used for all the vascular assessments.
A high-resolution ultrasound machine (Acuson XP128 with a
7-MHz linear probe) for image acquisition and a semiautomatic
edge detection software for postacquisition analyses (Medical
Imaging Applications, vascular research tools, version 5.6.7) was
used to measure endothelium-dependent and -independent vaso-
dilatations of the brachial artery, as previously described.16 After
10 minutes of rest, endothelium-independent dilatation was mea-
sured after sublingual administration of 25 μg of glyceryl trinitrate
(GTN), according to the same recording protocol.16 Brachial artery
dilation was calculated as a percentage change from baseline
to the peak diameters. A single examiner blinded to the RIPC or
sham procedures, acquired all vascular data. Analysis of the flow-
mediated dilatation (FMD) images was performed in a blinded
fashion. The sonographer attended a training session (London
Core Lab, London, United Kingdom) and completed a certification
process which involved 10 repeat scans with <2% variability in
%FMD. All the patients were assessed at the same time (morning)
of the day at each study visit (baseline, day 1, and day 7).
Inflammatory and Vascular Biomarkers
At baseline, 24 hours and 7 days after IPT fasting blood
samples were collected, immediately processed in several ali-
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quots and stored at −70 degrees. Measures of a broad panel
of inflammatory biomarkers were performed in blind fashion
at the end of the trial using multiplex high sensitivity assays
(Meso Scale Discovery, MD), including IL-1β (interleukin-1β),
IL-6, IL-8, IL-10, IL-12, INF-γ (interferon-γ), and TNF-α (tumor
necrosis factor-α) according to manufacturer’s instructions.
Serum hs-CRP (C-reactive protein) was measured by immuno-
turbidimetry (Cobas, Roche Diagnostic, Mannheim, Germany).
Soluble E-selectin, soluble P-selectin, sICAM-3 (soluble inter-
cellular adhesion molecule 3), and soluble thrombomodulin
were assayed with a multiplex assay (Meso Scale Discovery,
MD). Coefficient of variation for all assays (intra and inter) were
recorded and confirmed to be lower than 5%.
Oxidative Stress
In this study, we chose a cumulative oxidative test in
serum, d-ROMs (reactive oxygen metabolites) test, to estimate
the total amount of oxidative metabolites of each sample. This
test measures the serum concentration of hydroperoxides,
a class of chemical oxidant species belonging to the wider
group of d-ROMs.17 It has been previously used to measure
total levels of circulating oxidative markers in patients under-
going periodontal treatment, showing reliable and reproduc-
ible results.18 Further, mtROS (mitochondria reactive oxygen
species) were measured at each study visit in peripheral blood
mononuclear cells (PBMCs), isolated following standard pro-
cedures by density gradient centrifugation with Ficoll (Ficoll-
Paque PLUS, GE, United Kingdom). Mitochondrial oxidative
stress production was assessed by flow cytometry using
070
RIPC and Endothelial Function
the mitochondrial probes MitoSOX Red (Invitrogen, United
Kingdom) as previously described.19
Lipopolysaccharide Assay
Limulus amebocyte lysate was adopted for the detection of
endotoxin (QCL-1000, Lonza). According with this assay, gram-
negative bacterial endotoxin present in the samples catalyzes the
activation of a proenzyme in the limulus amebocyte lysate. The
initial rate of activation was determined by the concentration of
endotoxin present. This was measured photometrically at 405 to
410 nm, after which the reaction is stopped with stop reagent. The
correlation between the absorbance and the endotoxin concentra-
tion was linear in the 0.1 to 1.0 EU/mL range. The concentration
of endotoxin in a sample was calculated from the absorbance val-
ues of solutions containing known amounts of endotoxin standard.
Outcome Assessment
The primary outcome of this study was the difference in brachial
endothelial function assessed by FMD 24 hours following the
dental treatment between study groups. Secondary outcomes
included changes in the GTN-mediated dilatation (GTNMD), con-
centration of common inflammatory and circulating endothelial
markers, d-ROMs, lipopolysaccharide, and MitoSOX median fluo-
rescent intensity measured at 24 hours and at 7 days after IPT.
Statistical Analysis
Based on our previous study,11 we estimated that a minimum
of 22 per group patients should be enrolled into the study to
detect a 2% difference in FMD between the test and control
groups 24 hours after IPT (using an SD of the mean differ-
ence of 1.6% at a 2-sided α level of 5% and 90% power).
Accounting for a potential dropout rate of 10%, a final sample
size of 24 participants per group was recruited.
All are presented as mean values±SE or median and inter-
quartile range (25th to 75th percentile). Categorical variables
are shown as counts and percentages. Comparisons between
groups (RIPC versus placebo) at each time point were based
on the independent-samples Student t test or the nonparamet-
ric Mann-Whitney test for continuous variables and the χ2 test
for categorical variables.
Differences in vascular and inflammatory markers within
each group (RIPC or sham) and across study’s duration (base-
line, day 1, and day 7 after IPT) were initially assessed with
the nonparametric Kruskal-Wallis test. To control the inflation
of error rate in case of >2 comparisons among time points, we
implemented the Dunn test using rank sums with the Sidak
adjustment. Remaining comparisons between groups with
respect to main outcomes (FMD and GTNMD) and exposure
variables (inflammatory and oxidative markers) were based on
independent, prespecified hypotheses. Therefore, no further
correction for multiple comparisons was performed.
Subsequently, linear mixed models with random effects (ran-
dom intercept and random coefficient) and unstructured variance-
covariance matrix were implemented to test the effect of RIPC
versus the sham procedure on longitudinal changes in variables
of interest across the acute (baseline to D1) and the sustained
phase (baseline to D7) of the experiment. Demographic character-
istics (ie, age, sex, ethnicity, smoking, and body mass index) were
prespecified as covariates in the multivariable linear mixed models.
 Orlandi et al
An interaction term between exposure status and time (RIPC ver-
sus Sham*time) was used in the linear mixed model analysis to
assess the differential effect of the randomly allocated intervention
(RIPC versus placebo) on acute and prolonged changes in vascu-
lar and biochemical markers in comparison to placebo. For FMD
specifically, we also tested linear mixed models involving addi-
tional independent variables with repeated measurements such as
d-ROMs, hs-CRP (high sensitivity C-reactive protein), TNF-α, and
P-selectin to infer about potential temporal causality between
longitudinal changes in endothelial function and in inflam-
matory/oxidative molecules. To ensure normal distribution of
dependent variables, we performed inverse ranking normaliza-
tion20 before running the linear mixed models. Results of the linear
mixed model analysis are reported in the original scale for vascular
markers to facilitate understanding the magnitude of the effect.
We used generalized structural equation modeling to fit mul-
tivariate linear mixed models with random intercept and random
slope and test the overall interaction between RIPC and time
(baseline to D1) on simultaneous repeated measurements in 4
key inflammatory variables (hs-CRP, TNF-α, IL-6, and INF-γ).
We used the robust Huber/White/Sandwich estimator to derive
the variance-covariance matrix of the estimates.
Finally, we aimed to disentangle the direct and indirect effects
of the RIPC versus placebo on endothelial function, and we used
structural equation models. In detail, we assessed the mediating
effect of RIPC versus control on FMD through P-selectin, hs-
CRP, and TNF-α, and d-ROMs while controlling for the impact
of age, sex, body mass index, and ethnicity. Standardized esti-
mates were used in connecting paths. To address non-normality
of variables and validate the indirect effects, we used maximum
likelihood estimation with the robust estimator of the variance-
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covariance matrix (Huber/White/sandwich estimator). SE and
CI for the indirect effects were obtained through bootstrapping
with 200 replicates. The comparative fit index (≥0.90 indicates
acceptable fit) and the root mean square error of approxima-
tion (<0.06 indicates acceptable fit) were calculated to assess
model fit for our main SEM. Statistical analysis was performed
by STATA package, version 11.1 (StataCorp, College Station, TX).
RESULTS
A total of 49 patients were recruited from April to Octo-
ber 2013 and randomly allocated to the control group
(n=25) and to the remote ischemic conditioning group
(n=24; Figure 1).
Study participants were middle-aged (46±9 years)
with similar ethnicity and sex distribution, body mass
index, cardiovascular risk factors, levels of inflammatory
cytokines, periodontal status, FMD, and GTN-induced
vasodilation (Table 1). No serious adverse events were
recorded during the study. Table 2 reports changes in
vascular parameters and circulating biomarkers across
the study’s follow-up according to status of randomiza-
tion (RIPC versus control).
Vascular Function
Changes in the endothelial function (primary outcome)
from the baseline to the day 1 visits were different in the
071
RIPC and Endothelial Function
RIPC (RIPC×baseline to day 1 interaction=2.16% [95%
CLINICAL AND POPULATION
CI, 1.45–2.88], P<0.001; Figure 2A) compared with the
control group (unadjusted between-groups difference
STUDIES - VB
at day 1 P=0.04 and adjusted difference=1.85% [95%
CI, 0.463–3.24], P=0.009), suggesting that RIPC might
attenuate the endothelial dysfunction induced by the
acute inflammatory response following IPT. RIPC had a
similar impact on the changes of the GTNMD so that GTN
induced higher vasodilation in the RIPC (RIPC×baseline
to day 1 interaction=2.84% [95% CI, 1.13–4.55],
P=0.001) compared with the control group 24 hours post-
IPT (unadjusted between-groups difference P=0.057
and adjusted difference 4.33% [95% CI, 1.08–7.57],
P=0.009; Figure 2B). These vascular changes recovered
completely 7 days after IPT, when no differences between
RIPC and control groups were observed for endothelial-
dependent and -independent vasodilation (P>0.1 for
both interaction terms and between-groups differences,
Figure 2A and 2B). Furthermore, we observed a posi-
tive association between changes in FMD and GTNMD
(coefficient=0.214% [95% CI, 0.133–0.295], P<0.001).
Markers of Inflammation and Vascular
Activation
RIPC attenuated the IPT-induced increase in IL-1β
between the baseline and day 1 (RIPC×baseline to day
1=−1.57 [95% CI, −1.90 to −1.23, P<0.001) and thus,
the difference in IL-1β levels between groups (adjusted
difference as compared to the sham procedure at day
1=0.195 [95% CI, −0.937 to 1.33], P=0.735).
Among other inflammatory markers, in the RIPC group,
there was an attenuated rise in circulating E-selectin (P
for interaction [baseline to day 1]=0.056), sICAM-3 (P for
interaction [baseline to day 1]=0.092 and P for interaction
[baseline to day 7]=0.078) and s-thrombomodulin (P for
interaction [baseline to day 7]=0.06). IL-10 was increased
at day 1 in the placebo group as compared to the RIPC
(mean adjusted difference =0.762 [95% CI, 0.239–1.28],
P=0.004) but no statistically significant interaction of treat-
ment with changes in its levels was established. IL-12 was
increased at day 1 in the placebo group as compared to the
RIPC (mean adjusted difference =0.692 [95% CI, 0.129–
1.26], P=0.016) but no statistically significant interaction
of treatment with changes in this cytokine was established
(P for interaction [baseline to day 1]=0.376); in contrast,
RIPC differentially increased the IL-12 levels at the end of
the experiment (RIPC×baseline to day 7=1.43 [95% CI,
0.196–2.67], P=0.023). Importantly, by generalized struc-
tural equation modeling, we found a statistically significant
overall interaction between type of allocated treatment
before IPT and time (baseline to day 1) on changes of
the inflammatory array consisting of hs-CRP, TNF-α, IL-6,
and INF-γ (P=0.01). This suggests that RIPC was able to
attenuate the global systemic inflammatory response gen-
erated by the IPT compared with placebo.
 Orlandi et al RIPC and Endothelial Function

Table 1. Baseline Characteristics of the 2 Study Groups Oxidative Stress

CLINICAL AND POPULATION

Control (N=25) RIPC (N=24) Participants who received RIPC exhibited higher increase
STUDIES - VB

Age, y 47±9 45±9 in d-ROMs levels from baseline to day 1 (RIPC×baseline

Sex, male (%) 14 (56.0) 11 (45.8) to day 1=0.835 [95% CI, 0.255–1.42], P for interaction
Ethnicity, White (%) 15 (60.0) 15 (62.5) =0.005) and to day 7 (RIPC×baseline to day 7 =0.768
Body mass index, kg/m2 26.5±3.8 26.1±3.7 [95% CI, 0.185–1.35], P for interaction =0.01) when com-
Waist circumference, cm 92±8 93±8 pared with the patients in the controls group. PBMC isolated
Hip circumference, cm 106±8 105±6
from participants in the RIPC group revealed increased
Smoking, current (%) 7 (28.0) 8 (33.3)
mtROS production compared with the those in the control
group between baseline and day 1 (RIPC×baseline to day
SBP, mm Hg 120±16 118±10
1=0.666 [95% CI, 0.018–1.314], P for interaction =0.044).
DBP, mm Hg 77.58±8.51 76.43±8.08
TC, mmol/L 5.18±0.81 5.17±0.78
Lipopolysaccharide
TG, mmol/L 1.11±0.53 1.06±0.26
No substantial differences in plasma values of LPS in
hs-CRP, mg/L 2.05±2.19 1.74±1.72
serum of patients in the RIPC and the control group
IL-1β, pg/mL 0.35±0.2 0.21±0.17
were observed at all 3 time points (baseline, 24 hours
IL-6, pg/mL 1.15 (0.63–1.79) 0.92(0.38–2.00) and 7 days post-IPT, P>0.1 for all). RIPC was not related
IL-8, pg/mL 11.22±3.45 11.64±4.47 with differential changes in LPS across the study for the
IL-10, pg/mL 6.26±8.01 4.08±3.82 2 groups (P>0.1 for both interaction terms, baseline to
IL-12, pg/mL 0.66±0.64 0.74±0.48 24 hours and baseline to 7 days).
TNF-α, pg/mL 4.00±1.92 3.52±1.85
INF-γ, pg/mL 2.39±2.46 3.15±3.15 Interplay Between Inflammatory and Oxidative
sE-selectin, pg/mL 21.26±16.43 20.75±15.20 Markers
sP-selectin, pg/mL 122.89±62.12 114.92±43.91 By linear mixed model analysis, we found an inverse lin-
s-ICAM3, pg/mL 1.92±3.86 2.02±3.39 ear association of changes in TNF-α (coefficient=−0.071
s-TM, pg/mL 5.30±3.98 5.47±6.06 [95% CI, −0.141 to −0.002], P=0.044), IL-8 (coeffi-
d-ROMs (Carr/U) 455.04±89.17 383.60±98.05 cient=−0.033 [95% CI, −0.058 to −0.008], P=0.01),
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PBMC mtROS (MitoSOX, MFI) 26.63±11.54 24.05±10

and hs-CRP (coefficient=−0.026 [95% CI, −0.041 to
−0.011], P=0.001) with fluctuations in FMD during the
LPS, EU 0.88±0.59 1.09±0.91
study’s period after adjusting for age, sex, ethnicity, body
FMD, % 6.28±2.56 6.28±3.68
mass index, and changes in SBP (Figure 3). When all 3
GTNMD, % 17.40±7.14 19.52±7.64
inflammatory markers were forced in the same model, hs-
IPT time, min 144±25 128±24 CRP was the only biomarker to retain its association (coef-
PPD, cm 4.16±0.82 3.84±0.56 ficient=−0.019 [95% CI, −0.035 to −0.002], P=0.028)
REC, cm 0.85±0.86 0.86±0.82 with changes in FMD. We did not find evidence of con-
NPKTS, n 69.33±28.96 61.00±21.81 comitant changes in other circulating markers and fluctua-
FMPS, % 63.97±16.23 58.90±15.6 tions of endothelial function across the prespecified time
FMBS, % 49.86±21.97 50.05±16.04 points of the study (P>0.1 for all). Furthermore, changes
NTEETH, n 28.46±2.72 28.57±2.84
in GTNMD were inversely correlated with changes in
TNF-α (coefficient=−0.575 [95% CI, −0.898 to −0.252],
Values are expressed in mean±SD for continuous variables and number
(%) for categorical variables. DBP indicates diastolic blood pressure; d-ROMs,
P<0.001), IFN-γ (coefficient=−0.116 [95% CI, −0.223 to
reactive oxygen metabolites; FMBS, full-mouth gingival bleeding score; FMD, −0.0089], P=0.034), IL-12 (coefficient=−0.196 [95% CI,
flow-medicated dilation; FMPS, full-mouth dental plaque score; GTNMD, −0.348 to −0.044], P=0.011), IL-6 (coefficient=−0.091
glyceryl trinitrate-mediated dilatation; hs-CRP, high sensitivity C-reactive pro-
tein; IL-10, interleukin-10; IL-12, interleukin-12; IL-1β, interleukin-1β; IL-6,
[95% CI, −0.167 to −0.0154], P=0.018), IL-8 (coeffi-
interleukin-6; IL-8, interleukin-8; INF-γ, interferon-γ; IPT, intensive periodon- cient=−0.137 [95% CI, −0.239 to −0.0345], P=0.009).
tal therapy; LPS, lipopolysaccharides; mtROS, mitochondrial reactive oxygen
species; NPKTS, number of periodontal lesions with PPD >4 mm; NTEETH,
number of teeth; PPD, probing pocket depth; REC, recessions; RIPC, remote
Mediation Analysis
ischemic preconditioning; SBP, systolic blood pressure; s-TM, soluble throm- By structural equation models analysis, it was shown
bomodulin; TC, total cholesterol; TG, triglycerides; and TNF-α, tumor necrosis
factor-α. that while RIPC was a direct determinant of FMD
changes (β coefficient for the direct effect =1.67 [95%
CI, 0.342 to 2.999], P=0.014), no indirect effect through,
No changes related to type of allocated treatment hs-CRP, TNF-α, and IL-8 (β coefficient for the indirect
were observed for hs-CRP, TNF-α, INF-γ, IL-6, IL-8 fluc- effect =−0.31 [95% CI, −0.061 to 0.123], P=0.510)
tuations across the prespecified time points. was established.

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 Orlandi et al RIPC and Endothelial Function

Table 2. Changes in Vascular Indices and Circulating Biomarkers Across the Study’s Follow-Up

CLINICAL AND POPULATION

According to Status of Randomization (RIPC Versus Control)

Treatment Variable Baseline Day 1 Day 7 P value

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Control FMD 6.15 (4.21–8.34) 2.94 (1.95–5.06)*† 4.78 (4.12–6.75) <0.001
RIPC 5.03 (3.75–7.9) 3.89 (2.9–7.93) 5.02 (3.66–8.55) 0.471
Control GTNMD 16.9 (12.3–22.6) 14.2 (9.84–18.8) 13.8 (12.2–19) 0.309
RIPC 17.8 (13.8–25.5) 18 (14.8–24.8) 17.6 (14.8–23.8) 0.971
Control TNF–α 3.47 (2.45–5.46) 4.47 (3.47–7.74) 5.28 (3.52–6.27) 0.121
RIPC 3.16 (1.94–4.75) 3.86 (2.28–5.16) 3.67 (3.14–5) 0.29
Control hs-CRP 1.30 (0.9–2.7) 8 (4.5–14.2)* 2.15 (1.45–4.55) <0.001
RIPC 1.00 (0.6–2.8) 5.25 (3.3–8.5)* 1.85 (1.1–2.8) <0.001
Control IFN-γ 1.42 (0.50–4.44) 10.8 (2.54–26.7)* 2.83 (1.43–7.13) 0.012
RIPC 2.57 (0.66–4.68) 4.59 (1.88–11.1) 2.33 (1.33–3.48) 0.174
Control IL-1β 0.36 (0.21–0.50) 0.45 (0.22–0.76) 0.29 (0.11–0.51) 0.557
RIPC 0.25 (0.02–0.36) 1.27 (0.187–3.17) 0.21 (0.18–0.66) 0.661
Control IL-6 1.15 (0.63–1.79) 4.05 (2.2–7.19)* 1.56 (1.17–2.99) <0.001
RIPC 0.92 (0.38–2) 3.9 (1.95–5.43)* 1.43 (1.13–2.69) <0.001
Control IL-8 11.4 (8.04–13.9) 13.3 (7.86–17.6) 13.8 (7.89–18.6) 0.366
RIPC 11.7 (8.9–16.3) 9.43 (7.67–14.9) 11.9 (7.7–6.0) 0.614
Control IL-10 3.8 (1.92–6.61) 7.49 (4.92–11.6)*† 4.58 (3.4–9.17) 0.064
RIPC 2.77 (1.25–6.75) 3.86 (2.1–6.94) 4.67 (1.85–8.26) 0.532
Control IL-12 0.38 (0.19–0.83) 2.13 (1.11–4.2)*† 0.55 (0.26–1.31)† 0.004
RIPC 0.90 (0.46–1.03) 1.11 (0.57–1.61) 2.17 (1.13–3.22)* 0.061
Control s-ICAM3 1.14 (0.79–1.54) 1.05 (0.91–1.57) 1.28 (0.85–1.65) 0.793
RIPC 1.39 (0.83–1.78) 1.22 (0.93–1.49) 1.3 (1.14–1.51) 0.707
Control sE-selectin 17 (11.7–22.9) 20.2 (15–24.8) 17.5 (10.8–21.9) 0.459
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RIPC 15.9 (10.7–24.9) 18.7 (10.7–22.5) 14.4 (10.5–21.2) 0.552

Control sP-selectin 102 (85.1–142) 103 (64.3–148) 125 (76.6–141) 0.794
RIPC 102 (83.9–155) 80.6 (68–106) 91.1 (69.7–128) 0.163
Control s-TM 4.7 (3.2–5.79) 4.31 (3.45–5.9) 4.5 (3.45–5.27) 0.957
RIPC 4.15 (3.49–4.82) 4 (3.24–4.62) 3.57 (3.38–4.44) 0.383
Control d-ROMs 451 (409–493)† 470 (395–529) 436 (388–491) 0.804
RIPC 388 (326–439) 443 (393–525) 467 (366–544)* 0.038
Control PBMC mtROS 23.1 (19.1–28.6) 23 (17–31.5) 23.3 (17.6–26.7) 0.774
RIPC 23.1 (17.2–25.3) 25.6 (20.1–34.2) 21.7 (16.5–34.3) 0.353
Control Lymphocytes mtROS 19.6 (13.8–25.3) 18.5 (14.4–23.7) 18.9 (14.2–22.5) 0.966
RIPC 17.6 (12.7–23.9) 18.6 (14.1–33.8) 17.5 (13.2–30.5) 0.556
Control Monocytes mtROS 55.2 (33.7–100) 87.8 (37.3–140) 56.2 (41.8–91.4) 0.342
RIPC 44.6 (25.9–82) 83.5 (42–167)* 50 (30.8–108) 0.067

Values are expressed in mean (CI). d-ROMs indicates reactive oxygen metabolites; FMD, flow-medicated dilation; GTNMD, glyceryl
trinitrate-mediated dilatation; hs-CRP, high sensitivity C-reactive protein; IL-10, interleukin-10; IL-12, interleukin-12; IL-1β, interleukin-1β;
IL-6, interleukin-6; IL-8, interleukin-8; INF-γ, interferon-γ; LPS, lipopolysaccharides; mtROS, mitochondrial reactive oxygen species; RIPC,
remote ischemic preconditioning; s-TM, soluble thrombomodulin; and TNF-α, tumor necrosis factor-α.
*Statistically significant within-group difference from the reference category (baseline) after Sidak adjustment for multiple comparisons.
†Statistically significant between-groups difference for the same time point (baseline, 24 h, or day 7).

DISCUSSION inflammation and redox activity. The vascular benefits

This is the first clinical trial in humans showing that RIPC involved both endothelial-dependent and -independent
before a moderate inflammatory stimulus (induced by vasodilation, suggesting a protective effect not limited to
IPT) confers protection to the vasculature and that it the endothelium but involving also the smooth muscle
was associated with modulation of markers of systemic cells of the tunica media.

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 Orlandi et al
CLINICAL AND POPULATION
STUDIES - VB
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Figure 2. Mean flow and glycerlyl trinitrate mediated
dilatation during the study duration.
I bars represent SE. Data are for the 24 patients in the test group
and the 25 patients in the control-treatment group. A, Flow-
medicated dilation (FMD) % changes. Please notes values are
adjusted for age, sex, smoking, body weight, and ethnicity. P values
for the interaction terms baseline to 24 h (P<0.001). P values are
derived from linear mixed model analysis. B, Mean GTNMD (glyceryl
trinitrate-mediated dilatation) during the study duration. I bars
represent SE. Data are for the 24 patients in the test group and
the 25 patients in the control-treatment group. GTN % changes.
Please notes values are adjusted for age, sex, smoking, body weight,
and ethnicity. P values for the interaction terms Baseline to 24 h
(P=0.007). P values are derived from linear mixed model analysis.
The ability of RIPC to modulate inflammatory responses
has been previously investigated in humans with conflict-
ing results. Some evidence reported that RIPC induces
leukocyte inflammation gene expression,21 attenuates
systemic neutrophil activation,3 and alters neutrophil func-
tion.22 These changes occur within minutes after RIPC
and are even more pronounced after 24 hours.
By contrast, a lack of inflammatory modulation after
endotoxemia following LPS administration has been
described in a pilot experiment on healthy volunteers.23 IPT
represents a mixed infectious/inflammatory stimulus, as it
induces a transient bacteremia that is accompanied by an
increase in the circulating levels of inflammatory cytokines.
We previously documented modifications of the systemic
074
RIPC and Endothelial Function
endothelial function that track these changes of the inflam-
matory response.11 We now show that, in the same model,
RIPC is able to attenuate the rise of several inflammatory
markers (including hs-CRP, IL-1β, and TNF-α) during the
acute phase of the inflammatory response, supporting the
hypothesis that RIPC could modulate systemic inflamma-
tion. Furthermore, we report that this effect is accompanied
by a substantial attenuation of the endothelial dysfunction
commonly recorded 24 hours after IPT. The potential pro-
tective effect of RIPC against the inflammatory stimulus
generated by the IPT is confirmed by the reduced eleva-
tion of circulating P-selectin levels in the RIPC versus
placebo groups. Importantly, through a formal mediation
analysis, we also show for the first time that, while there
was a relationship between changes in FMD and the sys-
temic levels of some inflammatory cytokines, inflammation
is unlikely to mediate the impact of RIPC on the endothe-
lial function. Similarly, the lack of substantial differences
in LPS levels between groups at any time point during
the study suggests that also the bacterial dissemination
which follows the IPT is unlikely to account for its vascular
effects. However, LPS does not reflect the levels of oral
bacteria but could be subsequent to the far larger burden
represented by the gut microbiome.
In this experiment, we have also observed an improve-
ment in GTNMD in the RIPC group. A deterioration in
GTNMD following IPT was previously reported by our
group.11 GTN triggers vasodilation independently from
the vascular endothelium and represents the vascular
smooth muscle cell sensitivity to NO.24 A deterioration
in GTNMD might be the result of underlying vascular
smooth muscle dysfunction and possibly an impairment
in ions (Ca2+and K+)-mediated mechanisms regulating
vascular smooth muscle cell contractility caused by the
inflammatory response following IPT.25 This finding war-
rants further investigation in further experiments.
We further investigated the impact of RIPC on PBMCs
mitochondrial function assessed by superoxide production.
PBMCs comprise a heterogeneous population of leuko-
cytes including cells from the lymphoid system (predomi-
nantly T cells, B cells, and NK cells) and myeloid system
(mainly monocytes). Although ROS are produced by several
extracellular and intracellular processes, the mitochondria
represent one of the main sources of oxidants. A role of
mitochondria in the ischemia-reperfusion injury has been
previously hypothesized, suggesting that RIPC might pre-
serve cardiomyocyte mitochondrial function following isch-
emia-reperfusion.26 Furthermore, we have recently reported
that a lower PBMC mtROS production tracks the amelio-
ration of FMD of the brachial artery observed 6 months
following periodontal treatment.19 These data suggested a
potential role of mitochondria dysfunction in mediating the
endothelial effects of both the IPT and RIPC. Unexpect-
edly, we found a higher mtROS in PBMCs of the RIPC
compared with the placebo group 24 hours after IPT. The
capacity of RIPC to induce a pro-oxidant environment is
 Orlandi et al
Figure 3. Changes in hs-CRP (high sensitivity C-reactive
protein) and fluctuations in flow-medicated dilation (FMD)
during the study duration.
Please notes values are adjusted for age, sex, ethnicity, body mass
index (BMI), and changes in systolic blood pressure.
confirmed by the results of the d-ROMs test, showing that
subjects receiving RIPC had a more substantial increase
of d-ROMs than the control group 24 hours after IPT and
that this difference persisted a week later. It has been
documented that an excessive generation of ROS and
reactive nitrogen species within immune cells is linked to
diminished inflammasome activation and a reduced inflam-
matory response.27 Thus, we can speculate that an acute
rise of mtROS production in inflammatory cells might impair
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their proinflammatory cytokine secretion and, through this
mechanism, have a protective role on the endothelial func-
tion. This hypothesis could also explain the reduced cumu-
lative inflammatory response (hs-CRP, TNF-α, IL-6, and
INF-γ) observed in the RIPC group 24 hours after the
treatment. Other potential explanations of our results
relates to the capacity of specific ROS to act as signal-
ing mediators. d-ROMs are derivatives of reactive oxygen
metabolites and their quantification indirectly estimates
the total amount of hydroperoxides in serum representing
an index of oxidant capacity. Hydrogen peroxide has been
identified as a signaling mediator in the vasculature, hav-
ing positive effect on the endothelium-dependent vaso-
relaxation.28 Furthermore, there is evidence to suggest a
role of hydroperoxides in the endothelium-dependent vaso-
dilation through cyclooxygenase-1–mediated release of
prostaglandin E2. Finally, hydrogen peroxide acts directly on
smooth muscle by hyperpolarization through KCa channel
activation leading to relaxation.29 This would also explain the
improved GTN-induced vasorelaxation at 24 hours after IPT
observed in the RIPC compared with the placebo group.
The importance of an adequate ROS response to the RIPC
is confirmed by the loss of preconditioning protection when
cardiomyocytes are treated with antioxidants.30,31 Although
these findings might provide potential explanations to our
results, the unexpected nature of our results, the lack of data
describing the changes of PBMC mtROS production and
075
RIPC and Endothelial Function
its association with endothelial activation/protection after
CLINICAL AND POPULATION
an acute inflammatory stimulus impose a careful interpreta-
tion of our results and confirmation in future investigations.
STUDIES - VB
Our study has some limitations. The anti-inflammatory
effects of RIPC observed may be specific to the IPT
model and their relevance in other human models of acute
inflammation remains to be tested. Although we included
healthy participants with no other systemic conditions
known to impact on the endothelium (such as hyperten-
sion, heart failure, atherosclerosis, hypercholesterolemia,
and diabetes), we cannot rule out an alternative mecha-
nism of protection of RIPC on vascular dysfunction A dif-
ference in the levels of d-ROM at the baseline visit, could
confound the interpretation of the results, although the
overproduction of ROS following RIPC is confirmed by
the data on the mtROS. However, our study has several
strengths. The randomized design and masked assess-
ment contribute to high internal validity. Our group has
extensively characterized the inflammatory and oxida-
tive stress responses to IPT, making this a solid model to
study the complex interaction between inflammation and
endothelial function in humans. The presence of a sin-
gle-blind vascular examiner reduced the variability of our
vascular measures. Finally, data on a wide range of poten-
tial mediators of the benefits related to the RIPC on the
endothelial function were acquired and analyzed using a
robust statistical methodology to ascertain the potential
influence of many parameters on the link between RIPC,
inflammation, and vascular phenotype.
RIPC performed before an acute inflammatory stim-
ulus can modulate both acute inflammation and endo-
thelial cell activation. This resulted in an improvement of
endothelial function and was associated with a transient
increase in oxidative stress. A wide range of infective
disorders and iatrogenic procedures can cause severe
systemic inflammation. Acute systemic inflammation is
associated with an increase in the risk of cardiovascular
events that may persist for days or weeks. The present
study demonstrates that RIPC is tested as preven-tion
technique to protect the endothelium from the nega-tive
impact of an acute inflammatory response.
ARTICLE INFORMATION
Received April 14, 2021; accepted May 27, 2021.
Affiliations
Periodontology Unit, UCL Eastman Dental Institute and Hospital (M.O., Y.L., F.D.),
National Centre for Cardiovascular Prevention and Outcomes Institute of Car-
diovascular Science (S.M., D.B., S.T.C., J.D.), The Hatter Cardiovascular Institute
(D.Y., D.J.H.), Institute of Cardiovascular Science (A.H.), University College Lon-
don, United Kingdom. Internal Medicine Unit, University of Pisa, Italy (S.M.). Peri-
odontology Unit, Faculty of Odontology, University of Santiago de Compostela
and Medical-Surgical Dentistry Research Group (Y.L.) and Clinical Neurosciences
Research Laboratory (Y.L.), Health Research Institute of Santiago de Compostela,
Spain. School of Biomedical Engineering and Imaging Sciences, King’s College
London, St Thomas Hospital, United Kingdom (G.G.). Cardiovascular and Meta-
 Orlandi et al RIPC and Endothelial Function

bolic Disorders Program, Duke-National University of Singapore Medical School,

CLINICAL AND POPULATION

Singapore (D.J.H.). National Heart Research Institute Singapore, National Heart

Centre, Singapore (D.J.H.). Yong Loo Lin School of Medicine, National University
STUDIES - VB

Singapore, Singapore (D.J.H.). Cardiovascular Research Center, College of Medi-

cal and Health Sciences, Asia University, Taiwan (D.J.H.).

Acknowledgments
We thank Dr Jeanie Suvan, Banbai Hirani, Tiff Mellor, Chiara Curra, and Kasia
Niziolek for their invaluable assistance, the Eastman Dental Hospital for the valu-
able support in the recruitment, and Riccardo Latina for the design of the graphi-
cal abstract.

Sources of Funding
This study was funded thanks to a UCL Impact Award to Marco Orlandi partially
supported with a fellowship grant by Johnson and Johnson Consumer Services
EAME Limited and a fast-track grant to Francesco D’Aiuto from the UCL Bio-
medical Research Centre (CRDC - F189), and it was performed at UCL/UCLH
which receives support from the UCL Biomedical Research Centre who ob-
tained funding from the NIHR. Marco Orlandi holds a NIHR Clinical Lectureship
awarded by the National Institute for Health Research (NIHR). Y. Leira holds a
research contract with Fundación Instituto de Investigación Sanitaria de Santiago
de Compostela (fIDIS) and a Senior Clinical Research Fellowship supported by
the UCL Biomedical Research Centre who receives funding from the NIHR (NI-
HR-INF-0387). D.J. Hausenloy was supported by the British Heart Foundation
(CS/14/3/31002), the Duke-NUS Signature Research Programme funded by
the Ministry of Health, Singapore Ministry of Health’s National Medical Research
Council under its Clinician Scientist-Senior Investigator scheme (NMRC/CSA-
SI/0011/2017), Centre Grant, and Collaborative Centre Grant scheme (NMRC/
CGAug16C006). S. Chiesa holds a British Heart Foundation project grants
(PG/18/45/33814 and PG/19/31/34343). G. Georgiopoulos was supported
by a postdoctoral research fellowship from the Alexander S. Onassis Foundation.

Disclosures
None.
Downloaded from http://ahajournals.org by on April 17, 2024

076
 Long-term, regular remote ischemic preconditioning
improves endothelial function in patients with coronary
heart disease

Abstract

Remote ischemic preconditioning (RIPre) can prevent myocardial injury. The purpose of this study was to
assess the beneficial effects of long-term regular RIPre on human arteries.

Method

Forty patients scheduled for coronary artery bypass graft (CABG) surgery were assigned randomly to a
RIPre group (n=20) or coronary heart disease (CHD) group (n=20). Twenty patients scheduled for
mastectomy were enrolled as a control group. RIPre was achieved by occluding arterial blood flow 5 min
with a mercury sphygmomanometer followed by a 5-min reperfusion period, and this was repeated 4
times. The RIPre procedure was repeated 3 times a day for 20 days.

Assessment

In all patients, arterial fragments discarded during surgery were collected to evaluate endothelial function
by flow-mediated dilation (FMD), CD34+ monocyte count, and endothelial nitric oxide synthase (eNOS
expression). Phosphorylation levels of STAT-3 and Akt were also assayed to explore the underlying
mechanisms.

Result

Compared with the CHD group, long-term regular RIPre significantly improved FMD after 20 days (8.5±2.4
vs 4.9±4.2%, P,0.05) and significantly reduced troponin after CABG surgery (0.72±0.31 and 1.64±0.19,
P,0.05). RIPre activated STAT-3 and increased CD34+ endothelial progenitor cell counts found in arteries.

Conclusion

Long-term, regular RIPre improved endothelial function in patients with CHD, due to STAT-3 activation,
and this have led to an increase in endothelial progenitor cells.

077
Brazilian Journal of Medical and Biological Research (2015) 48(6): 568-576, http://dx.doi.org/10.1590/1414-431X20144452
ISSN 1414-431X

Long-term, regular remote ischemic preconditioning

improves endothelial function in patients
with coronary heart disease
Y. Liang, Y.P. Li, F. He, X.Q. Liu and J.Y. Zhang
Department of Cardiology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China

Abstract

Remote ischemic preconditioning (RIPre) can prevent myocardial injury. The purpose of this study was to assess the beneficial
effects of long-term regular RIPre on human arteries. Forty patients scheduled for coronary artery bypass graft (CABG) surgery
were assigned randomly to a RIPre group (n=20) or coronary heart disease (CHD) group (n=20). Twenty patients scheduled
for mastectomy were enrolled as a control group. RIPre was achieved by occluding arterial blood flow 5 min with a mercury
sphygmomanometer followed by a 5-min reperfusion period, and this was repeated 4 times. The RIPre procedure was
repeated 3 times a day for 20 days. In all patients, arterial fragments discarded during surgery were collected to evaluate
endothelial function by flow-mediated dilation (FMD), CD34+ monocyte count, and endothelial nitric oxide synthase (eNOS
expression). Phosphorylation levels of STAT-3 and Akt were also assayed to explore the underlying mechanisms. Compared
with the CHD group, long-term regular RIPre significantly improved FMD after 20 days (8.5±2.4 vs 4.9±4.2%, P,0.05) and
significantly reduced troponin after CABG surgery (0.72±0.31 and 1.64±0.19, P,0.05). RIPre activated STAT-3 and
increased CD34+ endothelial progenitor cell counts found in arteries. Long-term, regular RIPre improved endothelial function
in patients with CHD, possibly due to STAT-3 activation, and this may have led to an increase in endothelial progenitor cells.

Key words: Remote ischemic preconditioning; endothelial function; coronary heart disease; STAT-3

Introduction

Studies have shown that remote ischemic precondi-
tioning (RIPre) can reduce infarct size and decrease
ischemia-reperfusion injury, making it an attractive, novel
strategy to prevent myocardial injury (1,2). It has been
shown to be effective in reducing myocardial reperfusion
injuries in both experimental and clinical settings (3).
Recently, the application of RIPre for patients with acute
myocardial infarction during ambulance transit was shown
to reduce infarct size, but the mechanism is largely
unknown (4). Signal transducer and activator of transcrip-
tion-3 (STAT-3) activation is thought to play a cardiopro-
tective role during myocardial infarction by ischemic
preconditioning.
Patients with coronary heart disease (CHD) who
experience myocardial infarction exhibit endothelial dys-
function (5). This contributes to the pathogeneses of
atherosclerosis and its complications and is closely related
to cardiovascular events. RIPre before hospital admission
increases myocardial salvage and has a favorable safety
profile (6); however, the direct role of RIPre in regulating
human arterial function remains elusive.
The aims of the present study were twofold: to identify
the beneficial effects of long-term regular RIPre in human
arteries, especially with regard to endothelial function
protection, and to determine its potential mechanisms.
Material and Methods
Study design
Forty patients who had significant coronary arterial
stenosis and were scheduled for coronary artery bypass
graft (CABG) surgery were enrolled in the study. The
patients were randomly assigned to the RIPre (n=20) or
CHD group (n=20). Twenty patients with breast cancer
who were scheduled for mastectomy were included as a
control group. Exclusion criteria were the presence of an
acute coronary syndrome, heart failure, hypertrophic
cardiomyopathy, uncontrolled hypertension, serum crea-
tinine concentration .2 mg/dL, or anticipation of non-
compliance with the study protocol. The study objectives
and protocol were fully explained, and written informed
consent was obtained from all patients. This study was

Correspondence: Jinying Zhang: ,x201017liangying@126.com..

Received October 15, 2014. Accepted December 19, 2014. First published online April 28, 2015.

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078
RIPre improves endothelial function
approved by the regional Ethics Committee in accordance
with the standards of the Declaration of Helsinki.
RIPre was administered while upper arm blood
pressure was monitored 3 times a day for 20 days. A
blood pressure cuff was placed around the upper arm and
inflated to 200 mmHg to occlude arterial blood flow for
5 min. The cuff was then deflated to allow reperfusion. The
procedure was repeated 4 times at 5-min intervals. Limb
ischemia was confirmed by a change in the color of the skin
and a decrease in skin temperature. Following limb
reperfusion, the skin color returned to pink, and the skin
temperature returned to the baseline level. In the CHD
group, blood pressure was routinely measured three times
daily. After 20 days, all patients underwent CABG surgery,
and discarded artery fragments were collected following
the procedure for later analysis (7,8). Routine patient
treatment for CHD was maintained during the study in both
the RIPre and CHD groups. Antihypertensive treatment
was titrated to achieve a target blood pressure of ,130/80
or ,140/90 mmHg for patients with or without diabetes,
respectively, during the study period.
Measurement of flow-mediated vasodilation (FMD)
response
FMD response was measured at baseline and on day
20 and used as an index of endothelial function. Color
Doppler ultrasound measurement of the brachial artery
was performed in the resting state end-diastolic dimen-
sion, (D0), pneumatic compression to 250-300 mmHg
was maintained for 5 min, and 60-90 s after deflation,
reactive hyperemia of the brachial artery diastolic dia-
meter (D1) was determined. The color Doppler evaluation
of FMD is reported as the percent change in brachial
artery diameter and was calculated as follows: (D1– –D0)/
D0)6100. FMD values >10% and ,10% were consid-
ered to indicate normal vascular endothelial function and
endothelial dysfunction, respectively.
CD34+ monocyte count by flow cytometry
CD34+ monocyte and leukocyte counts were per-
formed in all patients at baseline and on day 20. A total of
10 mL blood was added to two tubes, and IgG antibody
was added to one tube as a control. Fluorescein
isothiocyanate (FITC)-conjugated CD34 and phycoery-
thrin (PE)-conjugated CD38 antibodies were added to the
remaining patient samples and incubated for 30 min in the
dark. Subsequently, 1 mL erythrocyte lysis buffer was
added, and after an additional 15 min in the dark,
phosphate-buffered saline was added to adjust the cell
concentration for flow cytometry (FACSCAN Becton
Dickinson, USA). The antibody positive rate was recorded
as the percentage of CD34+ leukocytes.
Arterial CD34+ monocytes by immunohistochemisty
Tissue sections were stained as previously described
(9). Cryosections of internal mammary artery fragments
www.bjournal.com.br
079
(7,8) were stained for CD34 (BD Pharmingen, USA),
counterstained with FITC-fluorescence (Sigma, USA) and
mounted with fluorescent mounting medium (Dako,
Denmark). Sections were examined using a Nikon E600
fluorescence microscope (Japan) and the Lucia Measure-
ment software 4.6 (India).
Endothelial nitric oxide synthase (eNOS) mRNA
expression by semiquantitative RT-PCR
Total RNA was extracted from arterial tissue samples
using the SV total RNA Isolation System (Promega, USA)
and diluted in 30 mL RNA-free water. The purity of RNA
can be confirmed by RNA electrophoresis. The RevertAid
First Strand cDNA Synthesis Kit (Thermo Scientific, USA)
was used to obtain 3 mg total RNA. The reaction mixture
contained 2 mL cDNA, 10 mL 26 PCR Taqmix, 1 mL
upstream and downstream primers, and 5 mL RNA
enzyme water, for a total volume of 20 mL. The primers
used were as follows: eNOS-specific (299 bp) sense, 59-
TTCCGGGGATTCTGGCAGGAG-39 and antisense, 59-
GCCAT-GGTAACATCGCCGCAG-39); glyceraldehyde
phosphate dehydrogenase (GAPDH)-specific (343 bp)
sense, 59-CTCTAAGGCTGTGGGCAAGGTCAT-39 and
antisense, 59-GAGATCCACCACCCTGTTGCTGTA-39.
The 35 PCR cycles were carried out as follows: 946C
for 5 min, 946C for 30 s, 656C for 45 s, and 726C for 30 s.
RT-PCR products were analyzed by 2% agarose gel
electrophoresis, visualized with ethidium bromide, and
quantified by densitometry. The results are reported as
the ratio of eNOS and GAPDH.
Western blotting
Fragments of arterial tissue obtained during CABG
surgery were used to measure protein phosphorylation by
immunoblotting for survival kinases (10). The excised
tissue was clamped between stainless steel tongs
precooled with liquid nitrogen, frozen in liquid nitrogen,
and stored at –806C. Approximately 100 mg powdered
arterial tissue was used for protein extraction. Frozen
tissue samples were homogenized on ice in 0.5-mL ice-
cold lysis buffer containing 30 mM HEPES, 20 mM KCl,
2.5 mM EGTA, 2.5 mM EDTA, 40 mM sodium fluoride,
4 mM sodium pyrophosphate, 1 mM sodium orthovana-
date, 10% glycerol, 1% Nonidet P-40, a phosphatase
inhibitor cocktail (Sigma), and a protease inhibitor cocktail
(Complete Mini; Roche Applied Science, Germany).
Following electrophoresis and blotting, membranes were
incubated overnight at 46C with antibodies against 705Tyr-
phospho-STAT-3, total STAT-3, total serine/threonine-
protein kinase (Akt), and 473Ser-phospho-Akt (Cell
Signaling Technology, USA). Beta-actin (Sigma) was
used as a loading control. After washing in Tris-buffered
saline and Tween-20, the membranes were incubated for
1 h at room temperature with horseradish peroxidase-
conjugated anti-human IgG secondary antibody (1:2000,
Santa Cruz Biotechnologies, USA), and the bound
Braz J Med Biol Res 48(6) 2015

antibody was detected using an enhanced chemilumines-
cence Western blotting kit (Santa Cruz Biotechnologies).
The densities of bands with appropriate molecular
masses (60 kDa for Akt and 80 kDa for STAT-3) were
semiquantitatively determined using a LAS-3000 mini
Lumino image analyzer (Fujifilm, Japan). Blood troponin
concentration was assayed in all patients after CABG
surgery using a diabody one-step sandwich enzyme
immunoassay (Access Immunoassay System; Beckman
Coulter, USA).
Statistical analysis
Data are reported as means±SD for continuous
variables or frequency percentages for categorical vari-
ables. The Kolmogorov-Smirnov test was used to
determine the normality of distribution for continuous
variables. Multiple comparisons were evaluated by one-
way ANOVA, and the differences between two groups
were analyzed by Student-Newman-Keuls post hoc test.
Either chi-square or Fisher’s exact tests were used to
compare differences between categorical variables.
Differences of continuous variables that were not normally
distributed were compared with Kruskal-Wallis or Mann-
Whitney U-tests. Differences were considered to be
statistically significant if the P value was less than 0.05.
All analyses were performed with the S-Plus statistical
package (Mathsoft Inc., USA).
Results
Patient characteristics
As shown in Table 1, the control, RIPre, and CHD
groups did not differ significantly in age, gender, body
mass index, blood pressure, or smoking status. At the
beginning of the study, the patients in RIPre and CHD
groups were well matched with respect to age, gender,
angina class, and cardiovascular risk factors. On echo-
cardiography, left ventricle (LV) end-diastolic dimensions
were similar in the two groups. In addition, there were no
differences between the RIPre and CHD groups in
medical treatment (Table 1). The use of beta blockers,
calcium channel blockers, and statins was similar
between the two groups. Coronary angiography showed
serious lesions in coronary vessels in all RIPre and
CHD patients, and all underwent CABG surgery
(Table 2). Hematologic analysis showed that the three
groups had similar white cell and mononuclear cell
counts (Table 3).
FMD and eNOS mRNA expression showed that long-
term regular RIPre improved arterial endothelial
function
RIPre can improve arterial endothelial function in
patients with CHD. At baseline, patients in both the CHD
and RIPre groups had lower FMD than controls
(FMD=12.8±3.5%, 4.6±3.2%, and 5.5±3.3% in the
Braz J Med Biol Res 48(6) 2015
080
Y. Liang et al.
control, RIPre and CHD groups, respectively; both
P,0.05 vs control; Table 2). FMD was significantly
improved in the RIPre group compared to the CHD group
30 days after treatment (8.5±2.4% vs 4.9±4.2%,
P,0.05). eNOS mRNA levels were measured by semi-
quantitative RT-PCR. Total cell RNA was isolated,
amplified, and then subjected to RT-PCR with eNOS-
and GAPDH-specific primers in separate reactions. As
shown in Figure 1, RIPre treatment significantly enhanced
eNOS mRNA expression compared with the CHD group
(n=20, P,0.05).
Interestingly, troponin expression was significantly
lower in the RIPre group than the CHD group after
CABG surgery (0.72±0.31 ng/L and 1.64±0.19 ng/L,
P,0.05), indicating that less cardiomyocyte necrosis
occurred during the procedure.
Long-term regular RIPre activated STAT-3
There were no significant differences in total Akt or
STAT-3 protein levels among the study groups. As
illustrated in Figures 2 and 3, phospho-Akt levels were
significantly enhanced in RIPre compared with control
group patients (P,0.05). While RIPre treatment was
more effective in increasing endothelial function than
medical treatment alone in the CHD group, Akt was
significantly more phosphorylated in RIPre than in CHD,
suggesting that another signaling pathway might play a
role in the enhanced cardioprotection afforded by this
treatment. When testing the Survivor Activating Factor
Enhancement (SAFE) pathway (11), phospho-STAT-3
levels were significantly increased following RIPre.
Interestingly, RIPre significantly increased phospho-
STAT-3 levels compared with the controls and CHD
patients. (P,0.05, Figure 3).
Long-term regular RIPre increased
immunohistochemical staining of endothelial
progenitor cells (CD34+) in arteries
The arterial CD34+ cells of patients in the RIPre group
exhibited increased immunofluorescence compared with
those of patients in the CHD group. The arterial CD34+
cells of patients in the control and CHD groups had the
highest and lowest immunofluorescence, respectively,
and the immunofluorescence observed in the RIPre group
was significantly higher than that in the CHD group
(n=20, P,0.05; Figure 4). The number of CD34+ cells
was significantly higher in the control group than in
patients in the CHD or RIPre group. At the beginning of
the study, the absolute number of CD34+ cells was
significantly different (2.35±0.72 cells/mL, 1.48±0.49
cells/mL and 1.56±0.37 cells/mL, P,0.05) in control
group patients than in patients in the CHD and RIPre
groups. On the other hand, no significant difference was
noted in the number of CD34+ cells in the RIPre and CHD
groups. On day 20, the numbers of CD34+ cells in the
RIPre and CHD groups were significantly different
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RIPre improves endothelial function

Table 1. Clinical, electrocardiographic and echocardiographic data of remote ischemic preconditioning

(RIPre) and coronary heart disease (CHD) patients.

Clinical data Control RIPre CHD

Age (years) 64 ± 10 64 ± 10 64 ± 10
Gender (male/female) 11/9 12/8 8/12
CCS class 0 2.4 ± 0.7 2.2 ± 0.6
NYHA functional class
II 0 10 10
III or IV 0 10 10
Hypertension 2 3/17 4/16
Diabetes 0 5/15 6/14
Hypercholesterolemia 1 13 12
BMI 23.85 ± 7.3 24.78 ± 6.8 23.95 ± 7.5
Current Smoking 7/13 8/12 6/10
Previous myocardial infarction 0 13 12
EF% 60 ± 12 60 ± 7 58 ± 10

Data are reported as means±SD or number. CCS: Canadian Cardiovascular Society; NYHA: New York
Heart Association; BMI: body mass index; EF%: ejection fraction.

(1.79±0.31 cells/mL vs 1.34±0.20 cells/mL, P,0.05;
Figure 5). Because the increase in endothelial progenitor
cells (EPCs) and improvement in endothelial function may
be correlated, we carried out a regression analysis that
revealed a slight but significantly positive correlation
between FMD and the number of CD34+ cells (r=0.46,
P,0.05).
Discussion
The present study showed that RIPre is effective in
improving endothelial function in patients with CHD and
that the mechanism may involve activation of the Akt and
STAT-3 pathways. The findings also indicated that
improvement in endothelial function was related to
increased EPCs in CHD.
Ischemic preconditioning experiments in animal mod-
els have demonstrated significant cardioprotective effects
and reductions in the size of myocardial infarcts. A recent
clinical study found that administering RIPre to patients
with acute myocardial infarction while still in transit in a
ambulance reduced infarct size (11).
Here we demonstrated that long-term, regular RIPre
had a cardioprotective effect. After CABG surgery, the

Table 2. Quantitative coronary angiographic data and medical treatment of remote ischemic
preconditioning (RIPre) and coronary heart disease (CHD) patients.

Characteristics RIPre (n=20) CHD(n=20)

Extension of disease
Single vessel disease (left main lesion) 8 (40%) 7 (35%)
Multivessel disease 12 (60%) 13 (65%)
Vessel disease
LAD involved 14 (70%) 13 (65%)
Cx involved 11 (55%) 10 (50%)
RCA involved 13 (65%) 14 (70%)
Medication
ACE inhibitors 16 (80%) 15 (75%)
Beta-blockers 19 (95%) 20 (100%)
Diuretics 4 (20%) 6 (30%)
Calcium antagonists 3 (15%) 5 (25%)
Statins 20 (100%) 20 (100%)

Data are reported as number and percent. LAD: left anterior descending artery; Cx: left circumflex artery;
RCA: right coronary artery; ACE: angiotensin-converting enzyme. There were no significant differences
between groups (chi-square test).

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081
 Y. Liang et al.

Table 3. Number of endothelial progenitor cells (CD34+), white cells and flow-mediated dilation (FMD) in
remote ischemic preconditioning (RIPre) and coronary heart disease (CHD) patients and the control
group.

Features Control RIPre CHD

Before After Before After

3
White cells (10 /mL) 9.86 ± 2.79 8.96 ± 2.87 7.93 ± 3.04 6.92 ± 1.67 7.34 ± 2.56
CD34+ (cells/mL) 2.35 ± 0.72 1.48 ± 0.49 1.79 ± 0.31*# 1.56 ± 0.37 1.34 ± 0.20*
FMD (%) 12.8 ± 3.5 4.6 ± 3.2 8.5 ± 2.4*# 5.5 ± 3.3 4.9 ± 4.2*

Data are reported as means±SD. * P,0.05 vs control; # P,0.05 vs before treatment (Student-Newman-
Keuls test).

mean troponin levels in the RIPre (0.72±0.31 ng/L) and
CHD groups (1.64±0.19 ng/L) were significantly differ-
ent, indicating that RIPre protected patients and reduced
myocardial cell damage. This finding is consistent with
previous reports (12,13). As most patients with CHD have
endothelial dysfunction, we propose that the cardiopro-
tective effect of long-term regular RIPre is related to
improved endothelial function.
This study provides some insight into the relationship
between regular RIPre and improved endothelial function
in CHD patients. Currently, echocardiography or arterial
FMD is used as a standard tool for the evaluation of
endothelium-dependent vasodilatation. In this study,
assessment of FMD, endothelial progenitor cells, CD34+ +
monocyte counts, and eNOS mRNA gene expression were
carried out to evaluate endothelial function (11), and we

Figure 1. A, Effects of remote ischemic preconditioning (RIPre)
on endothelial expression of endothelial nitric oxide synthase
(eNOS) mRNA as measured by semiquantitative RT-PCR. RIPre
caused an increase in eNOS mRNA expression. B, Relative
expression of eNOS mRNA (density ratio, eNOS:GADPH) is
reported as means±SE for n=20 in each group. *P,0.05 vs
CHD (Student-Newman-Keuls test). Note that the example does
not exactly reflect the mean value of all samples.
Figure 2. A, Representative Western blot showing phosphory-
lated serine-threonine protein kinase (p-Akt), total Akt, and actin
in arterial tissues. B, Ratio of p-Akt to Akt. Data are reported as
means±SE for n=20 in each group. RIPre: remote ischemic
preconditioning; CHD: coronary heart disease. *P,0.05 vs
control group; #P,0.05 vs CHD (Student-Newman-Keuls test).
Note that the immunoblot example does not exactly reflect the
mean value of all samples.

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082
RIPre improves endothelial function
Figure 3. A, Representative Western blot of phosphorylated
STAT-3 (p-STAT-3), total STAT-3, and actin in arterial tissue. B,
Ratio of p-STAT-3 to STAT-3. Data are reported as means±SE
for n=20 in each group. RIPre: remote ischemic preconditioning;
CHD: coronary heart disease. *P,0.05 vs control group;
#
P,0.05 vs CHD (Student-Newman-Keuls test). Note that the
immunoblots do not exactly reflect the mean values of all
samples.
found significant improvements consistent with previously
reported results (14). In the RIPre group, FMD improved
from 4.6±3.2% to 8.5±2.4%, but this change was not
www.bjournal.com.br
083
significant in the CHD group (5.5±3.3% vs 4.9±4.2%).
After treatment, FMD in the RIPre patients was close to
normal and significantly improved compared with the
CHD group. The observed increase in FMD demon-
strates that long-term regular RIPre significantly
improved endothelial function. However, these methods
test peripheral arteries rather than coronary vascular
endothelial function. To this end, we tested samples of
the internal thoracic artery obtained during surgery (7,8).
eNOS mRNA gene expression in artery samples (7,8)
was higher in the RIPre group than in the CHD group.
These results indicate that increased eNOS mRNA
gene expression resulted from improved endothelial
function (15).
Recently, ischemic preconditioning studies demon-
strated activation of STAT-3 as a part of the SAFE
pathway (16,17). Studies in animal models have con-
firmed that the cardioprotective effects of RIPre are due to
STAT-3 activation (18,19). The SAFE pathway is thought
to have a cardioprotective effect related to RIPre in
myocardial infarction (9,20), and its role has been
confirmed in patients after CABG surgery in whom
troponin values were significantly different between
patients in RIPre and CHD groups. This indicates that in
the event of myocardial infarction, long-term regular RIPre
can protect myocardial cells and reduce myocardial
necrosis. In this model, RIPre activates STAT-3 (SAFE
pathway), increases p-Akt, and then increases eNOS
mRNA gene expression, resulting in improved endothelial
function and FMD. Hence, we presume that the increase
in eNOS mRNA gene expression is correlated with the
activation of STAT-3 (SAFE pathway). However, as this
was a human experiment, blockers could not be applied to
test our hypothesis.
Figure 4. Representative micrographs of immu-
nofluorescence staining of arterial CD34+ cells in
the A, control group; B, remote ischemic pre-
conditioning (RIPre) group; and C, coronary heart
disease (CHD) group. Cells expressing hemato-
poietic progenitor cell antigen CD34 (CD34+)
were significantly reduced in the CHD group
compared with the control group but were
significantly increased by RIPre (P,0.05). D,
Relative fluorescence of EPC staining of
endothelial progenitor cells (EPCs); CD34+
staining. Data are reported as means±SE for
n=20 in each group. *P,0.05 vs control group;
#
P,0.05 vs RIPre group (Student-Newman-
Keuls test). Note that the example does not
exactly reflect the mean value of all samples.
Braz J Med Biol Res 48(6) 2015

Figure 5. A, Representative flow cytometry measurements of
CD34+ monocytes in blood before and after treatment in the A,
control group; B, remote ischemic preconditioning (RIPre) group;
and C, coronary heart disease (CHD) group. D, CD34+
monocytes in blood before and after treatment. Data are reported
as means±SE. *P,0.05 vs control group; #P,0.05 vs RIPre
group (Student-Newman-Keuls test).
Stem cell research in the past decade has shown that
EPCs, a population of bone marrow-derived cells,
circulate in the blood and modulate new vessel formation
and homeostasis in adults (21-23). In addition, low EPC
Braz J Med Biol Res 48(6) 2015
084
Y. Liang et al.
number is correlated with poor vascular function (24) and
is an independent risk factor for adverse events in CHD
patients (25,26). It follows that the number of circulating
EPCs may determine the capacity to repair vascular
damage and may be viewed as an index of vascular
health. The popular concept is that circulating EPCs are
protective, and their alteration may account for the
progression of endothelial dysfunction and atherosclero-
sis that are commonly seen in LV dysfunction and CHD
(27). The role of EPCs in endothelial dysfunction,
however, remains a matter of debate. Due to the
difficulties in their identification, limited information is
available about the normal range of EPCs in CHD. In
addition, earlier studies have been hampered by their
retrospective nature and by the lack of uniform criteria to
precisely identify EPCs. Studies have mainly focused on
cells positive for the CD34 endothelial marker, which is an
adhesion molecule expressed in hematopoietic stem cells
(28), because it is not expressed in mature endothelium
(29). Conversely, there are few data on the pathophysio-
logical significance of other subsets of stem/progenitor
cells. The levels of EPCs are decreased in coronary artery
disease, and animal models have shown that a decrease
in the endogenous pool of progenitor cells may accelerate
the course of atherosclerosis (30,31). Similarly, early
investigations showed that circulating EPCs are involved
in modulating LV remodeling processes that lead to heart
failure (32). It was recently reported that patients with
ischemic and nonischemic cardiomyopathy have a lower
number of EPCs than healthy individuals (33). In the
present study, immunohistochemistry of internal mam-
mary artery specimens (7,8) revealed that EPCs were
positive for the CD34 endothelial marker and that they
increased in CHD. Although this does not prove a causal
relationship, we found a significant positive correlation
between FMD and CD34+.
Accordingly, overall increases in stem cell number and
function have been proposed as a therapeutic means for
improving LV function in CHD patients. Stem and
progenitor cell circulation and pretreatment of cardiac
homing have been shown to reduce infarct size and
improve cardiac function.
In conclusion, long-term regular RIPre improved
endothelial function in patients with CHD, via STAT-3
activation (SAFE pathway), and have been associated
with an increase in arterial EPCs.
Acknowledgments
This research was supported by the First Affiliated
Hospital of Zhengzhou University.
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Seven-Day Remote Ischemic Preconditioning Improves Local and Systemic Endothelial Function and
Microcirculation in Healthy Humans

BACKGROUND

Ischemic preconditioning (IPC) protects tissue against ischemia-induced injury inside and outside ischemic
areas.

AIM

The purpose was to examine the hypothesis that daily IPC leads to improvement in endothelial function
and skin microcirculation not only in the arm exposed to IPC but also in the contralateral arm.

METHODS

Thirteen healthy, young, normotensive male individuals (aged 22±2 years) were assigned to 7-day daily
exposure of the arm to IPC (4×5 minutes).

ASSESSMENT

Assessment of brachial artery endothelial function (using flow-mediated dilation (FMD)) and forearm
microcirculation (cutaneous vascular conductance (CVC) at baseline and during local heating) was
performed before and after 7 days to examine the local (i.e., intervention arm) and remote (i.e., control
arm) effect of IPC. We repeated the assessment tests 8 days after the intervention (Post+8).

RESULTS

FMD increased after repeated IPC (P = 0.03) and remained significantly elevated at Post+8 in the
intervention (5.0±2.2%, 6.1±2.2%, and 6.6±2.3%) and contralateral arms (5.4±2.2%, 6.0±2.2%, and
7.5±2.2%). Forearm CVC also increased following repeated IPC (P = 0.006) and remained elevated at
Post+8 in both arms (intervention: 0.12±0.03, 0.14±0.04, 0.16±0.04 mV/mm Hg; contralateral: 0.14±0.04,
0.015±0.04, 0.17±0.07). No interaction between IPC arm and time was evident for FMD and CVC (both P
> 0.05). IPC intervention did not alter CVC responses to local heating (P > 0.05).

CONCLUSIONS

Daily exposure to IPC for 7 days leads to local and remote improvements in brachial artery FMD and
resting skin microcirculation that remain after cessation of the intervention and beyond the late phase of
protection. These findings have clinical relevance for micro- and macro-vascular improvements.

085
Original Article
Seven-Day Remote Ischemic Preconditioning Improves Local
and Systemic Endothelial Function and Microcirculation in
Healthy Humans
Helen Jones,1 Nicola Hopkins,1 Tom G. Bailey,1 Daniel J. Green,1,2 N. Timothy Cable,1 and
Dick H.J. Thijssen1,3
Background
Ischemic preconditioning (IPC) protects tissue against ischemia-
induced injury inside and outside ischemic areas. The purpose was
to examine the hypothesis that daily IPC leads to improvement in
endothelial function and skin microcirculation not only in the arm
exposed to IPC but also in the contralateral arm.
METHODS
Thirteen healthy, young, normotensive male individuals (aged
22 ± 2 years) were assigned to 7-day daily exposure of the arm to IPC
(4 × 5 minutes). Assessment of brachial artery endothelial function
(using flow-mediated dilation (FMD)) and forearm microcirculation
(cutaneous vascular conductance (CVC) at baseline and during local
heating) was performed before and after 7 days to examine the local
(i.e., intervention arm) and remote (i.e., control arm) effect of IPC. We
repeated the assessment tests 8 days after the intervention (Post+8).
RESULTS
FMD increased after repeated IPC (P = 0.03) and remained signifi-
cantly elevated at Post+8 in the intervention (5.0 ± 2.2%, 6.1 ± 2.2%,
Despite optimal (pharmacological) therapy and risk factor
management, cardio- and cerebrovascular diseases remain
the leading causes of mortality and morbidity in the Western
world.1 Consequently, innovative treatment strategies for
protecting against cardiovascular disease are required to
improve clinical outcomes. A cycle of repeated bouts of
ischemia followed by reperfusion over a short period of
time, commonly known as ischemic preconditioning (IPC),
has been shown to delay cardiac cell injury2 and protect
against myocardial and vascular damage.3 In addition to
the local protective effects of IPC,4 protection against tis-
sue damage has also been found in distant vascular areas
not directly exposed to the repeated ischemic stimuli.5,6 This
well-known phenomenon is commonly referred to as remote
Correspondence: Dick H.J. Thijssen (D.Thijssen@ljmu.ac.uk).
Initially submitted October 31, 2013; date of first revision November
20, 2013; accepted for publication January 2, 2014; online publication
March 13, 2014.
American Journal of Hypertension 27(7) July 2014
086
and 6.6 ± 2.3%) and contralateral arms (5.4 ± 2.2%, 6.0 ± 2.2%, and
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7.5 ± 2.2%). Forearm CVC also increased following repeated IPC
(P = 0.006) and remained elevated at Post+8 in both arms (intervention:
0.12 ± 0.03, 0.14 ± 0.04, 0.16 ± 0.04 mV/mm Hg; contralateral: 0.14 ± 0.04,
0.015 ± 0.04, 0.17 ± 0.07).
concLuSionS
Daily exposure to IPC for 7 days leads to local and remote
improve-ments in brachial artery FMD and resting skin
microcirculation that remain after cessation of the intervention and
beyond the late phase of protection. These findings have clinical
relevance for micro- and macrovascular improvements.
Keywords: blood pressure; cardiovascular risk; endothelial
function; hypertension; microcirculation; remote ischemic
preconditioning; vascularadaptation.
doi:10.1093/ajh/hpu004
IPC.6–8 These effects suggest that repeated IPC may represent
a potent, systemic stimulus for vascular adaptations.
Previous studies have demonstrated that acute exposure to
IPC leads to increased blood flow in conduit and resistance
vessel beds in distant areas (such as the contralateral limb)9
or organs (such as the heart)10,11 and also to enhanced cuta-
neous tissue oxygen saturation and arterial capillary blood
flow.12 Single IPC may acutely change endothelial function,13
although results are conflicting.4,8 Given these acute effects
of IPC, repeated exposure to IPC may induce beneficial
sustained vascular adaptations. Little is known about the
potential of repeated IPC to alter conduit and skin circula-
tory beds and whether such adaptations also occur in remote
vascular beds. Because the IPC stimulus is associated with
1Research Institute for Sport and Exercise Science, Liverpool John
Moores University, Liverpool, UK; 2School of Sport Science, Exercise and
Health, The University of Western Australia, Crawley, Western Australia,
Australia; 3Department of Physiology, RRadboud University Medical
Center, Nijmegen, The Netherlands.
© American Journal of Hypertension, Ltd 2014. All rights reserved.
For Permissions, please email: journals.permissions@oup.com

elevation of circulating growth factors and endothelial pro-
genitor cells,5,14 we hypothesize that repeated IPC will lead to
systemic changes in both conduit artery endothelial function
and skin microcirculation. Therefore, we examined whether
7 days of daily exposure to unilateral IPC leads to bilateral
adaptation of the brachial artery endothelial function and
forearm skin microcirculation in healthy, young men.
Previous studies have also revealed the presence of a late
phase of protection for IPC.8 This late phase begins 12–24
hours after IPC and lasts 3–4 days.15 To determine whether
repeated IPC improves endothelial function and cutaneous
microcirculation beyond the late phase of protection (of
the last IPC session), we assessed endothelial function and
forearm microcirculation 8 days after cessation of the IPC
intervention. We hypothesize that repeated IPC results in
adaptations, resulting in improved endothelial function and
elevated microcirculation that are still present 8 days after
cessation of the intervention.
METHODS
Participants
Thirteen healthy males (Table 1) volunteered to par-
ticipate in this uncontrolled, 7-day IPC intervention study.
Participants were recreationally active, were measured by a
self-report questionnaire, and typically engaged in low- (e.g.,
walking) and moderate-intensity (e.g., running and station-
ary cycling) aerobic activities (2–3 days/week). We excluded
smokers and participants with (a history of) cardiovascular
disease including hypertension, diabetes, or hypercholester-
olemia and participants who were on any medication. All
participants provided written informed consent before par-
ticipation. The study was approved by the institutional eth-
ics committee and adhered to the Declaration of Helsinki
(2000).
Experimental design
Participants in the IPC intervention group (i.e., 7 days of
daily IPC exposure) reported 3 times to our laboratory for
assessment of bilateral brachial artery endothelial function
Table 1. Descriptive characteristics of the male participants in the
intervention (n = 13)
Characteristics Intervention group
Age, y 21.5 ± 2.2
Height, cm 178.4 ± 4.8
Weight, kg 75.0 ± 4.6
BMI, kg/m2 22.8 ± 1.0
Systolic blood pressure, mm Hg 138 ± 6
Diastolic blood pressure, mm Hg 73 ± 6
Mean arterial blood pressure, mm Hg 95 ± 5
Pulse pressure, mm Hg 66 ± 7
Data are mean ± SD.
Abbreviation: BMI, body mass index.
087
Effect of 7-Day IPC on Endothelial Function
(using flow-mediated dilation (FMD)) and bilateral forearm
cutaneous vascular conductance (CVC) at rest and during
a local heating stimulus (Figure 1).16 Assessment of bilat-
eral brachial artery FMD and bilateral CVC responses was
performed before (Pre) and after (Post) 7 days of daily IPC
training and also 8 days after the cessation of the interven-
tion (Post+8 days). The IPC intervention consisted of daily
exposure to 4 bouts of 5-minute cuff occlusion in a single
arm, with the contralateral arm serving as a within-subject
control arm. We randomized and counterbalanced the arm
that received the IPC intervention between participants
(dominant vs. nondominant). Repeated measures were per-
formed at the same time of day to control for diurnal varia-
tion in endothelial function.17
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Experimental measures
Brachial artery endothelial function. Simultaneous
bilateral brachial artery endothelium–dependent function
was measured using the FMD technique.18 For this pur-
pose, participants were instructed to abstain from strenuous
exercise, caffeine, and alcohol ingestion for 24 hours before
reporting to the laboratory. Participants were also asked to
fast for 6 hours before each visit. Measurements were per-
formed in the supine position in a quiet, darkened, temper-
ature-controlled room. Measurement began after resting for
20 minutes, followed by assessment of heart rate and blood
pressure using an automated sphygmomanometer (GE Pro
300V2; Dinamap, Tampa, FL). To examine brachial artery
FMD, both arms were extended and positioned at an angle
of approximately 80° from the torso. A rapid inflation and
deflation pneumatic cuff (D.E. Hokanson, Bellevue, WA)
was positioned on each forearm, immediately distal to the
olecranon process to provide a stimulus to forearm ischemia.
A 10-MHz multifrequency linear array probe, attached to
a high-resolution ultrasound machine (T3000; Terason,
Burlington, MA), was then used to image the brachial
artery in the distal third of the upper arm. When an opti-
mal image was obtained, the probe was held stable and the
ultrasound parameters were set to optimize the longitudinal,
B-mode image of the lumen–arterial wall interface. Settings
were identical between all FMD assessments. Continuous
Doppler velocity assessments were also obtained using the
ultrasound machine and were collected using the lowest
possible isonation angle (always <60°). After baseline assess-
ments, the forearm cuffs were inflated (>200 mm Hg) for 5
minutes. Diameter and flow recordings resumed 30 seconds
before cuff deflation and continued for 3 minutes thereafter,
in accordance with recent technical specifications.19,20
Brachial artery diameter and blood flow ­analysis. Analysis
of brachial artery diameter was performed using custom-
designed edge-detection and wall-tracking software, which is
largely independent of investigator bias. Recent articles con-
tain detailed descriptions of our analysis approach.19,20 From
synchronized diameter and velocity data, blood flow (the
product of lumen cross-sectional area and Doppler velocity)
and shear rate (an estimate of shear stress without viscosity)
were calculated at 30 Hz. Within-day reproducibility of the
FMD possesses a coefficient of variation of 6.7%–10.5%.21
American Journal of Hypertension 27(7) July 2014
Jones et al.
5-min 5-min
occlusion reperfusion
Pre
-Brachial FMD
-Baseline CVC
-CVC-responses to
local heat
Figure 1. Protocol of the study. Every IPC session consisted of 4 periods of 5-minute arterial occlusion (through inflation of a blood pressure cuff around
the upper arm to 220 mm Hg) and 5-minute reperfusion. Bilateral assessment of brachial artery FMD and forearm CVC (baseline and in response to heat)
is performed not only before the IPC intervention (day 1; Pre) but also immediately after (day 8; Post) and 8 days after the intervention (day 15; Post+8).
Abbreviations: CVC, cutaneous vascular conductance; FMD, flow-mediated dilation; IPC, ischemic preconditioning.
Microvascular function measurements. Assessments
were performed in a quiet, temperature-controlled room
(23 ± 1 °C), with the participants positioned comfortably
on a bed. Both arms were positioned approximately 5 cm
above heart level, and the laser Doppler probes were posi-
tioned on the volar side of the forearm, approximately
5 cm below the elbow crest. The m easurement s ites f or
each of the laser Doppler probes were recorded, and efforts
were made to use the same places during the 3 consecu-
tive measurements. After an acclimatization period of 30
minutes, microvascular function was examined by record-
ing of local skin blood flow. To o btain a n i ndex o f s kin
blood flow, c utaneous r ed b lood c ell fl ux (i n mi llivolts)
was measured simultaneously in both forearms using a
laser Doppler flowmetry system (Model 413, Periflux 5001
System; Perimed, Stockholm, Sweden). Skin temperature
was controlled at the 2 measuring sites with local heating
units (Perimed 455; Perimed). To verify whether blood
pressure was stable throughout the experimental proto-
col, blood pressure was measured at 5-minute intervals
by an automated sphygmomanometer. After i nstrumen-
tation, red blood cell flux of b oth sites was monitored to
examine baseline skin blood flow. The temperature of the
local heating units at both sites was kept constant at 33 °C
during the baseline period. After b aseline r ecording f or
10 minutes, local heating protocol was performed simul-
taneously at both sides. Temperature of the local heating
units was increased at a rate of 0.5 °C every 5 seconds to a
temperature of 42 °C.16 The protocol was finished after
red
American Journal of Hypertension 27(7) July 2014
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Post Post+8
-Brachial FMD -Brachial FMD
-Baseline CVC -Baseline CVC
-CVC responses to -CVC responses to
local heat local heat
blood cell flux in both sites had reached a stable plateau
(approximately 30–40 minutes). Skin blood flow response
to this protocol is nitric oxide dependent.16
The laser Doppler probe signals were continuously moni-
tored by an online software chart recorder (PSW; Perimed).
CVC was calculated as laser Doppler flow (in millivolts)
divided by mean arterial pressure (mm Hg) to account
for any differences in blood pressure between the groups.
Analysis was performed as described elsewhere.22
IPC intervention. The 7-day IPC training protocol con-
sisted of daily exposure to 4 repetitions of inflating an upper
arm blood pressure cuff using a rapid cuff inflator (EC-20;
D.E. Hokanson) to 220 mm Hg for 5 minutes, followed by
deflation of 5 minutes (Figure 1). All sessions were supervised
by a researcher to ensure full compliance to the intervention.
Statistics
Statistical analyses were performed using SPSS 17.0 (SPSS,
Chicago, IL) software. Our primary outcome was endothe-
lial function as measured using the brachial artery FMD.
All FMD data were analyzed and presented as covariate
controlled for baseline artery diameter measured before the
introduction of hyperemia in each test; this approach is more
accurate for scaling changes in artery diameter than simple
percentage change.23 These data were analyzed using a 2-fac-
tor general linear model with repeated measures of IPC (2
levels: IPC arm and contralateral arm) and time (3 levels:
088
 Effect of 7-Day IPC on Endothelial Function

Pre, Post, and Post+8 days). Our primary outcome variable
for microvascular endothelial function was CVC. Similarly,
a 2-factor general linear model was used with the same
repeated measures factors to analyze the summary data (rest-
ing baseline, first peak, nadir, and plateau) during the heat-
ing protocol. Significant main effects or interactions were
followed up using the least significant difference method for
pairwise multiple comparisons.24 For all statistical tests, a
2-tailed comparison was used. All data are reported as mean
(±SD) unless stated otherwise, and statistical significance was
assumed at P < 0.05.
RESULTS
All 13 participants completed the IPC intervention
(Table 1), with 100% compliance to the IPC sessions (IPC on
dominant arm: n = 7).
Brachial artery endothelial function
A significant main effect of time was evident for bra-
chial artery FMD across the 15-day experiment (P = 0.03;
Figure 2). Brachial artery FMD increased during the 7-day

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Figure 2. Impact of repeated IPC on the vasculature. (a) Brachial artery FMD (%) and (b) resting (baseline) forearm CVC before (Pre), after (Post), and
8 days after (Post+8) the 7-day daily IPC ­intervention in the IPC (open circles) and contralateral arm (i.e., rIPC; solid squares) of healthy volunteers (n = 13).
Error bars represent SE. *Post hoc significantly different from day 0. Abbreviations: CVC, cutaneous vascular conductance; FMD, flow-mediated dilation; IPC,
ischemic preconditioning.

American Journal of Hypertension 27(7) July 2014

089
Jones et al.

IPC intervention (P = 0.03). FMD in both arms remained sig-
nificantly elevated at Post+8 (7.0 ± 1.5%) compared with Pre
(5.2 ± 1.5%) (P < 0.01; Figure 2). The main effect of IPC or
the IPC × time interaction was not significant (P > 0.05). No
changes across time or differences between the arm (i.e., IPC
or contralateral arm) were evident in diameter, time-to-peak
diameter, or shear rate area under the curve (P > 0.05; Table 2).
Forearm microvascular function
Mean arterial pressure significantly decreased across
the 15-day time frame (Table 3). Because of these changes
in blood pressure, we have taken CVC as the primary out-
come measure. A significant main effect of time was evident
in forearm CVC during baseline (rest) across the 15-day
with Pre (0.13 ± 0.04 mV/mm Hg) (P = 0.01; Table 3). The
IPC × time interaction and the main effect of IPC were not
significant (P > 0.05).
Local heating. The local heating protocol induced the
typical pattern of an initial peak, followed by a nadir and a
subsequent plateau in skin flux, which was present during
all assessments for all participants in both arms and during
all time points. No significant main effects of time, IPC,
or interactions between these factors were evident for flux,
mean arterial pressure, or CVC at the first peak, the nadir,
or the plateau points during heating (P > 0.05) (Table 3).
Discussion

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experiment (P = 0.006) (Table 3). Forearm CVC increased by Our study provides a number of observations. First, 7-day
0.03 ± 0.03 mV/mm Hg during the 7-day IPC intervention daily exposure to unilateral IPC of the arm results in sig-
(P = 0.001) (Table 3). Forearm CVC remained significantly nificant improvements in conduit artery endothelial func-
elevated at Post+8 (0.16 ± 0.04 mV/mm Hg) when compared tion not only in the arm exposed to repeated IPC, but also in

Table 2. Brachial artery FMD before (Pre) and immediately after (Post) the IPC intervention and 8 days after (Post+8) cessation of the
intervention in healthy volunteers (n = 13, intervention and contralateral arms)

Intervention arm Contralateral arm Two-way ANOVA P values

Intervention group Pre Post Post+8 Pre Post Post+8 IPC Time IPC × time

Diameter, mm 3.8 ± 0.6 3.9 ± 0.4 3.9 ± 0.3 3.9 ± 0.5 3.8 ± 0.4 3.7 ± 0.4 0.88 0.42 0.38
FMD, % 5.0 ± 2.2 6.1 ± 2.2 6.6 ± 2.3* 5.4 ± 2.2 6.0 ± 2.2 7.5 ± 2.2* 0.41 0.03 0.71
Shear AUC, 103 17.9 ± 12.9 13.6 ± 6.2 16.4 ± 8.1 12.8 ± 5.9 13.1 ± 8.3 16.6 ± 8.6 0.32 0.56 0.47
Time to peak 56 ± 31 47 ± 21 58 ± 35 52 ± 25 49 ± 23 50 ± 18 0.65 0.48 0.37

Data are mean ± SD. Bold numbers represent significant main effects (P < 0.05).
Abbreviations: ANOVA, analysis of variance; AUC, area under the curve; FMD, flow-mediated dilation; IPC, ischemic preconditioning.
*Post hoc significantly different from Pre (P < 0.05).

Table 3. Forearm skin responses at baseline and during local heating in the IPC and contralateral arms before (Pre), immediately after
(Post), and 8 days after (Post+8) the intervention in healthy volunteers (n = 13, intervention and contralateral arms)

Intervention arm Contralateral arm Two-way ANOVA P values

Intervention group Pre Post Post+8 Pre Post Post+8 IPC Time IPC × time

Flux, mV 11 ± 3 13 ± 4 14 ± 3 13 ± 4 15 ± 6 16 ± 6 0.03 0.09 0.93

MAP, mm Hg 93 ± 5 90 ± 6* 88 ± 7* — — — — 0.002 —
CVC, mV/mm Hg 0.12 ± 0.03 0.14 ± 0.04* 0.16 ± 0.04* 0.14 ± 0.04 0.15 ± 0.04* 0.17 ± 0.07* 0.21 0.006 0.78
Local heating
 First peak, flux 176 ± 17 165 ± 18 188 ± 19 159 ± 14 171 ± 15 170 ± 9 0.30 0.62 0.46
 First peak, CVC 1.88 ± 0.18 1.87 ± 0.20 2.23 ± 0.21 1.82 ± 0.13 2.12 ± 0.14 1.98 ± 0.09 0.89 0.22 0.16
 Nadir, flux 159 ± 17 144 ± 21 169 ± 19 135 ± 13 151 ± 19 148 ± 10 0.26 0.62 0.39
 Nadir, CVC 1.68 ± 0.18 1.61 ± 0.23 1.96 ± 0.23 1.41 ± 0.14 1.59 ± 0.24 1.58 ± 0.12 0.09 0.35 0.39
 Plateau, flux 222 ± 19 202 ± 21 237 ± 28 193 ± 13 203 ± 19 195 ± 11 0.10 0.77 0.54
 Plateau, CVC 2.34 ± 0.18 2.28 ± 0.26 2.73 ± 0.32 2.05 ± 0.24 2.28 ± 0.23 2.28 ± 0.15 0.10 0.73 0.43

Data are mean ± SD.

Abbreviations: ANOVA, analysis of variance; CVC, cutaneous vascular conductance; IPC, ischemic preconditioning; MAP, mean arterial
pressure.
*Post hoc significantly different from Pre (P < 0.05).

American Journal of Hypertension 27(7) July 2014

090

the contralateral arm. Second, we observed a higher forearm
CVC in both forearms. These findings imply that 7-day IPC
results in both local and remote improvements in conduit
artery endothelial function and forearm CVC. Third, we
found that these bilateral improvements in conduit artery
endothelial function and forearm CVC persist up to 8 days
after intervention. These observations suggest that IPC is an
intervention capable of improving endothelial function and
skin microcirculation both locally and systemically and that
these beneficial effects remain present for at least 7 days after
the cessation of the IPC stimulus.
IPC is typically used to prevent endothelial dysfunc-
tion induced by ischemia-reperfusion injury.4,7,8,25 A single
bout of IPC may also acutely change endothelial function,13
although results are conflicting.4,8 Furthermore, preliminary
evidence suggests that repeated IPC improves resistance
artery endothelial–dependent vasodilation.14 We extend
this knowledge with the observation that 1 week of daily
IPC leads to a significant improvement in conduit artery
endothelial function. The potent vascular effects of daily
IPC are further emphasized by the increased forearm CVC.
Although these observations provide evidence for vascular
adaptations after IPC throughout the vascular tree, we add
the novel observation that these vascular adaptations are
also present in remote areas. Indeed, a simultaneous increase
in endothelial function and CVC was found in the contralat-
eral arm, i.e., the arm not directly exposed to IPC. Thus, our
data support the use of IPC to induce local and systemic
improvements in conduit artery endothelial function and
skin microcirculation in humans.
Our study was not designed to examine mechanisms
that relate to the local and remote effects of 7 days of IPC.
Therefore, we can only speculate about the potential under-
lying mechanisms. Shear stress represents a major physi-
ological stimulus responsible for improvement in conduit
artery function26 and skin microcirculation.27 Although
episodic increases in shear stress may contribute (partly) to
the adaptations found in the intervention arm, it is unlikely
that shear represents the principle mechanism responsible
for the remote effects of IPC because elevations in shear in
remote areas are only modest. Systemic stimuli or circulat-
ing markers activated by IPC more likely explain the remote
improvement in conduit artery endothelial function. For
example, IPC leads to an increase in vascular endothelial
growth factor and endothelial progenitor cells,14 which may
improve endothelial function in remote areas.28 Indeed, tis-
sue ischemia29 and shear,30 both prominent during IPC, are
key stimuli for endothelial progenitor cell release from bone
marrow into the circulation. An alternative explanation
relates to reduced levels of oxygen free radicals and inflam-
mation because IPC improves antioxidative defence mecha-
nisms (e.g., increased superoxide dismutase activity) and/
or lowers the generation of oxidative stress.4,31,32 Although
future studies should further examine the exact mechanisms
by which IPC improves the vasculature, it is likely that sys-
temic pathways contribute to the effects.
Because the late phase of protection coincides with our
postintervention measurements, the increased FMD and
CVC after the 7-day IPC intervention may relate to the
late phase of protection of the last IPC bout rather than a
091
Effect of 7-Day IPC on Endothelial Function
“true” adaptation. To control for this effect, we repeated
postintervention measurements (Post+8 days). We found
that the increase in brachial artery and skin microcircula-
tion endothelial function remained significantly elevated in
both arms 8 days after cessation of the IPC intervention.
This finding infers that repeated IPC induces sustained
local and remote functional adaptation of the conduit artery
endothelium. The late phase of protection is thought to be
induced by de novo synthesis of cardioprotective proteins,
such as upregulation of inducible nitric oxide synthase.33
These changes may contribute to a higher NO production
and improved conduit artery FMD, i.e., a response that is
mediated, at least partly, through NO.34
An interesting observation in our study is the significant
decline in blood pressure after IPC. A recent study also
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reported a blood pressure–lowering effect of IPC in normo-
tensive participants after 3 days of daily IPC.35 Interestingly,
many of the suggested mechanisms of (remote) IPC, such
as hormonal5 and neuronal effects by the autonomic nerv-
ous system,8 also contribute to the regulation of the sys-
temic blood pressure. Previous data report an inverse
relation between brachial artery FMD and blood pressure.36
Accordingly, the enhanced (systemic) endothelial function
in our study may contribute to the lower blood pressure in
our participants. Alternatively, methodological aspects may
explain the decline in the blood pressure. First, we have
adopted a single measure of blood pressure rather than
repeated measurements. Secondly, anxiety before the pre-
intervention may have affected measurements and overesti-
mated preintervention values.
Limitations. We did not examine endothelium-independ-
ent vasodilation. However, we26 and others14 have demon-
strated that endothelium-independent function typically
does not change over time in healthy participants. Thus, it is
unlikely that the improvement in endothelial function in our
study is explained by changes in endothelium-independent
dilation. Second, we were not able to examine the peak CVC,
which some authors use to normalize skin CVC responses.
However, peak CVC responses typically do not change across
time. Finally, we did not include a control group within the
study to control for potential bias in our study.
Clinical relevance. Endothelial dysfunction is an impor-
tant event in the atherosclerotic cascade and predicts car-
diovascular endpoints.37,38 Our data demonstrate that IPC,
which represents a simple, virtually cost-free, and nonin-
vasive intervention, improves endothelial function in the
intervention and contralateral arms in healthy individuals.
Although speculative, these local and systemic effects of
IPC on endothelial function may have clinical relevance,
especially in individuals with impaired micro- or mac-
rovessel functions (e.g., type 2 diabetes), where vascular
function is a primary target to improve cardiovascular
risk and associated complications. Whether repeated IPC
improves micro- and macrovessel functions in individuals
with endothelial dysfunction, or in those with conditions
that require cardiovascular risk management, is currently
unknown, and this should be the subject of future research.
Furthermore, such studies should also examine how long
adaptations in endothelial function and skin microcircula-
tion remain preserved.
American Journal of Hypertension 27(7) July 2014
Jones et al.

In conclusion, 7 days of daily exposure to IPC leads to

local (intervention arm) and systemic (contralateral arm)
improvements in conduit artery endothelial function and
elevation of resting skin microcirculation, which are present
beyond the late phase of protection of IPC. These
findings have clinical relevance because improving
endothelial function independently prevents
cardiovascular morbidity and mortality.38 Furthermore,
repeated IPC increased resting microcirculation by
approximately 30%, a clinically relevant improvement,
especially because the increase was similar in a remote
vascular bed not exposed to IPC. Our findings highlight
the potential for IPC as a simple, safe, and feasible
therapeutic tool.

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ACKNOWLEDGMENTS

D.H.J.T. is recipient of the E. Dekker stipend (Netherlands

Heart Foundation, 2009T064). D.J.G. is funded by the
Australian Research Council (DP 130103793). H.J. and
N.H. contributed equally to this work.

DISCLOSURE

The authors declared no conflict of interest.

American Journal of Hypertension 27(7) July 2014

092
 Ischemic Preconditioning Improves Microvascular endothelial
Function in Remote Vasculature by Enhanced Prostacyclin
Production

BACKGROUND:

The mechanisms underlying the effect of preconditioning on remote microvasculature remains

undisclosed. The primary objective was to document the remote effect of ischemic preconditioning on
microvascular function in humans. The secondary objective was to test if exercise also induces remote
microvascular effects.

METHODS AND RESULTS:

A total of 12 healthy young men and women participated in 2 experimental days in a random counter-
balanced order. On one day the participants underwent 4×5 minutes of forearm ischemic preconditioning,
and on the other day they completed 4×5 minutes of hand-grip exercise. On both days, catheters were
placed in the brachial and femoral artery and vein for infusion of acetylcholine, sodium nitroprusside, and
epoprostenol.

ASSESSMENT:

Vascular conductance was calculated from blood flow measurements with ultrasound Doppler and
arterial and venous blood pressures. Ischemic preconditioning enhanced (P<0.05) the remote vasodilator
response to intra-arterial acetylcholine in the leg at 5 and 90 minutes after application.

RESULT:

The enhanced response was associated with a 6-fold increase (P<0.05) in femoral venous plasma
prostacyclin levels and with a transient increase (P<0.05) in arterial plasma levels of brain-derived
neurotrophic factor and vascular endothelial growth factor. In contrast, hand-grip exercise did not
influence remote microvascular function.

CONCLUSIONS:

These findings demonstrate that ischemic preconditioning of the forearm improves remote microvascular
endothelial function and suggest that one of the underlying mechanisms is a humoral-mediated
potentiation of prostacyclin formation.

093
 Journal of the American Heart Association

ORIGINAL RESEARCH

Ischemic Preconditioning Improves

Microvascular Endothelial Function
in Remote Vasculature by Enhanced
Prostacyclin Production
Nicolai Rytter , MSc; Howard Carter, PhD; Peter Piil, PhD; Henrik Sørensen, MD, DMSc; Thomas Ehlers, MD;
Frederik Holmegaard, MSc; Christoffer Tuxen, MSc; Helen Jones, PhD; Dick Thijssen, PhD;
Lasse Gliemann , PhD; Ylva Hellsten , DMSc

BACKGROUND: The mechanisms underlying the effect of preconditioning on remote microvasculature remains undisclosed. The
primary objective was to document the remote effect of ischemic preconditioning on microvascular function in humans. The
secondary objective was to test if exercise also induces remote microvascular effects.

METHODS AND RESULTS: A total of 12 healthy young men and women participated in 2 experimental days in a random counter-
balanced order. On one day the participants underwent 4×5 minutes of forearm ischemic preconditioning, and on the other
day they completed 4×5 minutes of hand-grip exercise. On both days, catheters were placed in the brachial and femoral
artery and vein for infusion of acetylcholine, sodium nitroprusside, and epoprostenol. Vascular conductance was calculated
from blood flow measurements with ultrasound Doppler and arterial and venous blood pressures. Ischemic preconditioning
enhanced (P<0.05) the remote vasodilator response to intra-arterial acetylcholine in the leg at 5 and 90 minutes after applica-
tion. The enhanced response was associated with a 6-fold increase (P<0.05) in femoral venous plasma prostacyclin levels and
Downloaded from http://ahajournals.org by on April 18, 2024

with a transient increase (P<0.05) in arterial plasma levels of brain-derived neurotrophic factor and vascular endothelial growth
factor. In contrast, hand-grip exercise did not influence remote microvascular function.

CONCLUSIONS: These findings demonstrate that ischemic preconditioning of the forearm improves remote microvascular en-
dothelial function and suggest that one of the underlying mechanisms is a humoral-mediated potentiation of prostacyclin
formation.

Key Words: ischemic preconditioning ■ microvascular endothelial function ■ platelets ■ prostacyclin ■ vasodilation

R
epeated bouts of ischemia followed by reperfu-
sion, known as ischemic preconditioning, is a
procedure that originally was shown to reduce
myocardial infarct size in animals1 and subsequently
also to protect against vascular injury.2 The protective
effect was demonstrated to be remote, that is, effective
also in organs not directly exposed to ischemia.3,4 In
addition, ischemic preconditioning has been reported
to induce improved vascular endothelial function of
remote conduit arteries in human studies.5,6 These
findings suggest a systemic vascular effect of ischemic
preconditioning, which could be related to either neu-
ral or humoral signaling.7 Interestingly, a single session
of exercise has recently also been shown to induce a
remote cardioprotective effect,8,9 similar to that of isch-
emic preconditioning, but whether exercise can lead
to improved remote microvascular function remains
unclear.

Correspondence to: Ylva Hellsten, DMSc, Department of Nutrition, Exercise and Sports, University of Copenhagen, Universitetsparken 13, DK-2100
Copenhagen Ø, Denmark. E-mail: yhellsten@nexs.ku.dk
Supplementary Materials for this article are available at https://www.ahajo​urnals.org/doi/suppl/​10.1161/JAHA.120.016017
For Sources of Funding and Disclosures, see page 11.
© 2020 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley. This is an open access article under the terms of the Creative
Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use
is non-commercial and no modifications or adaptations are made.
JAHA is available at: www.ahajournals.org/journal/jaha

094
J Am Heart Assoc. 2020;9:e016017. DOI: 10.1161/
 Rytter et al
CLINICAL PERSPECTIVE
What Is New?
• This is the first study to explore the acute effect
of ischemic preconditioning on remote microcir-
culation in humans.
• We demonstrate that the vasodilator response
to intra-arterial acetylcholine is enhanced in the
leg after ischemic preconditioning applied at the
arm.
• The enhanced vasodilatory responsiveness to
acetylcholine in remote vascular tissue is asso-
ciated with increased prostacyclin production.
What Are the Clinical Implications?
• The findings suggest that ischemic precondi-
tioning may be an effective treatment strategy to
improve remote microvascular endothelial func-
tion and endogenous prostacyclin production,
which are essential steps in the prevention of
cardiovascular morbidity and mortality.
Nonstandard Abbreviations and Acronyms
BDNF brain-derived neutrophic factor
6-keto PGF1α 6-keto prostaglandin F1α
VEGF vascular endothelial growth factor
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Although the abovementioned effects of remote
ischemic preconditioning are likely to be of important
clinical relevance,10,11 there are several critical aspects
that remain to be resolved. First, it is unknown whether
ischemic preconditioning improves not only macro-
vascular but also microvascular function, and thereby
the control of vascular resistance. Second, the mech-
anism by which ischemic preconditioning induces its
beneficial effects on the vasculature remains to be
identified. Further knowledge of mechanisms would
bring us closer to the development of a pharmaco-
logical intervention that can simulate or potentiate the
effect. Third, it is unknown whether an acute exercise
session can induce a beneficial effect on the remote
microvasculature. This knowledge would provide fur-
ther insight into the mechanisms of preconditioning.
In this study, we address these 3 aspects.
The observation that vascular protection can be
transferred through blood from a preconditioned an-
imal to a naïve animal emphasizes that remote isch-
emic preconditioning is mediated through humoral
signaling via plasma or cells such as platelets.12
Platelets are likely candidates for conveying signal-
ing molecules to remote parts of the vasculature as
they can take up and release compounds where
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J Am Heart Assoc. 2020;9:e016017. DOI: 10.1161/
Ischemic Preconditioning and Vascular Function
needed.13,14 In animal studies, platelets have been
found to carry and release serotonin, BDNF (brain-de-
rived neurotrophic factor) , and VEGF (vascular endo-
thelial growth factor), which are all compounds that
can modulate vascular tone by promoting the forma-
tion of vasodilators, including prostacyclin, in vascu-
lar endothelium.15–19 Combined with our findings that
prostacyclin is an important regulator of vascular tone
in humans20,21 and with findings from animal models
showing that acute ischemic preconditioning im-
proves endogenous formation of prostaglandins,22,23
these data suggest that preconditioning could in part
be mediated through this pathway.
The primary objective of the present study was to
document the remote effect of ischemic preconditioning
on microvascular endothelial function in humans as as-
sessed by vasodilatory responsiveness to intra-arterial
acetylcholine. Moreover, we aimed to elucidate if en-
dogenous formation of prostacyclin was involved in the
microvascular response to ischemic preconditioning. A
secondary objective was to assess whether a remote
microvascular effect could be achieved by exercise pre-
conditioning. We hypothesized that forearm ischemic
preconditioning would improve remote microvascular
function as determined in the leg and that this effect
would be mediated via enhanced endogenous prosta-
cyclin formation. In addition, it was hypothesized that a
session of hand-grip exercise would improve remote mi-
crovascular function.
METHODS
Because of the sensitive nature of the data collected for
this study, requests to access the data set from quali-
fied researchers trained in human subject confidential-
ity protocols may be sent to the corresponding author.
Participants
A total of 12 healthy young participants (7 men, 5
women) were included in the study (Table S1). All
participants were nonsmokers, showed no signs
of cardiovascular diseases, and were not taking
any chronic medications. The study was approved
by the Ethics Committee of the Capital Region of
Copenhagen (H-18000977), and all procedures were
carried out in accordance with the latest guidelines
of the Declaration of Helsinki. Written and oral in-
formed consent was obtained from all participants
before final enrollment.
Experimental Design
Study Overview
The experimental design included 2 separate visits to
the laboratory in randomized order, where the subjects
 Rytter et al
Figure 1. Experimental protocol.
Vertical arrows refer to sampling of muscle biopsies for gene expression analysis (M), arterial blood for isolation of platelets and
platelet-free plasma (B), and venous plasma for analysis of vasoactive compounds (P).
underwent either (1) ischemic preconditioning or (2)
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hand-grip exercise (Figure 1). Measurements of micro-
vascular function were conducted, and blood samples
were drawn, simultaneously in 1 arm and 1 leg.
To address the primary aim (to elucidate whether
ischemic preconditioning induces a remote effect
on microvascular function), the vasodilator response
to acetylcholine was determined in the leg at base-
line and after ischemic preconditioning of the arm.
Measurements in the arm were conducted simulta-
neously to determine the local effect.
To explore the potential mechanisms underlying the
effect of the intervention, plasma prostacyclin levels
were determined in blood samples obtained before
and after acetylcholine infusion given that acetylcho-
line stimulates prostacyclin formation.
To further evaluate mechanisms underlying
changes in microvascular function, serotonin, BDNF,
and VEGF were determined in platelets and plate-
let-free plasma obtained before and after ischemic
conditioning. In addition, skeletal muscle biopsies
were obtained from the leg to assess a potential in-
fluence of ischemic preconditioning on the expres-
sion of vascular proteins and to assess whether
changes related to microvascular function occurred
at the transcriptional level.
To address the secondary aim (to determine the
effect of hand-grip exercise on remote microvascular
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J Am Heart Assoc. 2020;9:e016017. DOI: 10.1161/
Ischemic Preconditioning and Vascular Function
function for comparison with that of the ischemic inter-
vention), the same experimental protocol as for isch-
emic preconditioning was conducted with hand-grip
exercise.
Intervention
Participants were instructed to refrain from physi-
cal activity as well as caffeine and alcohol intake for
24 hours before the experiments and to arrive in the
laboratory at 8 am after a light breakfast (similar be-
tween visits).
The ischemic preconditioning protocol consisted of
4 repetitive bouts of forearm occlusion, which were ac-
complished by rapidly inflating a pneumatic cuff (E20,
D.E. Hokanson Inc., Bellevue, WA) to 220 mm Hg for
5 minutes, followed by rapid deflation and 5 minutes of
rest. The hand-grip exercise protocol was conducted as 4
intermittent bouts, where participants performed 5 min-
utes of dynamic hand-grip exercise (SH5001, Saehan
Corp., Masan, South Korea) with 30 contractions per
minute, followed by 5 minutes of rest. The workload was
based on the participants’ maximal voluntary isometric
contraction and set to ensure that the participants were
barely able to complete the 150 contractions in 1 bout.
All sessions of ischemic preconditioning and hand-grip
exercise were supervised by the same person, ensuring
consistency between experiments.
 Rytter et al
Experimental Protocol
Under aseptic conditions and local anesthesia (li-
docaine, 20 mg mL−1; Astra Zeneca, Cambridge,
UK), catheters (20G, Arrow International, Reading,
PA) were inserted in the femoral artery and vein 2 to
4 cm below the inguinal ligament of the experimen-
tal leg. Under guidance of ultrasound, the tip of the
catheter was placed in same position in the common
femoral artery. A catheter was inserted in the brachial
artery (20G, arterial cannula; Becton, Dickinson and
Company, Sandy, UT) and in the basilic vein (18G, pe-
ripheral catheter; Becton, Dickinson and Company)
of the experimental arm. A 3-way cock was placed
on the arterial line so that infusions and blood pres-
sure measurements could be performed simultane-
ously. In the supine position with the experimental
arm extended laterally, participants received simulta-
neous intra-arterial (brachial and femoral) infusion of
acetylcholine (10 and 25 µg min−1 L tissue volume −1;
miochol-E, Bausch & Lomb Inc., Bridgewater, NJ),
sodium nitroprusside (3 µg min−1 L tissue volume −1;
Hospira Inc., Lake Forest, IL), and epoprostenol
(25 and 50 µg min−1 L tissue volume −1; Flolan, GSK
Pharma, Brentford, UK).
On the first visit in the laboratory, leg and fore-
arm tissue volumes were estimated by anthropomet-
ric measures. The infusion doses were determined
based on these measures. Infusions were separated
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by at least 15 minutes, and each dose was infused
for ≈3 minutes. Measurements (blood flow and blood
pressure) were performed in the last minute of respec-
tive infusion.
The entire infusion protocol was performed before
(pre), 5 minutes after (post+5), and 90 minutes after
(post+90) ischemic preconditioning or hand-grip exer-
cise (Figure 1).
Venous blood samples were collected at rest
and during the highest dose of acetylcholine at pre
and post+5. On the experimental day involving isch-
emic preconditioning, arterial blood for the isolation
of platelets and platelet-free plasma was collected
before (pre), in the arm immediately after (post arm)
and in the leg 10 minutes after (post leg) the last cuff
deflation.
In addition, skeletal muscle biopsies were obtained
from the m. vastus lateralis before (pre) and 2.5 hours
after (post) ischemic preconditioning (Figure 1).
Blood and Tissue Sampling
Venous Plasma
Venous blood was drawn in EDTA anticoagulated tubes
and immediately centrifuged (5 minutes at 4000g and
4°C), and aliquots of plasma were stored at −80°C until
later analysis.
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J Am Heart Assoc. 2020;9:e016017. DOI: 10.1161/
Ischemic Preconditioning and Vascular Function
Platelets and Platelet-Free Plasma
Arterial blood was drawn in citrate-anticoagulated
tubes and kept at room temperature until centrifuged
(10 minutes at 200g and 20°C) to obtain platelet-rich
plasma. The platelet-rich plasma was layered on top of
a gradient density medium (iodixanol solution, OptiPrep,
Sigma-Aldrich, St. Louis, MO) and centrifuged (15 min-
utes at 450g and 20°C), after which a fraction of
isolated platelets and platelet-poor plasma was col-
lected. To obtain plasma completely free of platelets,
the platelet-poor plasma was centrifuged (15 minutes
at 450g and 20°C) once more on the density gradient
medium, and a fraction of the plasma was collected as
platelet-free plasma. In all collected samples, platelets
were counted using a hematology analyzer (XP-300;
Sysmex, Kobe, Japan). Isolated platelets were lysed in
a fresh batch buffer (Meso Scale Diagnostics Tris lysis
buffer, Meso Scale Diagnostics, Rockville, MD) on ice
before being centrifuged (10 minutes at 10 000g and
4°C), and the supernatant was kept as platelet lysate.
All samples were stored at −80°C until later analysis.
Skeletal Muscle Biopsies
Under local anesthesia (lidocaine, 20 mg mL−1), skel-
etal muscle biopsies were obtained using the percu-
taneous needle biopsy technique with suction.24 The
biopsies were immediately frozen in liquid nitrogen and
stored at −80°C until further analysis.
Measurements
Hemodynamics
Arterial blood flow (leg, femoral arterial blood flow;
forearm, brachial arterial blood flow) was measured
with ultrasound Doppler (Vivid E9, GE Healthcare,
Chicago, IL) equipped with a linear probe (L9) operat-
ing at an imaging frequency of 4/8 MHz and a Doppler
frequency of 4.2 MHz. In the femoral artery, the site of
measurement was distal to the inguinal ligament but
above the bifurcation into the superficial and profound
femoral branch to avoid turbulence from the bifurca-
tion. For measurements of brachial arterial flow, the
probe was positioned approximately half way up the
upper arm. All recordings were obtained at the low-
est possible insonation angle but always below 60°.
Sample volume was maximized by choosing the wid-
est section of the artery, and recordings were made
without interference of the vascular wall. A low-velocity
filter (<1.8 m s−1) rejected noise caused by turbulence
at the vascular wall. Doppler traces and B-mode im-
ages were recorded continuously and averaged over
≈30 seconds. Arterial diameter was assessed during
systole from B-mode images for each Doppler re-
cording. Intra-arterial and venous blood pressure and
heart rate were monitored with transducers (Pressure
 Rytter et al
Monitoring Set, Edwards Lifesciences, Irvine, CA) po-
sitioned at the level of the heart. Vascular conduct-
ance was calculated by use of the following formula:
Vascular conductance
=
arterial blood flow
(mean arterial blood pressure − mean venous blood pressure)
Vasoactive Compounds in Venous Plasma
Plasma levels of the stable metabolite of prostacyclin,
6-keto prostaglandin F1α (PGF1α), and norepinephrine
were measured with enzyme immunoassay kits (6-
keto PGF1α, Cayman Chemical Co., Ann Arbor, MI; nor-
epinephrine; LDN, Nordhorn, Germany) in accordance
with the manufacturer’s instructions.
Serotonin, VEGF, and BDNF in Platelets and
Platelet-Free Plasma
The content of serotonin in platelets and platelet-free
plasma was measured with an enzyme immunoassay
kit (IBL International, Hamburg, Germany) in accord-
ance with the manufacturer’s instructions, and VEGF
and BDNF content were measured with an electro-
chemiluminescent assay kit (U-PLEX platform, Meso
Scale Diagnostics).
Gene Expressions in Skeletal Muscle Biopsies
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Total RNA was isolated from the muscle biopsies using
a TRIzol reagent (Invitrogen, Carlsbad, CA) in accord-
ance with the manufacturer’s instructions. First-strand
cDNA was synthesized from 1 μg of total RNA by
SuperScript II reverse transcriptase (Invitrogen) as de-
scribed previously.25 The mRNA content of cyclooxy-
genase 1 (Hs00377726_m1), prostacyclin synthase
(Hs00153133_m1), and endothelial NO synthase
(Hs00167166_m1) was determined by real-time re-
verse transcription polymerase chain reaction (ABI
PRISM 7900 Sequence Detection System, Applied
Biosystems, Foster City, CA). The cDNAs were ampli-
fied using the TaqMan gene expression assays from
Applied Biosystems. For each sample, the amount of
target gene mRNA was normalized to GAPDH mRNA
content. The effect of the interventions on the level of
GAPDH mRNA was determined statistically, and no sig-
nificant effect was found with ischemic preconditioning.
Statistical Analysis
A priori sample size determination was performed based
on previous data for the primary outcome, the difference
in vasodilatory responsiveness to intra-arterial acetyl-
choline with ischemic preconditioning.26 Significance
level was set to P<0.05 at a power level of 0.8. To detect
a significant difference of 5.0±5.0 mL min−1 mm Hg−1 in
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J Am Heart Assoc. 2020;9:e016017. DOI: 10.1161/
Ischemic Preconditioning and Vascular Function
vascular conductance with acetylcholine infusion, 12
participants were needed. Statistical analyses were per-
formed with R (version 3.4.1, R Foundation for Statistical
Computing, Vienna, Austria) using the interface RStudio
(version 1.1.463, RStudio Team, Boston, MA).
To estimate limb and intervention-specific differ-
ences in the vasodilator response to intra-arterial in-
fusions, a linear mixed model approach27 was used
with “time” (pre, post+5, post+90) and “infusion dose”
(dose1, dose2) as fixed factors. Likewise, a linear mixed
model was used for differences in baseline hemody-
namics, venous plasma norepinephrine, and 6-keto
PGF1α levels and platelet and platelet-free plasma levels
of serotonin, VEGF, and BDNF with “time” (pre, post+5,
post+90) as the fixed factor. For all analysis, partici-
pants were specified as a repeated factor and identifier
of random variation. Model checking as well as homo-
geneity of variance and normal distribution were con-
firmed through residual and Q-Q plots. To control for
type I error regarding multiple statistical testing on the
response to intra-arterial infusions and venous plasma
prostacyclin levels, a Bonferroni correction was applied
within the family of statistical tests by multiplying the P
value with the number of statistical tests performed (e.g.,
the vasodilator response to intra-arterial acetylcholine,
which was analyzed in 4 separate models). If significant
main effects or interactions were observed, the Tukey
honestly significant difference post hoc procedure was
used to detect all pairwise differences performed with
multiple comparisons,28 and adjusted P values are re-
ported. A paired Student t test was used to estimate dif-
ferences in mRNA levels with ischemic preconditioning.
Two participants (1 man and 1 woman) failed to at-
tend their second visit but completed the initial visit.
Consequently, the data presented for all measure-
ments before and after the hand-grip exercise include
only 10 of the 12 participants. Furthermore, because
of technical difficulties with sample preparation, data
presented for changes in mRNA levels with ischemic
preconditioning and data presented for platelet and
platelet-free plasma levels include 11 of the 12 partici-
pants (clarified in figure labels). The influence of sex and
timing of the visits were initially evaluated statistically
with a linear mixed model approach; however, no effect
of either variable on “time” was found (absence of sig-
nificant interaction). Data are presented as mean±SEM.
RESULTS
Effect of Ischemic Preconditioning on
Local and Remote Microvascular Function
Baseline
Mean arterial blood pressure, blood flow, and vascular
conductance in the femoral and brachial arteries were
unaltered by ischemic preconditioning (Table S2).
 Rytter et al Ischemic Preconditioning and Vascular Function

Leg and Forearm Microvascular Endothelial Cell
Responsiveness to Acetylcholine Infusion
In the leg, the vasodilator response to intra-arterial
acetylcholine was increased (P<0.05) at post+5 and
post+90 compared with pre following ischemic pre-
conditioning (Figure 2). In the forearm, no differences
were detected in vasodilatory responsiveness to in-
tra-arterial acetylcholine after ischemic precondition-
ing. All changes in blood flow, vascular conductance,
mean arterial blood pressure, and venous blood pres-
sure for both extremities are presented in Table S3.
Leg and Forearm Microvascular Smooth Muscle
Cell Responsiveness to Epoprostenol and
Sodium Nitroprusside Infusions
In the leg and forearm, no differences were detected
in vasodilatory responsiveness to intra-arterial epo-
prostenol or sodium nitroprusside following ischemic
preconditioning (Figure 2; Table S4). Baseline values
and all changes in blood flow, vascular conduct-
ance, mean arterial blood pressure, and venous
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blood pressure for both extremities are presented in
Tables S4 and S5.
Venous Plasma 6-keto PGF1α and
Norepinephrine Levels Before and After
Ischemic Preconditioning
Baseline
In the leg, no differences were detected in venous
plasma 6-keto PGF1α or norepinephrine levels with is-
chemic preconditioning. In the forearm, venous plasma
6-keto PGF1α levels were increased (P<0.05) at post+5
compared with pre, whereas venous plasma norepi-
nephrine levels were increased (P<0.05) at post+5 and
post+90 compared with pre following ischemic pre-
conditioning (Table S2).
Changes in Venous Plasma 6-keto PGF1α Levels
With Acetylcholine Infusion
In the leg, the change in venous plasma 6-keto PGF1α
levels from baseline to the highest dose of acetylcholine

Figure 2. Leg and forearm vasodilator response to intra-arterial acetylcholine and epoprostenol before and after ischemic
preconditioning of the forearm.
Absolute changes from baseline in leg and forearm vascular conductance with arterial infusion of acetylcholine (A and B) and
epoprostenol (C and D). Data are presented as mean±SEM (n=12). *Significantly different from pre.

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 Rytter et al Ischemic Preconditioning and Vascular Function

Figure 3. Changes in leg and forearm venous plasma 6-keto PGF1α levels with intra-arterial acetylcholine.
Absolute changes from baseline to the highest dose of acetylcholine in venous plasma levels of 6-keto PGF1α before and after
ischemic preconditioning of the forearm (n=12) (A) and hand-grip exercise (n=10) (B). Data are presented as mean±SEM. *Significantly
different from pre. 6-keto PGF1α, 6-keto prostaglandin F1α.

was increased (P<0.05) at post+5 compared with Platelet-free plasma levels of BDNF and VEGF were
pre following ischemic preconditioning, whereas no increased (P<0.05) at post arm compared with pre
change was observed in the forearm venous plasma and post leg, whereas serotonin levels were unal-
6-keto PGF1α level (Figure 3). tered with ischemic preconditioning (Figure 4).

Effect of Ischemic Preconditioning on Skeletal Muscle mRNA Levels Before and

Serotonin, BDNF, and VEGF in Platelets After Ischemic Preconditioning
and Platelet-Free Plasma
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Endothelial NO synthase, prostacyclin synthase, and cy-

In platelets, no differences were detected in either ser- clooxygenase 1 mRNA levels in skeletal muscle were sim-
otonin, BDNF, or VEGF with ischemic preconditioning. ilar before and after ischemic preconditioning (Figure 5).

Figure 4. Platelet content and platelet-free plasma levels of serotonin, VEGF, and BDNF before and after ischemic
preconditioning of the forearm.
Platelet content (A through C) and platelet-free plasma levels (D through F) of serotonin, VEGF, and BDNF. Data are presented as
mean±SEM (n=11). *Significantly different from pre; #significantly different from post+5. BDNF, brain-derived neurotrophic factor; and
VEGF, vascular endothelial growth factor.

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 Rytter et al
Figure 5. Skeletal muscle mRNA levels of eNOS, PGIS,
and COX1 before and after ischemic preconditioning of the
forearm.
mRNA levels of eNOS, PGIS, and COX1 in skeletal muscle
homogenates from m. vastus lateralis. Data are presented as
mean±SEM (n=11). COX1, cyclooxygenase 1; eNOS, endothelial
nitric oxide synthase; and PGIS, prostacyclin synthase.
Effect of Hand-Grip Exercise on Local
and Remote Microvascular Function
Baseline
Mean arterial blood pressure in the femoral and bra-
chial arteries remained unaltered by hand-grip exer-
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cise. In the leg, blood flow and vascular conductance
were similar after hand-grip exercise, whereas in the
forearm blood flow and vascular conductance were
increased (P<0.05) at post+5 compared with pre and
post+90 (Table S2).
Leg and Forearm Microvascular Endothelial Cell
Responsiveness to Acetylcholine Infusion
In the leg, no differences were detected in vaso-
dilatory responsiveness to intra-arterial acetylcho-
line with hand-grip exercise. In the forearm, the
vasodilator response to intra-arterial acetylcholine
was increased (P<0.05) at post+5 and post+90
compared with pre following hand-grip exercise
(Figure 6). All changes in blood flow, vascular
conductance, mean arterial blood pressure, and
venous blood pressure for both extremities are
presented in Table S3.
Leg and Forearm Microvascular Smooth Muscle
Cell Responsiveness to Epoprostenol and
Sodium Nitroprusside Infusions
In the leg and forearm, no differences were detected in
vasodilatory responsiveness to intra-arterial epopros-
tenol or sodium nitroprusside with hand-grip exercise
(Figure 6; Table S4). Baseline values and all changes
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J Am Heart Assoc. 2020;9:e016017. DOI: 10.1161/
Ischemic Preconditioning and Vascular Function
in blood flow, vascular conductance, mean arterial
blood pressure, and venous blood pressure for both
extremities are presented in Tables S4 and S5.
Venous Plasma 6-keto PGF1α and
Norepinephrine Levels Before and After
Hand-Grip Exercise
Baseline
In the leg and forearm, no differences were detected in
venous plasma 6-keto PGF1α or norepinephrine levels
with hand-grip exercise (Table S2).
Changes in Venous Plasma 6-keto PGF1α Levels
With Acetylcholine Infusion
In the leg and forearm, no differences were detected
in changes in venous plasma 6-keto PGF1α levels from
baseline to the highest dose of acetylcholine with
hand-grip exercise (Figure 3).
DISCUSSION
The principal finding of this study was that an acute
bout of ischemic preconditioning performed on the
arm induced a sustained improvement in micro-
vascular endothelial function in the leg, that is, a
remote effect, as indicated by an enhanced vaso-
dilator response to arterial acetylcholine infusion.
The enhanced acetylcholine induced response
in leg vascular conductance was paralleled by a
greater increase in venous plasma prostacyclin
levels, suggesting that the increased sensitivity to
acetylcholine was related to enhanced formation
of prostacyclin. The enhancement in prostacyclin
levels was in turn associated with transiently in-
creased platelet-free plasma levels of VEGF and
BDNF, compounds known to stimulate endothelial
prostacyclin formation. Our findings demonstrate,
for the first time in humans, that ischemic precondi-
tioning leads to improved remote microvascular en-
dothelial function, an effect that may be related to
increased prostacyclin production. Hand-grip exer-
cise did not significantly alter remote microvascular
endothelial function. This finding indicates a differ-
ent mechanistic effect of exercise versus ischemic
preconditioning and underlines the unique effect of
ischemic preconditioning for promoting remote vas-
cular protection.
Effect of Ischemic Preconditioning on
Remote Microvascular Function
Remote ischemic preconditioning was originally
shown to prevent vascular damage associated with
 Rytter et al Ischemic Preconditioning and Vascular Function
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Figure 6. Leg and forearm vasodilator response to intra-arterial acetylcholine and epoprostenol before and after hand-grip
exercise.
Absolute changes from baseline in leg and forearm vascular conductance with arterial infusion of acetylcholine (A and B) and
epoprostenol (C and D). Data are presented as mean±SEM (n=10). *Significantly different from pre; #significantly different from post+5.

prolonged tissue ischemia,4 indicating that the pro-
cedure may induce a systemic stimulus leading to
functional adaptations in vascular endothelium. We
demonstrate that acute exposure to ischemic pre-
conditioning of the arm enhances the acetylcholine-
induced change in leg vascular conductance. The
effect was present within 5 minutes after the pro-
cedure and persisted for at least 90 minutes. The
enhanced vasodilator response was not paralleled
by changes in smooth muscle cell sensitivity, as in-
dicated by an unaltered responsiveness to the NO
donor, sodium nitroprusside, and the prostacyclin
analog, epoprostenol, emphasizing that the improve-
ment was localized to the endothelium. In support
of an improvement at the endothelial level, the ace-
tylcholine-induced increase in plasma prostacyclin
levels was higher in the leg after ischemic precon-
ditioning, suggesting that the procedure potenti-
ated prostacyclin production. This finding is in line
with the proposition that prostacyclin is important
for vascular protection induced by remote ischemic
preconditioning22,23 and that targeting prostacyclin
formation may play a central role in treating patients
with endothelial dysfunction.29
Increased prostacyclin production by ischemic
preconditioning could be explained by an enhanced
receptor sensitivity to acetylcholine, a higher sub-
strate availability or, alternatively, increased enzyme
activity in the prostacyclin system. In the current
study, we examined local and systemic alterations in
3 compounds known to enhance endothelial prosta-
glandin formation: serotonin, BDNF, and VEGF.16,30–33
These compounds can be transported both in plate-
lets and in plasma, and therefore these fractions
were analyzed separately. The purpose was to as-
sess whether the remote effect of the ischemic pre-
conditioning intervention was associated with an
increase in these compounds either by platelets or
in platelet-free plasma. We observed increased lev-
els of BDNF and VEGF in platelet-free plasma after
ischemic preconditioning, indicating that these com-
pounds could have been transported in plasma to the

102
J Am Heart Assoc. 2020;9:e016017. DOI: 10.1161/
 Rytter et al
remote vasculature and influenced the prostacyclin
production, although further evidence for this possi-
bility is clearly needed. Transport of BDNF and VEGF
by platelets could not be verified as the level of these
compounds in the platelet fraction remained unal-
tered after the ischemic preconditioning. Combined,
our findings suggest that ischemic preconditioning
leads to an improvement in remote microvascular en-
dothelial function through humoral signaling, leading
to increased prostacyclin production.
Sustained Effect of Remote Ischemic
Preconditioning
Previous studies have documented a sustained ef-
fect of ischemic preconditioning: a second window,
lasting for up to 48 hours after the procedure.34 Such
a sustained effect could be attributed to alterations
at the transcriptional level. We therefore determined
the mRNA levels of prostacyclin synthase, cyclooxy-
genase 1, and endothelial NO synthase in skeletal
muscle biopsies obtained before and 2.5 hours after
ischemic preconditioning. A potential increase in
mRNA levels of proteins related to the formation of
vasoactive compounds is also of interest in relation
to the observed beneficial effect of a period of daily
treatment with ischemic preconditioning on vascular
function.35–37 No significant alterations in mRNA lev-
els were, however, detected for either of the vasodi-
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lator enzymes, and the present study was not able
to confirm the hypothesis that the sustained effect
of ischemic preconditioning would be attributed to
the upregulation of these vasodilator enzymes. It is,
however, worth noting that endothelial NO synthase
mRNA was numerically higher after ischemic pre-
conditioning in 9 of the 11 participants, which may
indicate that ischemic preconditioning can lead to
upregulation of endothelial NO synthase.
It should also be clarified that our finding does not
exclude that an increased mRNA expression could
occur at a later time than measured here or that other
vasoactive proteins could be influenced.
Effect of Ischemic Preconditioning on
Local Microvascular Function
Ischemic preconditioning was not found to influence
the vasodilator response to acetylcholine locally in the
forearm, a finding that may have been related to an
opposing negative effect of the procedure attributed
to local ischemia. Ischemia reperfusion can lead to an
increased formation of reactive oxygen species in skel-
etal muscle,38 which readily remove NO and interfere
with prostacyclin formation.39,40 Thus, the availability of
these vasoactive compounds may have been reduced
locally in the arm, counteracting the enhanced vasodi-
latory capacity observed in the leg.
103
J Am Heart Assoc. 2020;9:e016017. DOI: 10.1161/
Ischemic Preconditioning and Vascular Function
Effect of Exercise Preconditioning on
Local and Remote Microvascular Function
We examined the functional effect of exercise pre-
conditioning to assess whether exercise could also
induce a remote effect. Previous studies have re-
ported that exercise can induce a preconditioning ef-
fect similar to that of ischemic preconditioning at the
macrovascular level,8 however, it is unclear whether
exercise improves remote microvascular vasodilatory
responsiveness. The remote microvascular response
to acetylcholine infusion was not detectably altered by
hand-grip exercise, whereas an improved response
was found locally in the arm. Smooth muscle sensitiv-
ity to NO and prostacyclin was not significantly altered
locally in the arm, suggesting that primarily endothe-
lial function was improved locally by the exercise. The
improved vasodilator response to acetylcholine in the
arm was not paralleled by a greater change in plasma
prostacyclin levels during arterial acetylcholine infu-
sion. This finding suggests that the mechanism medi-
ating vascular changes with exercise may be different
from that of ischemic preconditioning.
The lack of effect of hand-grip exercise on remote
microvascular responsiveness and prostacyclin for-
mation indicates that the previously observed effect
of exercise preconditioning on remote endothelial
injury8 is not likely caused by changes in microvas-
cular endothelial function. The explanation for the
difference between exercise and ischemia can only
be speculated on, but it appears plausible that the
observed remote signaling specifically is depen-
dent on an ischemic insult that cannot be induced
by dynamic hand-grip exercise. Overall, our current
observations suggest that the mechanism by which
ischemic preconditioning influences vascular func-
tion is different from that of exercise. The precise
mechanism underlying the local effect of exercise re-
mains to be elucidated.
Study Limitations
To assess the acute effect of remote ischemic pre-
conditioning on microvascular function, infusions of
the vasoactive compounds were repeated 3 times
during the experimental day. The sequence of drugs
was kept the same in the protocols to keep the time
after the interventions constant. It is our
experience from many years of conducting infusion
with these same drugs that the short-term
infusions used do not impact the vasodilatory
re-sponse of other subsequent drugs. Moreover,
the lack of influence of previously infused drugs
was verified by the clear difference in remote and
local vasodilator responses on the 2 experimental
days
 Rytter et al Ischemic Preconditioning and Vascular Function

involving ischemic preconditioning and hand-grip ex- Supplementary Materials

ercise, respectively. Tables S1–S5

The improved sensitivity to acetylcholine after

ischemic preconditioning could, in addition to in-
creased prostacyclin production, also have been
due to an enhanced formation of NO. As NO is short
lived, venous plasma levels can only be indirectly as-
sessed as the stable metabolites nitrite and nitrate.
However, because nitrite and nitrate are rather insen-
sitive markers, limited changes in formation would
not have been possible to detect in plasma. As a re-
sult, we cannot exclude the possibility that a poten-
tiation of NO formation contributed to the improved
vasodilator response to acetylcholine after ischemic
preconditioning. It should also be mentioned that
signaling proteins may be carried by microvesicles
or cells in the blood41; however, this was not studied
in the current investigation and thus their role cannot
be ruled out.

Conclusions
The present study demonstrates for the first time
in humans that ischemic preconditioning of the
forearm leads to an improved vasodilator response
to arterial acetylcholine infusion in the leg and to
an associated increase in formation of prostacy-
clin. This finding indicates that the remote effect
of ischemic preconditioning is related to a poten-
Downloaded from http://ahajournals.org by on April 18, 2024

tiation of the prostacyclin system. Collectively,

these findings suggest that ischemic
preconditioning is an effective new treatment
strategy in conditions associated with
microvascu-lar endothelial dysfunction.

ARTICLE INFORMATION
Received January 22, 2020; accepted June 18, 2020.

Affiliations
From the Section of Integrative Physiology, Department of Nutrition, Exercise
and Sports (N.R., H.C., P.P., H.S., T.E., F.H., C.T., L.G., Y.H.), Department
of Anesthesia, Centre for Cancer and Organ Diseases Rigshospitalet,
Copenhagen, Denmark (H.S.); Research Institute for Sport and Exercise
Sciences, Liverpool John Moores University, Liverpool, United Kingdom
(H.J., D.T.); and Department of Physiology, Radboud Institute for Health
Sciences, Nijmegen, The Netherlands (D.T.).

Acknowledgments
The technical assistance of Gemma Kroos is gratefully acknowledged.

Sources of Funding
The study was funded by The Independent Research Fund Denmark and
by The Danish Ministry of Culture Fund for Sports Research. Howard Carter
was supported by a grant from the Danish Diabetes Academy supported by
the Novo Nordisk Foundation.

Disclosures
None.

104
J Am Heart Assoc. 2020;9:e016017. DOI: 10.1161/
Supplemental Material

105
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 Table S1. Participant characteristics.

Variable
Age (years) 23 ± 1
Height (cm) 175 ± 3
Body mass (kg) 71 ± 2
VO2 max (ml O2 min-1) 3263 ± 218
VO2 max (ml O2 min-1 kg-1) 48 ± 2
Systolic blood pressure (mmHg) 112 ± 3
Diastolic blood pressure (mmHg) 67 ± 2
Hemoglobin (mmol L ) -1 8.6 ± 0.2
Thrombocytes (109 L-1) 277 ± 16
Glycated hemoglobin (mmol L )-1 5.3 ± 0.1
-1
Total cholesterol (mmol L ) 4.2 ± 0.4page s
High-density lipoprotein (mmol L-1) 1.4 ± 0.2
-1
Low-density lipoprotein (mmol L ) 2.6 ± 0.3
-1
Triglycerides (mmol L ) 1.0 ± 0.1
Ischemic Hand-grip exercise
Female sex hormones
preconditioning (n=5) (n=4)
-1
Estrogen (nmol L ) 0.17 ± 0.03 0.22 ± 0.13
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Progesterone (nmol L-1) 0.60 ± 0.00 0.63 ± 0.03

VO2 max, maximal oxygen uptake. Data are presented as mean ± SEM (n=12).

106
 Table S2. Baseline hemodynamics and venous plasma 6-keto prostaglandin F 1α and norepinephrine levels.
ANOVA P ANOVA P
Leg Ischemic preconditioning (n=12) Hand-grip exercise (n=10)
values values
Variable Pre Post+5 Post+90 Time Pre Post+5 Post+90 Time
Leg blood flow (ml
276 ± 42 306 ± 34 349 ± 47 P=0.8704 330 ± 43 321 ± 36 351 ± 40 P=1.0
min-1)
Leg vascular
conductance (ml min- 3.6 ± 0.5 4.0 ± 0.4 4.4 ± 0.6 P=1.0 4.4 ± 0.5 4.1 ± 0.4 4.6 ± 0.5 P=1.0
1 mmHg-1)

Mean arterial
81 ± 2 80 ± 2 81 ± 2 P=1.0 79 ± 2 80 ± 2 79 ± 2 P=0.9532
pressure (mmHg)

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Venous pressure
3±1 2±0* 2±0* P<0.0001 4±1 3±0 3±1 P=1.0
(mmHg)
Heart rate (bpm) 63 ± 3 63 ± 3 61 ± 2 P=1.0 63 ± 3 63 ± 3 62 ± 4 P=1.0

107
Venous
norepinephrine (nmol 0.9 ± 0.1 1.1 ± 0.2 1.0 ± 0.1 P=1.0 0.9 ± 0.1 0.9 ± 0.1 0.9 ± 0.1 P=1.0
-1
l )
Venous 6-keto PGF1α
61 ± 4 59 ± 7 - P=1.0 75 ± 4 72 ± 6 - P=1.0
(pg ml-1)
ANOVA P ANOVA P
Forearm Ischemic preconditioning (n=12) Hand-grip exercise (n=10)
values values
Variable Pre Post+5 Post+90 Time Pre Post+5 Post+90 Time
Forearm blood flow
94 ± 18 60 ± 8 81 ± 12 P=0.0762 79 ± 15 146 ± 17 * 92 ± 13 # P<0.0001
(ml min-1)
Forearm vascular P<0.0001
conductance (ml min- 1.3 ± 0.3 0.8 ± 0.1 1.0 ± 0.2 P=0.0947 1.0 ± 0.2 1.9 ± 0.2 * 1.2 ± 0.2 #
1 mmHg-1)
 Mean arterial 80 ± 2 79 ± 2 80 ± 2 P=0.6088 78 ± 2 78 ± 2 78 ± 2 P=1.0
pressure (mmHg)

Venous pressure 3±0 2±1 2±0 P=0.2116 4±1 3±0 2±1* P=0.0030
(mmHg)

Venous
1.0 ± 0.1 1.4 ± 0.1 * 1.3 ± 0.1 * P=0.0040 1.0 ± 0.1 1.1 ± 0.2 1.0 ± 0.1 P=1.0
norepinephrine
(nmol l-1)
- P=0.0084 -

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Venous 6-keto PGF1α 56 ± 5 76 ± 10 * 77 ± 8 60 ± 5 P=0.2277
(pg ml-1)

108
Data are presented as mean ± SEM. * denotes post-hoc significantly different from Pre within intervention; # denotes post-hoc significantly
different from Post+5 within intervention.
 Table S3. Absolute changes in hemodynamics with intra-arterial infusion of acetylcholine.
Leg Ischemic preconditioning (n=12) ANOVA P values Hand-grip exercise (n=10) ANOVA P values
Time x Time x
Variable Pre Post+5 Post+90 Time Pre Post+5 Post+90 Time
Dose Dose
Δ LBF (Ach; 10 1206 ± 1152 ± 1259 ± 1118 ± 1216 ±
-1 -1
914 ± 121
µg min L ) 132 * 185 * 184 199 200
P=0.0163 P=1.0 P=1.0 P=1.0
Δ LBF (Ach; 25 1214 ± 1709 ± 1513 ± 1495 ± 1440 ± 1551 ±
-1
µg min L )-1 168 178 * 201 * 230 253 243
Δ MAP (Ach; -3.7 ± 0.8 -3.8 ± 1.0
-1 -1
-1.9 ± 0.5 -3.6 ± 0.9 -4.4 ± 1.1 -3.5 ± 0.9
10 µg min L ) * *
P=0.0027 P=1.0 P=0.6056 P=1.0
Δ MAP (Ach; -3.6 ± 0.7 -5.0 ± 1.2
-2.0 ± 0.6 -2.2 ± 1.0 -4.3 ± 1.4 -2.8 ± 1.0

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25 µg min-1 L-1) * *
Δ VP (Ach; 10
-0.2 ± 0.4 0.1 ± 0.1 0.1 ± 0.3 0.1 ± 0.2 0.0 ± 0.3 -0.2 ± 0.2
µg min-1 L-1)
P=0.2930 P=1.0 P=1.0 P=1.0

109
Δ VP (Ach; 25
-0.4 ± 0.4 0.4 ± 0.3 0.0 ± 0.3 -0.1 ± 0.3 -0.3 ± 0.4 -0.2 ± 0.3
µg min-1 L-1)
Forearm Ischemic preconditioning (n=12) ANOVA P values Hand-grip exercise (n=10) ANOVA P values
Time x Time x
Variable Pre Post+5 Post+90 Time Pre Post+5 Post+90 Time
Dose Dose
Δ FBF (Ach; 10 118 ± 19
-1 -1
77 ± 14 68 ± 12 96 ± 13 46 ± 12 91 ± 19 *
µg min L ) *
P=0.6141 P=1.0 P<0.0001 P=1.0
Δ FBF (Ach; 25 185 ± 19 157 ± 27
99 ± 19 127 ± 18 134 ± 24 98 ± 14
µg min-1 L-1) * *
Δ MAP (Ach;
-2.8 ± 0.7 -3.4 ± 0.9 -4.3 ± 0.9 -3.7 ± 0.6 -4.6 ± 0.8 -3.5 ± 0.9
10 µg min-1 L-1)
P=0.0853 P=1.0 P=0.7396 P=1.0
Δ MAP (Ach;
-3.7 ± 0.5 -4.1 ± 0.9 -5.9 ± 0.9 -3.3 ± 0.8 -5.2 ± 1.1 -3.7 ± 1.1
25 µg min-1 L-1)
 Δ VP (Ach; 10 0.2 ± 0.2 0.6 ± 0.2
0.5 ± 0.3 0.2 ± 0.3 0.1 ± 0.4 0.5 ± 0.3
µg min-1 L-1) *
Δ VP (Ach; 25 0.4 ± 0.3 1.0 ± 0.2 *
0.5 ± 0.3 0.7 ± 0.3 0.3 ± 0.4 1.0 ± 0.4
µg min-1 L-1) *
*
LBF, leg blood flow; FBF, forearm blood flow; MAP, mean arterial pressure; VP, venous pressure; Ach, acetylcholine. Data are presented as
mean ± SEM. * denotes post-hoc overall significantly different from Pre within intervention.

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110
 Table S4. Absolute changes in hemodynamics with intra-arterial infusion of sodium nitroprusside.
Leg Ischemic preconditioning (n=12) ANOVA P values Hand-grip exercise (n=10) ANOVA P values
Time x Time x
Variable Pre Post+5 Post+90 Time Pre Post+5 Post+90 Time
Dose Dose
Baseline LVC
4.5 ± 0.5
(ml min-1 3.2 ± 0.4 3.6 ± 0.4 P<0.0001 - 4.3 ± 0.5 3.3 ± 0.4 3.9 ± 0.4 P=0.2958 -
-1
*#
mmHg )
Δ LVC (SNP; 3 11.9 ± 12.1 ± 12.3 ±
9.6 ± 1.1 10.6 ± 1.3 9.3 ± 1.3 P=0.4828 - P=1.0 -
µg min-1 L-1) 1.0 1.4 1.9
Baseline LBF 362 ± 38
-1
252 ± 28 285 ± 36 P<0.0001 - 321 ± 38 248 ± 27 301 ± 36 P=0.3524 -
(ml min ) *#

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Δ LBF (SNP; 3 814 ±
-1 -1
651 ± 77 714 ± 83 626 ± 85 P=0.8164 - 740 ± 60 810 ± 98 P=1.0 -
µg min L ) 124
Baseline MAP 78.7 ± 78.7 ± 79.9 ±
81.7 ± 2.1 80.2 ± 1.9 81.3 ± 2.0 P=0.9896 - P=1.0 -

111
(mmHg) 3.2 2.8 2.1
Δ MAP (SNP; 3
-7.8 ± 0.7 -7.8 ± 0.8 -7.5 ± 0.9 P=1.0 - -9.0 ± 1.3 -7.3 ± 0.6 -7.4 ± 1.3 P=1.0 -
µg min-1 L-1)
Baseline VP 1.5 ± 0.4 1.6 ± 0.5 2.4 ± 0.4 2.7 ± 0.5
2.8 ± 0.4 P<0.0001 - 3.9 ± 0.7 P=0.0035 -
(mmHg) * * * *
Δ VP (SNP; 3
-0.3 ± 0.3 0.0 ± 0.3 0.1 ± 0.3 P=1.0 - -0.5 ± 0.5 -0.2 ± 0.2 -0.1 ± 0.2 P=1.0 -
µg min-1 L-1)
Forearm IPC (n=12) ANOVA P values Hand-grip exercise (n=10) ANOVA P values
Time x Time x
Variable Pre Post+5 Post+90 Time Pre Post+5 Post+90 Time
Dose Dose
Baseline FVC
(ml min-1 1.3 ± 0.2 1.0 ± 0.1 1.0 ± 0.1 P=0.7328 - 1.3 ± 0.3 1.2 ± 0.1 1.1 ± 0.2 P=1.0 -
mmHg-1)
 Δ FVC (SNP; 1.7 ± 0.3 1.7 ± 0.3 1.8 ± 0.3 P=1.0 - 1.6 ± 0.3 2.0 ± 0.2 1.6 ± 0.1 P=1.0 -
3
µg min-1 kg-
1)
Baseline FBF 96 ± 17 77 ± 10 82 ± 9 P=0.7612 - 94 ± 20 90 ± 14 87 ± 16 P=1.0 -
(ml min-1)

Δ FBF (SNP; 102 ± 18 107 ± 19 116 ± 18 P=1.0 - 90 ± 16 120 ± 16 99 ± 7 P=0.8980 -

3
µg min-1 L-
1)
Baseline 80.5 ± 2.1 79.5 ± 1.8 80.5 ± 2.0 P=1.0 - 78.0 ± 3.2 78.0 ± 2.7 78.8 ± 1.9 P=1.0 -
MAP

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(mmHg)
Δ MAP (SNP; -9.6 ± 0.7 -9.5 ± 0.8 -9.4 ± 1.0 P=1.0 - -10.7 ± -8.9 ± 0.6 -8.5 ± 1.4 P=1.0 -
3 1.2

112
µg min-1 L-
1)
Baseline VP 2.9 ± 0.5 2.0 ± 0.6 1.5 ± 0.5 P=0.0024 - 3.6 ± 0.6 2.5 ± 0.6 2.2 ± 0.6 P<0.0001 -
(mmHg) * * * *

Δ VP (SNP; 3 -0.6 ± 0.2 0.1 ± 0.2 0.5 ± 0.1 P<0.0001 - -0.5 ± 0.3 0.0 ± 0.2 0.0 ± 0.3 P=0.1502 -
µg min-1 L- * *
1)

LVC, leg vascular conductance; FVC, forearm vascular conductance; LBF, leg blood flow; FBF, forearm blood flow; MAP, mean arterial
pressure; VP, venous pressure; SNP, sodium nitroprusside. Data are presented as mean ± SEM. * denotes post-hoc significantly different
from Pre within intervention; # denotes post-hoc significantly different from Post+5 within intervention.
 Table S5. Absolute changes in hemodynamics with intra-arterial infusion of epoprostenol.
Leg Ischemic preconditioning (n=12) ANOVA P values Hand-grip exercise (n=10) ANOVA P values
Time x Time x
Variable Pre Post+5 Post+90 Time Pre Post+5 Post+90 Time
Dose Dose
Baseline LVC
4.9 ± 0.8
(ml min-1 3.2 ± 0.4 3.9 ± 0.5 P=0.0136 - 4.4 ± 0.5 3.9 ± 0.5 4.3 ± 0.3 P=1.0 -
-1
*
mmHg )
Baseline LBF 400 ± 73
-1
247 ± 32 310 ± 40 P=0.0155 - 326 ± 38 290 ± 36 325 ± 29 P=1.0 -
(ml min ) *
Δ LBF (Epo; 25
243 ± 39 255 ± 50 187 ± 47 286 ± 44 268 ± 58 216 ± 42
µg min-1 L-1)
P=1.0 P=1.0 P=1.0 P=1.0

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Δ LBF (Epo; 50
352 ± 50 339 ± 53 334 ± 68 389 ± 45 416 ± 60 365 ± 61
µg min-1 L-1)
Baseline MAP 78.2 ± 77.9 ± 78.9 ±
81.3 ± 2.1 81.2 ± 1.8 82.1 ± 1.7 P=1.0 - P=1.0 -

113
(mmHg) 2.5 2.3 2.0
Δ MAP (Epo;
-2.3 ± 0.9 -4.2 ± 0.9 -3.1 ± 0.7 -1.6 ± 0.9 0.3 ± 1.3 -2.0 ± 0.7
25 µg min-1 L-1)
P=0.3572 P=1.0 P=0.5364 P=1.0
Δ MAP (Epo;
-4.5 ± 1.1 -5.6 ± 0.9 -4.1 ± 0.9 -3.6 ± 1.2 -2.5 ± 1.4 -2.5 ± 1.0
50 µg min-1 L-1)
Baseline VP 2.1 ± 0.4 1.8 ± 0.4
3.4 ± 0.4 P<0.0001 - 4.4 ± 0.8 3.0 ± 0.5 3.2 ± 0.7 P=0.1879 -
(mmHg) * *
Δ VP (Epo; 25 -0.4 ± 0.2 0.0 ± 0.2
-1 -1
-0.4 ± 0.2 -0.1 ± 0.1 0.0 ± 0.2 -0.2 ± 0.3
µg min L ) * #
P=0.1600 P=1.0 P=0.0347 P=1.0
Δ VP (Epo; 50 -0.5 ± 0.2 0.0 ± 0.2
-1 -1
-0.2 ± 0.2 0.0 ± 0.2 0.2 ± 0.2 0.1 ± 0.3
µg min L ) * #
Forearm IPC (n=12) ANOVA P values Hand-grip exercise (n=10) ANOVA P values
Time x Time x
Variable Pre Post+5 Post+90 Time Pre Post+5 Post+90 Time
Dose Dose
 Baseline FVC
(ml min-1 1.1 ± 0.2 1.0 ± 0.1 1.0 ± 0.1 P=1.0 - 1.2 ± 0.2 1.2 ± 0.2 0.9 ± 0.2 P=1.0 -
mmHg-1)
Baseline FBF
83 ± 14 78 ± 11 79 ± 9 P=1.0 - 88 ± 16 91 ± 15 70 ± 12 P=1.0 -
(ml min-1)
Δ FBF (Epo; 25
70 ± 10 72 ± 6 58 ± 8 73 ± 14 84 ± 10 78 ± 13
µg min-1 L-1)
P=0.5784 P=1.0 P=1.0 P=1.0
Δ FBF (Epo; 50
106 ± 12 103 ± 9 96 ± 9 109 ± 16 116 ± 16 113 ± 13
µg min-1 L-1)
Baseline MAP 77.6 ± 77.3 ± 78.6 ±
80.5 ± 2.1 80.5 ± 1.8 81.6 ± 1.6 P=1.0 - P=1.0 -
(mmHg) 2.4 2.2 1.9

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Δ MAP (Epo;
-4.0 ± 1.1 -6.0 ± 0.9 -4.6 ± 0.8 -3.2 ± 0.9 -1.3 ± 1.4 -3.6 ± 0.7
25 µg min-1 L-1)
P=0.1672 P=1.0 P=0.7300 P=1.0
Δ MAP (Epo;
-7.4 ± 1.1 -8.7 ± 0.9 -6.8 ± 0.9 -6.4 ± 0.9 -5.2 ± 1.5 -5.2 ± 1.1

114
50 µg min-1 L-1)
Baseline VP
3.3 ± 0.4 2.2 ± 0.5 2.0 ± 0.6 P=0.1100 - 3.1 ± 0.6 2.7 ± 0.6 2.2 ± 0.7 P=0.0757 -
(mmHg)
Δ VP (Epo; 25
0.0 ± 0.5 0.3 ± 0.2 0.1 ± 0.2 0.6 ± 0.3 0.3 ± 0.1 0.4 ± 0.3
µg min-1 L-1)
P=0.8820 P=1.0 P=0.9056 P=1.0
Δ VP (Epo; 50
0.0 ± 0.5 0.6 ± 0.2 0.3 ± 0.2 0.7 ± 0.4 0.3 ± 0.3 0.8 ± 0.4
µg min-1 L-1)

LVC, leg vascular conductance; FVC, forearm vascular conductance; LBF, leg blood flow; FBF, forearm blood flow; MAP, mean arterial
pressure; VP, venous pressure; Epo, epoprostenol. Data are presented as mean ± SEM. * denotes post-hoc overall significantly different
from Pre within intervention; # denotes post-hoc overall significantly different from Post+5 within intervention.