35
Selective Loss of Microvascular Endothelial
Function in Human Hypercholesterolemia
David M. Gilligan, MB; Victor Guetta, MD; Julio A. Panza, MD; Carlos E. Garcia, MD;
Arshed A. Quyyumi, MD; Richard 0. Cannon III, MD
With the technical assistance of Diane A. Badar, RN, and Crescence M. Kilcoyne, RN
Background Endothelial dysfunction is increasingly recog-
nized as an early and important feature of vascular disease.
Endothelium-dependent vasodilation is impaired in humans
with hypercholesterolemia, although it is unknown whether
this defect is selective for some pathways of nitric oxide
production or indicates a more generalized abnormality of
endothelial function. The aim of this study was to further
elucidate the nature of endothelial dysfunction in human
hypercholesterolemia by comparing vascular responses of ag-
onists that use different signal transduction pathways to acti-
vate production of nitric oxide.
Methods and Results Forearm flow was measured in 12
hypercholesterolemic patients (total cholesterol, 286±35
mg/dL [mean±SD]) aged 50±11 years and in 12 healthy
subjects (total cholesterol, 173±27 mg/dL) aged 48±7 years
using strain-gauge plethysmography and brachial artery drug
infusions. The endothelium-dependent vasodilators used were
acetylcholine (7.5, 15, and 30 ,g/min), which uses a pertussis
toxin-sensitive signal transduction pathway, and bradykinin
(100, 200, and 400 ng/min), which uses a pertussis toxin-
insensitive signal transduction pathway to activate nitric oxide
production. Sodium nitroprusside (0.8, 1.6, and 3.2 gg/min)
was used to test endothelium-independent vasodilation. The
nowledge of the role of the endothelium in
K controlling vascular tone has expanded greatly
in the past decade.1-3 In response to a large
number of agonists and shear stress, the endothelium
releases vasodilating substances, the most important of
which is nitric oxide, produced from L-arginine by the
enzyme nitric oxide synthase.4,5 A large body of evi-
dence now shows that endothelial dysfunction is not
only associated with established atherosclerosis in hu-
mans but is also detectable when only risk factors for
atherosclerosis, such as hypertension and hypercholes-
terolemia, are present.6-"1 This endothelial dysfunction
is probably an early and important feature in the
development of and ultimately in the clinical manifes-
tations of vascular disease.12 A detailed understanding
of the pathophysiology of endothelial dysfunction may
therefore lead to better understanding of and therapy
for atherosclerosis.
In vitro studies indicate that hypercholesterolemia
inhibits the production or biological activity of nitric
Received January 1, 1994; revision accepted March 24, 1994.
From the Cardiology Branch, National Heart, Lung, and Blood
Institute, National Institutes of Health, Bethesda, Md.
Correspondence to Richard 0. Cannon III, MD, Bldg 10, Rm
7B15, National Institutes of Health, Bethesda, MD 20892.
X 1994 American Heart Association, Inc.
Downloaded from http://circ.ahajournals.org/ by guest on March 20, 2015
maximum flow in response to acetylcholine was markedly
impaired in patients compared with healthy subjects (8.0±5.1
versus 17.5±7.7 mL* min-1'* 100 mL-1, P=.002). However, the
maximum forearm flow in response to bradykinin was similar
in the two groups (13.0±4.5 versus 16.2±5.5 mL. min-1l 100
mL-1, P=.14), as was the maximum flow in response to sodium
nitroprusside (7.0±2.8 versus 8.4±2.2 mL. min-1l 100 mL-1,
P=.13). N0-Monomethyl-L-arginine, an inhibitor of nitric ox-
ide synthesis, reduced the maximum forearm vasodilation
induced by bradykinin to the same extent in patients and in
healthy subjects (-29±8% versus -32±6% reduction in peak
flow, P=.80), with similar maximum flows in response to
bradykinin (9.2±4.0 versus 10.4±2.6 mL. min-l. 100 mL-1,
P=.35).
Conclusions Hypercholesterolemic patients are capable of
normal nitric oxide bioavailability in response to bradykinin.
Impairment of microvascular endothelial vasodilator function
in human hypercholesterolemia is selective, and the defect
occurs at the level of the acetylcholine receptor or its signal
transduction pathway. (Circulation. 1994;90:35-41.)
Key Words * cholesterol * bradykinin * acetylcholine .
nitric oxide * G proteins
oxide from endothelial cells of large and small arteries
of animals.13-'9 Recent studies in human hypercholes-
terolemia have shown that the endothelium-dependent
vasodilator responses to acetylcholine or methacholine
and to substance P are impaired in hypercholesterol-
emic patients78101120-23 and that this impairment is at
least in part mediated by a reduced activity of nitric
oxide.22 However, the precise mechanism underlying
this defective endothelial function remains unclear.
Studies of large arteries ex vivo have shown that the
impaired responses to endothelium-dependent vasodi-
lators may be selective. For example, relaxation of
arterial rings from hypercholesterolemic animals or
atherosclerotic humans were found to be impaired in
response to acetylcholine, 5 -hydroxytryptamine, ATP,
thrombin, histamine, and substance p.24-29 In contrast,
the responses to other endothelium-dependent vasodi-
lators such as bradykinin, ADP, norepinephrine, and
A23187 were unaffected, as were responses to endothe-
lium-independent vasodilators.
However, all of these studies were performed on
arterial rings ex vivo, primarily from animal models of
atherosclerosis, and did not assess the impact of endo-
thelium-dependent agonists on blood flow, which pri-
marily reflects microvascular vasomotion. Accordingly,
the present study was designed to determine if impaired
 36 Circulation Vol 90, No 1 July 1994
endothelium-dependent vasodilation in human hyper-
cholesterolemia is due to a selective defect in intracel-
lular pathways linking receptor activation to nitric oxide
production or indicates a generalized abnormality in
endothelial vasodilator function at the level of the
microcirculation in vivo.
Methods
Study Population
Hypercholesterolemic Patients
Patients aged from 18 to 75 years with fasting total choles-
terol levels of .250 mg/dL at the time of screening were
eligible for study. Twelve asymptomatic hypercholesterolemic
patients (five men and seven women aged 50±11 years)
without known atherosclerotic cardiovascular disease were
enrolled. Their lipid profile showed a total cholesterol of
286±35 mg/dL, high-density lipoprotein (HDL) cholesterol of
47±7 mg/dL, triglycerides of 148±62 mg/dL, and low-density
lipoprotein (LDL) cholesterol of 209±38 mg/dL. All patients
were free from hypertension, diabetes, or any other systemic
disease and were not receiving any medication or hormone
replacement therapy. Patients had not taken any cholesterol-
lowering agents within the previous 2 months or any antioxi-
dant vitamin supplements in the preceding 6 months. All
patients had a normal physical examination, resting ECG,
chest radiography, and a normal symptom-limited exercise test
using the standard Bruce protocol.
Healthy Subjects
Twelve healthy volunteer subjects (six men and six women
aged 48±7 years) were also studied. Their lipid profile showed
a total plasma cholesterol of 173 ±27 mg/dL, HDL cholesterol
of 51±12 mg/dL, triglycerides of 78±24 mg/dL, and LDL
cholesterol of 108±28 mg/dL. These subjects were screened by
clinical history, ECG, and blood chemistry to ensure the
absence of cardiovascular or other systemic disease, and they
were not taking any kind of medication, vitamin, or hormone
replacement therapy.
Protocol
The study was approved by the National Heart, Lung, and
Blood Institute Review Board, and all subjects gave written
informed consent. Because bradykinin stimulates the produc-
tion of vasodilating prostanoids,30 all study participants were
administered 2 324-mg aspirin tablets daily for 7 days before
and on the morning of the study to inhibit vascular prostaglan-
din synthesis.31 Alcohol, caffeine, and smoking were prohibited
for 24 hours before the study. A cannula (13/4-in, 20-gauge,
Arrow) was inserted into the brachial artery of the nondomi-
nant arm. A blood sample for lipid profile was obtained.
Forearm blood flow studies were performed using strain-gauge
plethysmography as previously described for our laboratory.9
Briefly, a mercury-filled Silastic strain gauge connected to a
plethysmograph (model EC4, DE Hokanson) that was con-
nected to a chart recorder (Pharmacia LKB, Biotechnology)
was calibrated to measure forearm volume changes. A rapid
cuff inflator (model E10, DE Hokanson) was used to occlude
venous outflow from the extremity, and a wrist cuff was
inflated to 50 mm Hg above systolic pressure 1 minute before
each measurement to exclude the hand circulation. Forearm
blood flow was expressed as milliliters per minute per 100 mL
of forearm volume. Brachial artery pressure was measured
directly from the intra-arterial catheter (model 90308,
Spacelabs). Forearm vascular resistance was calculated as the
mean arterial pressure divided by the forearm blood flow and
is expressed as units.
An intra-arterial infusion of 5% dextrose solution was begun
at 1 mL/min and continued throughout drug infusions. Basal
measurements were obtained 3 minutes after starting the
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infusion. Forearm blood flow was then measured during
intra-arterial infusions of acetylcholine chloride (Sigma Chem-
ical) at 7.5, 15, and 30 ,ug/min; bradykinin (Sigma Chemical) at
100, 200, and 400 ng/min; and sodium nitroprusside at 0.8, 1.6,
and 3.2 jig/min. Infusion rates (0.25, 0.5, and 1 mL/min) were
identical for each drug. Each dose of drug was infused for 5
minutes, and forearm blood flow was measured in the last 2
minutes of each dose. The order of administration of these
drugs was randomized, and a 30-minute rest period ensued
between infusions. After an additional 30-minute rest period,
an infusion of N0-monomethyl-L-arginine (L-NMMA, Calbi-
ochem), an inhibitor of nitric oxide synthesis, replaced the 5%
dextrose infusion at a rate of 4 gmol/min (1 mL). Five minutes
later, resting measurements were repeated, and the dose-
response curves to bradykinin were repeated during concom-
itant L-NMMA infusion.
Statistical Analysis
Differences between two mean values were compared by
paired or unpaired Student's t test, as appropriate. The
forearm flow response to acetylcholine, bradykinin, bradykinin
and L-NMMA in the two groups were compared by ANOVA
for repeated measures using a multiple linear regression
model that included dummy variables to correct for between-
subject variability.32 Because the baseline vascular resistances
differed between hypercholesterolemic patients and healthy
subjects, the relative forearm vascular resistance responses to
these same agonists were analyzed in an identical manner.
Data in the text are expressed as mean+ 1 SD. Error bars on
the figures represent ± 1 SEM. A two-tailed P value of <.05
was accepted as indicating statistical significance.
Results
Vasodilator Responses to Acetylcholine,
Bradykinin, and Sodium Nitroprusside
The forearm vasodilator response to acetylcholine was
significantly lower in hypercholesterolemic patients com-
pared with healthy subjects (Fig 1), with a maximum
forearm flow of 8.0+5.1 mL. min`1100 mL-1 in patients
compared with 17.5 +7.7 mL - min`1 100 ml-- in healthy
subjects (P=.002) (Fig 2). The relative fall iP forearm
vascular resistance with acetylcholine from a -seline of
44.0±26.8 U for patients and 38.4+13.0 U for healthy
subjects was markedly depressed in patients compared
with normal subjects (Fig 1). However, the forearm vaso-
dilator response to bradykinin was similar in both groups
(Fig 3), with the maximum forearm flow of 13.0±4.5
mL. min` * 100 mL-1 in patients versus 16.2±5.5
mL* min` * 100 mL`1 in healthy subjects (Fig 2). The
relative fall in forearm vascular resistance with bradykinin
from a baseline of 52.0±30.8 U for patients and 36.3± 12.9
U for healthy subjects likewise was similar for the two
groups (Fig 3). The maximum flows in response to acetyl-
choline and bradykinin were similar for healthy subjects
(17.5±7.7 versus 16.2±5.5 mL* min` * 100 mL-1, P=.32).
In contrast, the maximum flow in response to acetylcho-
line was significantly lower than the maximum flow in
response to bradykinin in hypercholesterolemic patients
(8.0±5.1 versus 13.0±4.5 mL * min` * 100 mL-1, P=.01).
The forearm flow response to sodium nitroprusside
was significantly greater for healthy subjects than for
patients (Fig 4), although the maximum forearm flow
was similar (7.0±2.2 mL* min'* 100 mL`1 in patients
versus 8.4±2.2 mL * min'. 100 mL-1 in healthy sub-
jects, P=.13). The relative fall in forearm vascular
resistance with sodium nitroprusside from a baseline of
 Gilligan et al Endothelial Function in Hypercholesterolemia 37

o-o Normal Subjects Acetylcholine Response

*-* Hypercholesterolemic Patients 30 r

25 1
20
- 20
3: E
0 o

15 LL0
C) ._7
E
W-
o E
0 0 E 10
0
c 10 5
0
,o
0
i-. 0
5
Bradykinin Response
30 r
0 L

Baseline 7.5 15.0 30.0 25 h

P=NS
3 20-
oc)
0 u- 0
0 - 15-
0 0 r-
tn ) ,E
Cd0 mf t-

CD -20
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(D
1._ 5

0 0
E
-40
0
Baseline Maximum Baseline Maximum
h-
0 Co Flow Row
to (D -60 Normal Subjects Hypercholesterolemic
0) Paffents
FIG 2. Plots of maximum forearm blood flows in response to
c0s
-80 acetylcholine (top) and to bradykinin (bottom) are shown for
0 0 healthy subjects and hypercholesterolemic patients.
0
LL -100
7.5 15.0 30.0 flow, -29+8% versus -32+6%, P=.80), with the max-
imum forearm flow of 9.2±4.0 mL * min'1 100 mL-' in
Acetylcholine. patients versus 10.5±2.6 mL. min- * 100 mL`' in
healthy subjects (Fig 5). There were no differences in
(ug/min) the reduction in forearm flow or the increase in vascular
FIG 1. Plots of forearm blood flow (top) and relative fall in resistance during simultaneous bradykinin and
forearm vascular resistance from baseline (bottom) in response L-NMMA infusions compared with infusion of brady-
to serial doses of acetylcholine in 12 healthy subjects (o) and 12
hypercholesterolemic patients (.). Values represent mean and kinin alone for the two groups (Fig 6).
SEM. The P values refer to comparison of the two curves by
ANOVA for repeated measures. Discussion
The present study confirms that forearm vascular
45.5±15.7 U for patients and 33.4±12.5 U for healthy response to the endothelium-dependent vasodilator
subjects was similar for the two groups (Fig 4). acetylcholine is depressed in human hypercholesterol-
emia.19-22 We have previously shown that nitric oxide
Effect of Nitric Oxide Inhibition on bioavailability is depressed in response to acetylcholine
Bradykinin Response in hypercholesterolemic patients.22 In the present study,
L-NMMA resulted in decreases in basal forearm flow the forearm vascular response to bradykinin, an endo-
in healthy subjects (2.4±0.8 to 2.1±0.6 mL min-1 100 thelium-dependent vasodilator that uses a different
mL-1 and in hypercholesterolemic patients (2.4±1.1 to signal transduction pathway than that of acetylcholine,
1.8±0.7 mL- min-1 * 100 mL-1) and in increases in vascu- was similar in hypercholesterolemic patients and
lar resistance (52.4±46.6 to 56.0±22.4 U for hypercholes- healthy subjects. Although the maximum forearm flows
terolemic patients and 38.5+9.3 to 45.9±13.3 U for in response to acetylcholine and bradykinin were similar
healthy subjects). There was no difference between the in healthy subjects, in hypercholesterolemic patients the
change in basal forearm flow (P=.53) or in forearm maximum flow in response to bradykinin was signifi-
resistance (P=.70) in response to L-NMMA between cantly greater than the maximum flow in response to
hypercholesterolemic patients and healthy subjects. acetylcholine. The impact of L-NMMA, an antagonist
L-NMMA resulted in a similar reduction in forearm of nitric oxide production, on basal forearm flow and on
flow response to bradykinin in hypercholesterolemic forearm vascular responses to bradykinin was similar in
patients and in healthy subjects (reduction in maximum hypercholesterolemic patients and in healthy subjects.
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38 Circulation Vol 90, No 1 July 1994

0-0 Normal Subjects o-o Normal Subjects

_- Hypercholesterolemic Patients ~ Hypercholesterolemic Patients
20 r
20
p=0.073
15 E
15 [
0 E 0
0
p=0.005
0 *0 0
E-
0 0
10
I-, 10 I
100 ._ 0
,o
0 E
0 E
t-.
'E 5
5
a/,"

0l O
Baseline 100 200 400 Baseline 0.8 1.6 3.2

0 0
CD 0
0 cJ
C.
c
ci 0
c. CD 0

0 ii>i -20 C.
c
_ -20
0
0 0u co
0 0

cr. .0

E -40
0 (c 0

C)
(D
Co
m
0
m -60 p=0.56 0 E
CD
-40
-60
0 4-
0
0
0
m) -80 0 -80
(D
L-
b..

0
U.
-100
100 200 400
Bradykinin
(ng/min)
FIG 3. Plots of forearm blood flow (top) and relative fall in
forearm vascular resistance from baseline (bottom) in response
to serial doses of bradykinin in the healthy subjects and hyper-
cholesterolemic patients. Values represent mean and SEM.
The maximum forearm flow in response to the endothe-
lium-independent vasodilator sodium nitroprusside was
similar for healthy subjects and hypercholesterolemic
patients, as was the relative decrease in forearm vascu-
lar resistance, indicating unimpaired vascular smooth
muscle responsiveness in hypercholesterolemic patients.
These findings indicate a selective impairment in the
acetylcholine receptor-activated signal transduction
pathway in hypercholesterolemic patients, with preser-
vation of normal biological availability of nitric oxide via
alternative pathways as evidenced by similar decreases
in forearm flow and increases in resistance in response
to bradykinin after inhibition of nitric oxide production
for patients and healthy subjects.
Several groups have previously reported that the
endothelium in large arteries from hypercholesterol-
emic animals may respond to certain endothelium-
dependent vasodilator agonists.23-25 Shimokawa et a128
showed that the pertussis toxin-sensitive Gi protein-
Downloaded from http://circ.ahajournals.org/ by guest on March 20, 2015
-100
0.8 1.6 3.2
Sodium Nitroprusside
(pg/min)
FIG 4. Plots of forearm blood flow (top) and relative fall in
forearm vascular resistance from baseline (bottom) in response
to serial doses of sodium nitroprusside in the healthy subjects
and hypercholesterolemic patients. Values represent mean and
SEM.
dependent signal transduction pathway linked to adenyl
cyclase is impaired in hypercholesterolemia with early
atherosclerosis at a time when other signal transduction
mechanisms are intact. Bradykinin is an endogenous,
locally acting hormone that has been demonstrated to
have an endothelium-dependent vasodilator action,
with the bradykinin receptor linked by a pertussis
toxin-insensitive Gq protein-dependent signal trans-
duction pathway to phospholipase C.33 In our study, we
found that endothelium-dependent vasodilation to
bradykinin is preserved in the microcirculation of hy-
percholesterolemic patients at a time when the acetyl-
choline response is markedly depressed. Thus, the data
indicate that the depression of endothelial responsive-
ness in human hypercholesterolemia without clinically
apparent atherosclerotic disease is selective for certain
agonists and signal transduction pathways and is not
generalized.
 Gilligan et al Endothelial Function in Hypercholesterolemia 39

20

30 16
lL -

0 c 12
00

E WEE 8
0 E
0) -

0
LL 4

V 70 r
n)
c

CK
(U
(0 56 -
U)

G :) 42 1
:3 ._

g _ 28 I
>

E 14
I)
0
0
LL nn
Baseline 100 200 400 Baseline 100 200 400

Bradykinin (ng/min) Bradykinin (ng/min)

NORMAL SUBJECTS HYPERCHOLESTEROLEM IC

PATI ENTS
FIG 5. Plots of forearm blood flow (top) and resistance (bottom) in response to serial doses of bradykinin during intra-arterial infusion
of D5W (o) and NG_monomethyi-L-arginine (L-NMMA, *) in healthy subjects and hypercholesterolemic patients. Values represent mean
and SEM.

Agonist stimulation of the endothelium results in
production of endothelium-derived relaxing factors, in-
cluding nitric oxide, prostacyclin, and endothelium-
derived hyperpolarizing factor.12 One possible mecha-
nism by which the endothelium-dependent vasodilation
to bradykinin was preserved in our hypercholesterol-
emic patients was that the release of other endotheli-
um-derived relaxing factors was increased to compen-
sate for an impairment of nitric oxide production,
despite administration of aspirin to inhibit prostacyclin
production. However, L-NMMA increased basal resis-
tance and inhibited the vasodilator response to brady-
kinin to a similar degree in patients and in healthy
subjects, indicating that hypercholesterolemic patients
had normal nitric oxide bioavailability in the basal state
and in response to bradykinin. We have previously
shown that the contribution of nitric oxide to the
acetylcholine-mediated vasodilator response is de-
pressed in hypercholesterolemic patients compared
with healthy subjects.22 Thus, depression in nitric oxide
bioavailability appears to be selective to acetylcholine
and not bradykinin signal transduction pathways in
hypercholesterolemic patients.
Minor et a129 reported increased nitrogen oxide pro-
duction in atherosclerotic rabbit aorta with evidence for
degradation of nitric oxide to biologically inactive com-
pounds. Because we did not measure production of
biologically active and inactive nitrogen oxide com-
pounds in our study, we cannot exclude the possibility of
increased production of nitric oxide in the hypercholes-
terolemic patients, balanced with its partial degradation
by the action of superoxide anions. Thus, it is possible
that the normal basal and bradykinin-stimulated nitric
oxide bioavailability in hypercholesterolemic patients
indicated by our data represents a balance between
increased production and degradation of nitric oxide.
Certainly, our data are incompatible with decreased
basal and bradykinin-stimulated production of nitric
oxide in hypercholesterolemic patients. Our study dif-
fered from that of Minor et al in that we studied a
vascular bed probably free of atherosclerosis, albeit
vulnerable to the effects of hypercholesterolemia, in a
group of patients free of clinically apparent atheroscle-
rosis. Accordingly, studies of vascular responses to
bradykinin and L-NMMA in atherosclerotic arteries of
humans (eg, the coronary circulation) and studies of the
effect of inhibitors of superoxide anion production on
endothelium-dependent relaxation would be of interest.
The data from this study are compatible with a
selective defect of endothelium-dependent vasodilation
and nitric oxide bioavailability at the level of the
pertussis toxin-sensitive Gi protein-dependent signal
transduction pathway in the microcirculation of hyper-
cholesterolemic patients. Potential mechanisms respon-
sible for endothelial dysfunction in hypercholesterol-
emia have been reviewed recently,34 including inhibition
of nitric oxide production by oxidatively modified LDL,
an effect linked to lysolecithin in the oxidized particle,3

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40 Circulation Vol 90, No 1 July 1994

o-o Normal Subjects 5. Palmer RM, Ashton DS, Moncada S. Vascular endothelial cells
synthesize nitric oxide from L-arginine. Nature. 1988;333:664-666.
*-a Hypercholesterolemic Patients 6. Ludmer PL, Selwyn AP, Shook TL, Wayne RR, Mudge GH,
Alexander RW, Ganz P. Paradoxical vasoconstriction induced by
0 F acetylcholine in atherosclerotic coronary arteries. N Engl J Med.
1986;315:1046-1051.
7. Drexler H, Zeiher AM. Endothelial function in human coronary
10 F
p=0.49 arteries in vivo: focus on hypercholesterolemia. Hypertension. 1991;
18(suppl II):II-90-II-99.
a, 8. Zeiher AM, Drexler H, Wollschlager H, Just H. Modulation of
(D coronary vasomotor tone in humans: progressive endothelial dys-
z
20 function with different early stages of coronary atherosclerosis.
0
C)
Circulation. 1991;83:391-401.
9. Panza JA, Quyyumi AA, Brush JE Jr, Epstein SE. Abnormal endo-
0) 30 F thelium-dependent vascular relaxation in patients with essential
0~
hypertension. N EnglJMed. 1990;323:22-27.
10. Creager MA, Cooke JP, Mendelsohn ME, Gallagher SJ, Coleman
IL 40 F
SM, Loscalzo J, Dzau VJ. Impaired vasodilation of forearm
resistance vessels in hypercholesterolemic humans. J Clin Invest.
1990;86:228-234.
11. Vita JA, Treasure CB, Nabel EG, McLenachan JM, Fish D, Yeung
50 j
AC, Vekshtein VI, Selwyn AP, Ganz P. Coronary vasomotor
100 200 400 response to acetylcholine relates to risk factors for coronary artery
disease. Circulation. 1990;81:491-497.
12. Dzau VJ, Gibbons GH, Cooke JP, Omoigui N. Vascular biology
p=0.63 and medicine in the 1990s: scope, concepts, potentials, and per-
100 F
spectives. Circulation. 1993;87:705-719.
13. Freiman PC, Mitchell GD, Heistad DD, Armstrong ML, Harrison
DG. Atherosclerosis impairs endothelium-dependent vascular
._ 2 80 relaxation to acetylcholine and thrombin in primates. Circ Res.
Z 1986;58:782-789.
14. Verbeuren TJ, Jordaens FH, Zonnekeyn LL, Van Hove CE, Coene
60 M-C, Herman AG. Effect of hypercholesterolemia on vascular
reactivity in the rabbit: I. Endothelium-dependent and endotheli-
um-independent contractions and relaxations in isolated arteries of
control and hypercholesterolemic rabbits. Circ Res. 1986;58:
CD
C.) 40 552-564.
c
C 15. Habib JB, Bossaler C, Wells S, Williams C, Morrisett JD, Henry
h-
a) 0) PD. Preservation of endothelium-dependent vascular relaxation in
CL CO) 20 cholesterol-fed rabbit by treatment with the calcium channel
blocker PN 200110. Circ Res. 1986;58:305-309.
16. Andrews HE, Bruckdorfer KR, Dunn RC, Jacobs M. Low-density
0 lipoproteins inhibit endothelium-dependent relaxation in rabbit
100 200 400 aorta. Nature. 1987;327:237-239.
17. Osborne JA, Siegman MJ, Sedar AW, Mooers SU, Lefer AM. Lack
Bradykinin of endothelium-dependent relaxation in coronary resistance
arteries of cholesterol-fed rabbits. Am J Physiol. 1989;256:C-591-
(ng/min) C-597.
18. Seilke FW, Armstrong ML, Harrison DG. Endothelium-dependent
FIG 6. Plots of changes in the vascular response to bradykinin vascular relaxation is abnormal in the coronary microcirculation of
induced by NG_monomethyl-L-arginine (L-NMMA) expressed as atherosclerotic primates. Circulation. 1990;81:1586-1593.
percent decrease in forearm flow and percent increase in 19. Kuo L, Davis MJ, Cannon MS, Chilian WM. Pathophysiological
vascular resistance in healthy subjects and in hypercholesterol- consequences of atherosclerosis extend into the coronary micro-
emic patients. Values represent mean and SEM. circulation: restoration of endothelium-dependent responses by
L-arginine. Circ Res. 1992;70:465-476.
20. Creager MA, Gallagher SJ, Girerd XJ, Coleman SM, Dzau VJ,
and conversion of nitric oxides to biologically inactive Cooke JP: L-Arginine improves endothelium-dependent vasodi-
nitrogen oxide compounds by the action of superoxide lation in hypercholesterolemic humans. J Clin Invest. 1992;90:
anions.29 These findings give incentive to further study 1248-1253.
of how lipoproteins interfere with endothelial cell mem- 21. Chowienczyk PJ, Watts GF, Cockcroft JR, Ritter JM. Impaired
endothelium-dependent vasodilation of forearm resistance vessels
brane signaling and provide an impetus to develop in hypercholesterolemia. Lancet. 1992;340:1430-1432.
specific interventions to prevent or correct these ad- 22. Casino PR, Kilcoyne CM, Quyyumi AA, Hoeg JM, Panza JA. The
verse effects on endothelial vasodilator function. role of nitric oxide in the endothelium-dependent vasodilation of
hypercholesterolemic patients. Circulation. 1993;88:2541-2547.
References 23. Casino PR, Kilcoyne CM, Cannon RO, Giuligan DM, Quyyumi
AQ, Panza JA. Impaired endothelium-dependent relaxation in
1. Furchgott RF, Zawadzki JV. The obligatory role of the endothelial patients with hypercholesterolemia extends beyond the muscarinic
cells in the relaxation of arterial smooth muscle by acetylcholine. receptor. Circulation. 1993;88(suppl):I-618. Abstract.
Nature. 1980;288:373-376. 24. Bossaler C, Habib GB, Yamamoto H, Williams C, Wells S, Henry
2. Cocks TM, Angus JA. Endothelium-dependent relaxation of PD. Impaired muscarinic endothelium-dependent relaxation and
coronary arteries by noradrenaline and serotonin. Nature. 1983; cyclic guanosine 5-monophosphate formation in atherosclerotic
305:627-630. human coronary artery and rabbit aorta. J Clin Invest. 1987;79:
3. Furchgott RF. Role of endothelium in responses of vascular 170-174.
smooth muscle. Circ Res. 1983;53:557-573. 25. Shimokawa H, Vanhoutte PM. Impaired endothelium-dependent
4. Palmer RM, Ferrige AG, Moncada S. Nitric oxide release accounts relaxation to aggregating platelets and related vasoactive sub-
for the biological activity of endothelium-derived relaxing factor. stances in porcine coronary arteries in hypercholesterolemia and
Nature. 1987;317:524-526. atherosclerosis. Circ Res. 1989;67:900-914.

Downloaded from http://circ.ahajournals.org/ by guest on March 20, 2015

 Gilligan et al Endothelial Function in Hypercholesterolemia 41

26. Cohen RA, Zitnay KM, Haudenschild CC, Cunningham LD.
Loss of selective endothelial cell vasoactive functions caused by
hypercholesterolemia in pig coronary arteries. Circ Res. 1988;63:
903-910.
27. Komori K, Shimokawa H, Vanhoutte PM. Hypercholesterolemia
impairs endothelium-dependent relaxations to aggregating
platelets in porcine iliac arteries. J Vasc Surg. 1989;10:318-325.
28. Shimokawa H, Flavahan NA, Vanhoutte PM. Loss of endothelial
pertussis toxin-sensitive G protein function in atherosclerotic
porcine coronary arteries. Circulation. 1991;83:652-660.
29. Minor RLJ, Myers PR, Guerra R, Bates JN, Harrison DG. Diet-
induced atherosclerosis increases the release of nitrogen oxides
from rabbit aorta. J Clin Invest. 1990;86:2109-2116.
30. Hong SL. Effect of bradykinin and thrombin on prostacyclin syn-
thesis in endothelial cells from calf and pig aorta and human
umbilical vein. Thromb Res. 1980;18:787-795.
31. Weksler BB, Pett SB, Alonso D, Richter RC, Stelzer P, Subra-
manian V, Tack-Goldman K, Gay WA. Differential inhibition by
aspirin of vascular and platelet prostaglandin synthesis in athero-
sclerotic patients. N Engl J Med. 1983;308:800-805.
32. Glantz SA, Slinker BK. Repeated measures. In: Glantz SA, Slinker
BK, eds. Primer of Applied Regression and Analysis of Variance.
New York: McGraw-Hill; 1990:381-463.
33. Flavahan NA, Shimokawa H, Vanhoutte PM. Pertussis toxin
inhibits endothelium-dependent relaxation to certain agonists in
porcine coronary arteries. J Physiol. 1989;408:549-560.
34. Flavahan NA. Atherosclerosis or lipoprotein-induced endothelial
dysfunction: potential mechanisms underlying reduction in EDRF/
nitric oxide activity. Circulation. 1992;85:1927-1938.
35. Kugiyama K, Kerns SA, Morrisett JD, Roberts R, Henry PD.
Impairment of endothelium-dependent relaxation by lysolecithin
in modified low-density lipoproteins. Nature. 1990;344:160-162.

Downloaded from http://circ.ahajournals.org/ by guest on March 20, 2015

 Selective loss of microvascular endothelial function in human hypercholesterolemia.
D M Gilligan, V Guetta, J A Panza, C E García, A A Quyyumi and R O Cannon, 3rd

Circulation. 1994;90:35-41
doi: 10.1161/01.CIR.90.1.35
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