Circulation Research

PERIPHERAL VASCULAR DISEASE COMPENDIUM

New Directions in Therapeutic Angiogenesis and

Arteriogenesis in Peripheral Arterial Disease
Brian H. Annex , John P. Cooke

ABSTRACT: The prevalence of peripheral arterial disease (PAD) in the United States exceeds 10 million people, and PAD is a
significant cause of morbidity and mortality across the globe. PAD is typically caused by atherosclerotic obstructions in the
large arteries to the leg(s). The most common clinical consequences of PAD include pain on walking (claudication), impaired
functional capacity, pain at rest, and loss of tissue integrity in the distal limbs that may lead to lower extremity amputation.
Patients with PAD also have higher than expected rates of myocardial infarction, stroke, and cardiovascular death. Despite
advances in surgical and endovascular procedures, revascularization procedures may be suboptimal in relieving symptoms,
and some patients with PAD cannot be treated because of comorbid conditions. In some cases, relieving obstructive
disease in the large conduit arteries does not assure complete limb salvage because of severe microvascular disease.
Despite several decades of investigational efforts, medical therapies to improve perfusion to the distal limb are of limited
benefit. Whereas recent studies of anticoagulant (eg, rivaroxaban) and intensive lipid lowering (such as PCSK9 [proprotein
convertase subtilisin/kexin type 9] inhibitors) have reduced major cardiovascular and limb events in PAD populations, chronic
ischemia of the limb remains largely resistant to medical therapy. Experimental approaches to improve limb outcomes have
included the administration of angiogenic cytokines (either as recombinant protein or as gene therapy) as well as cell
therapy. Although early angiogenesis and cell therapy studies were promising, these studies lacked sufficient control groups
and larger randomized clinical trials have yet to achieve significant benefit. This review will focus on what has been learned
to advance medical revascularization for PAD and how that information might lead to novel approaches for therapeutic
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angiogenesis and arteriogenesis for PAD.

Key Words: cytokines ◼ metabolism ◼ microvessels ◼ permeability ◼ stem cells

THE PERVASIVE AND PERSISTENT
PROBLEMS OF PERIPHERAL ARTERIAL
DISEASE
Systemic atherosclerosis remains the number one
cause of morbidity and mortality in the western world
and is the major cause of peripheral arterial dis-
ease (PAD), causing obstructions in blood flow in
one or more of the major leg arteries.1–4 Estimates
place the prevalence of PAD at over 14 million in the
United States and >200 million people worldwide.1–3,5
In patients, PAD is still defined by the finding of an
ankle-brachial blood pressure index of 0.90 or less.5–7
Although the risk factors for the development of PAD
overlap those of coronary artery and cerebrovascular
disease, diabetes and smoking have been and remain
the 2 strongest age-adjusted risk factors for the devel-
opment of PAD.1,5,7 Patients with PAD continue to suf-
fer excessively high risks of heart attack and stroke
when compared with patients without PAD.8–10
The 2 major symptomatic manifestations of PAD are
intermittent claudication (IC) and critical limb ischemia
(CLI).1,8,11 IC is the most prevalent clinical manifestation in
patients with PAD and is defined by the presence of calf
or buttock muscle discomfort produced by exercise that is
relieved by rest.12 CLI is the most severe manifestation of
PAD and is characterized by one or more of the following:
leg pain occurring at rest, ulceration, and gangrene.11,13

Correspondence to: Brian H. Annex, MD, J. Harold Harrison, MD, Joseph C. “Rusty” Walter and Carol Walter Looke Presidential Distinguised Chair, Chair,
Department of Medicine, Medical College of Georgia, Augusta University, 1120 15th St BI 5076, Augusta, GA 30912, Email bannex@augusta.edu or John P.
Cooke, MD, Department of Cardiovascular Sciences, Houston Methodist Research Institute, 6670 Bertner Ave, Mail Stop R10-South, Houston, TX 77030, Email
jpcooke@houstonmethodist.org
For Sources of Funding and Disclosures, see page 1953.
© 2021 American Heart Association, Inc.
Circulation Research is available at www.ahajournals.org/journal/res

1944   June 11, 2021 Circulation Research. 2021;128:1944–1957. DOI: 10.1161/CIRCRESAHA.121.318266

 Annex and Cooke Vascular Regeneration in PAD

with PAD are similar to the impairment seen in patients

DISEASE COMPENDIUM
PERIPHERAL VASCULAR
Nonstandard Abbreviations and Acronyms with advanced congestive heart failure.14,15
Currently, medical therapies used to treat patients
ACE angiotensin-converting enzyme with PAD include antiplatelet and antithrombotic agents,
ARB angiotensin receptor blocker statins to lower cholesterol, antihypertensive therapy
CCR2 C-C chemokine receptor 2 with ACE (angiotensin-converting enzyme) inhibitors or
CLI critical limb ischemia ARB (angiotensin receptor blocker) or β-blockers, blood
CXCR-4 C-X-C chemokine receptor type 4 glucose control, and smoking cessation. These thera-
Del-1 developmental endothelial locus 1
pies were largely established from treatments used for
patients with coronary artery disease. In patients with
EC endothelial cell
PAD, these agents may prevent heart attack and stroke,
eNOS endothelial nitric oxide synthase
but clinical cardiovascular event rates in patients with
FGF fibroblast growth factor PAD remain high, and even new anticoagulant therapies
G-CSF granulocyte-colony stimulating factor and intensive lipid-lowering therapies have not consis-
GM-CSF granulocyte macrophage-colony stimu- tently altered the progression of disabling claudication
lating factor or limb loss.1,9,10 These medical therapies do not improve
HGF hepatocyte growth factor lower limb perfusion in patients with PAD.16 Surgical or
HIF-1 hypoxia-inducible factor 1 endovascular procedures may be suboptimal in reliev-
HLI hindlimb ischemia ing symptoms and may not be indicated because of the
HSS hypoxia and serum starvation extent of disease or because of comorbid conditions.
IC intermittent claudication Finally, even after successful large vessel revasculariza-
ICAM intercellular adhesion molecule tion, residual microvascular disease may well limit the
IL interleukin effectiveness of therapies in patients with PAD.11,13,16 Still
iNOS inducible nitric oxide synthase
today PAD effects both legs and life. Few areas in cardio-
vascular medicine are more in need of new therapeutic
iPSCs induced pluripotent stem cells
modalities than PAD.
lncRNA long noncoding RNA
PAD peripheral arterial disease
PCSK-9 proprotein convertase subtilisin/kexin
ANGIOGENESIS, ARTERIOGENESIS,
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type 9
PDGF platelet-derived growth factor
PFK-1 phosphofructokinase 1
SDF-1 stromal-derived factor 1
TNF tumor necrosis factor
VEGF vascular endothelial growth factor
Of critical importance, classic claudication symptoms
actually occur in only a minority of patients with PAD,
and this has been shown in those with a confirmed low
ankle-brachial blood pressure index.6,7,10 Despite its high
disease prevalence, the low ankle-brachial blood pres-
sure index is not sufficiently used, and because symp-
toms may be atypical or absent in many patients, PAD
often goes unrecognized and undertreated.2–4 Of critical
importance is that all patients with PAD, even those who
lack symptoms, have significant disability with limita-
tions in walking capacity, daily functional activities, and
exercise performance.14,15 The clinical course of patients
with PAD is strikingly different based upon whether the
patients present with IC or CLI. Patients with CLI suffer
rates of death and major amputation of 30% or more by
1-year, whereas over a 5-year period, only 1.0% to 3.3%
of IC patients will require amputation.1,8–10 Even in the
absence of CLI, the functional limitations seen in patients
VASCULOGENESIS, AND PAD
Within the adult, the overriding purpose of the vasculature
is to regulate blood flow (delivery of oxygen and nutrients
along with the removal of toxins) to organs to meet the
metabolic demands of active cells. The central problem in
PAD is the limitation of perfusion to the muscle and soft
tissue of the leg(s). It would, therefore, seem rational to
attempt to improve perfusion in PAD by enhancing vascu-
lar regeneration and function. Angiogenesis is defined as
the growth and proliferation of blood vessels from exist-
ing vascular structures.17 Angiogenesis is, of course, criti-
cal in embryonic development. In adults, angiogenesis is
required for tissue repair or remodeling, as well as resto-
ration of tissue perfusion.18 Angiogenesis can be physi-
ologically regulated such as in the response to exercise
training. A major process in angiogenesis is the sprout-
ing of endothelial cells (ECs) from preexisting capillaries
under the regulation of angiogenic factors such as VEGF
(vascular endothelial growth factor) generated by isch-
emic tissue (Figure 1). The sprouting ECs migrate, pro-
liferate, and form lumens while other processes that may
help to generate a functional microvasculature include
intussusception of existing capillaries,17,18 the incorpo-
ration of circulating endothelial progenitors,19,20 and the
generation of cytokines by circulating angiogenic cells.21

Circulation Research. 2021;128:1944–1957. DOI: 10.1161/CIRCRESAHA.121.318266 June 11, 2021   1945

 Annex and Cooke
DISEASE COMPENDIUM
PERIPHERAL VASCULAR
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Figure 1. Processes to restore perfusion in peripheral arterial
disease (PAD).
In angiogenesis, new microvasculature is derived from the sprouting
of preexisting capillaries under the influence of VEGF (vascular
endothelial growth factor), FGF (fibroblast growth factor), and other
angiogenic cytokines. Arteriogenesis is the positive remodeling
of preexisting collaterals when blood flow is redirected through
these channels. The positive remodeling of these channels is due,
in part, to the mobilization of monocytes by G-CSF (granulocyte-
colony stimulating factor) and vascular expression of monocyte
chemotactic protein, the ligand for the monocyte CCR2 (C-C
chemokine receptor type 2). Circulating angiogenic cells (CACs)
are also mobilized by G-CSF to participate in adult vasculogenesis.
These cells express CXCR-4 (C-X-C chemokine receptor type 4),
the receptor for SDF-1 (stromal-derived factor 1) (Continued )
1946   June 11, 2021 Circulation Research. 2021;128:1944–1957. DOI: 10.1161/CIRCRESAHA.121.318266
Vascular Regeneration in PAD
Pathological angiogenesis may support another
pathological process and examples would include
tumor angiogenesis, atherosclerotic plaque neovas-
cularization, or age-related macular degeneration. In
each of these tobacco-related conditions, nicotinic
acetylcholine receptors on the endothelium are stimu-
lated by exogenous nicotine to proliferate, migrate, and
form a microvasculature that supports the underlying
pathological process.22,23 Another form of pathologi-
cal angiogenesis is when the angiogenic response to
ischemia is inadequate to meet tissue demands, and
this most commonly occurs in the settings of coronary
artery disease and PAD. Here, hypoxia in heart and
leg muscles due to poor tissue perfusion activates the
transcription factor HIF-1 (hypoxia-inducible factor 1)
as its subunit HIF-1α has a degradation domain that
is dependent upon oxygen tension.24 With low oxygen
levels, HIF-1α is stabilized, translocates to the nucleus,
and activates target genes including VEGF, FGF (fibro-
blast growth factor), HGF (hepatocyte growth factor),
PDGF (platelet-derived growth factor), Del-1 (develop-
mental endothelial locus 1), angiopoietins, and matrix
metalloproteinases to facilitate angiogenesis.25 Notably,
exogenous administration of each of these genes or the
proteins that they encode, have been shown to improve
limb blood flow in animal models of PAD,26–31 but as
discussed below, have failed in randomized clinical trials
to significantly ameliorate PAD.
The remodeling of preexisting collateral channels is
termed arteriogenesis. In their normal state, these col-
lateral channels are narrow, high resistance vessels, and
they provide little blood flow to their distal tissue bed.
However, when a major conduit becomes obstructed,
blood flow is redirected through the collateral channels
(Figure 1) which causes alterations in vascular wall shear
stress. This hemodynamic stimulus provokes an increase
in the diameter and wall thickness of the collateral chan-
nels, with proliferation of vascular cells and turnover of
the vascular matrix.32,33 Other factors involved in arterio-
genesis include chemokines and adhesion molecules,
such as ICAM (intercellular adhesion molecule), CCR2
(C-C chemokine receptor 2), Delta-like 1, SDF-1 (stro-
mal-derived factor 1), which recruit monocytes that then
facilitate the positive remodeling by elaborating metal-
loproteinases and cytokines..34–37 Whereas macrophages
are the cell most linked to arteriogenesis, recent data
have also linked inflammatory (referred to as M1-like)
Figure 1 Continued. that is expressed by the ischemic tissue.
Most CACs are of hematopoietic origin and contribute to restoration
of perfusion by secreting angiogenic cytokines. Rare CACs are
of endothelial lineage and can incorporate into the newly forming
vessels. Transdifferentiation of fibroblasts to endothelial cells may
also contribute to angiogenesis. In the setting of inflammatory
signaling, increased DNA accessibility permits fluidity of cell fate,
and environmental influences such as hypoxia activate transcription
factors determining endothelial lineage.
 Annex and Cooke
macrophages in processes that negatively impact angio-
genesis and which may be a target for therapeutics.38–40
Inflammatory macrophages in ischemic tissue may be a
source for circulating cytokines (ie, TNF [tumor necrosis
factor]-α and IL [interleukin]-6) that can trigger acute
cardiovascular events.39,41
Adult vasculogenesis refers to the mobilization of
circulating cells with angiogenic potential from the
bone marrow into the circulation by factors, such as
G-CSF (granulocyte-colony stimulating factor) and
GM-CSF (granulocyte macrophage-colony stimulating
factor). These CD34+ circulating angiogenic cells may
also express CD133, VEGFR2, and CXCR-4 (C-X-C
chemokine receptor type 4; Figure 1).42–45 Under the
influence of SDF-1 (which binds to the chemokine
receptor CXCR-4), angiogenic cells are recruited to
the ischemic tissue.46,47 Bone marrow-derived angio-
genic cells are largely of hematopoietic lineage and
largely improve perfusion by secreting cytokines and
metalloproteinases, although a rare fraction may differ-
entiate into mature endothelium in vivo.19,48,49 It is also
possible that angiogenic cytokines cause mature ECs
in other nonischemic organs to be mobilized into the
circulation, home to ischemic tissue, and contribute to
angiogenesis.19,50
In patients with PAD, there is some degree of
endogenous arteriogenesis (and angiogenesis), but
the capacity of the processes even when active are
limited in their ability to restore limb perfusion to nor-
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mal levels.51 In patients with claudication, capillary
density is often reduced and is related to a patients’
functional capacity.52 Notably, an increase in capillary
density is observed in the limb skeletal muscle of PAD
patients with exercise training, which is still today the
cornerstone of therapy in patients with PAD.53 Moving
from concept to the clinic, we hope to harness blood
vessel growth to improve perfusion to ischemic tis-
sue, an approach which has been termed therapeutic
angiogenesis or medical revascularization. Perhaps,
a better term is vascular regeneration, which encom-
passes angiogenesis, arteriogenesis, and vasculo-
genesis, the combination of which processes may be
necessary for substantial benefit. Investigation into
therapeutic angiogenesis dates back to the work of
the late Isner et al,54 who administered by intraarte-
rial infusion a plasmid vector encoding VEGF to treat
a 70-year-old female with midfoot 3-vessel runoff
occlusion and digit gangrene. Their work lacked a suf-
ficient control group to draw conclusions but stimu-
lated a great number of clinical trials of therapeutic
angiogenesis involving thousands of patients, with
varying experimental protocols, agents, and modes
of delivery. Regrettably, these studies have failed to
deliver significantly positive results, as previously
reviewed.16,55–57 Thus, new approaches are needed for
vascular regeneration in PAD.
Circulation Research. 2021;128:1944–1957. DOI: 10.1161/CIRCRESAHA.121.318266
Vascular Regeneration in PAD
NEW DIRECTIONS FOR POTENTIAL
DISEASE COMPENDIUM
PERIPHERAL VASCULAR
THERAPEUTIC SOLUTIONS IN PAD
Angiogenesis Independent of VEGF
To date a substantial majority of the completed human
therapeutic angiogenesis trials, in whole or in part, sought
to activate the well described, canonical, VEGF-VEGFR2/
Akt-1/eNOS (endothelial nitric oxide synthase) pathway;
where increases in bioavailable nitric oxide is a critical
mediator.58–60 Promoting angiogenesis independent of
this pathway may be important for humans with PAD as
these patients frequently have comorbid conditions (even
diabetes which is a frequently causes and modifies PAD)
which can limit the generation, or bioavailability, of NO.16
Preclinical models of PAD remain an invaluable tool for
testing the potential efficacy and bioactivity of putative
therapeutic agents as well as to identify novel therapeu-
tics. Using established differences in the extent of perfu-
sion recovery and necrosis that occurs across informative
inbred mouse strains and congenic lines, Dokun et al61
were the first to identify a quantitative trait locus on the
short arm of mouse chromosome 7 (termed LSq-1) that
contained potential gene(s) that were sufficient to deter-
mine the extent of perfusion recovery and tissue loss after
hindlimb ischemia (HLI). This was an unbiased approach
and had the advantage of allowing us to look for genes
that modified the course of PAD in the setting of compara-
ble occlusions.16,61 More importantly, this strategy had the
advantage of not requiring that we preselect for known
angiogenesic or arteriogenic genes or pathways. One of
the first candidates to emerge from this approach was the
IL-21R (IL-21 receptor) which, resides at/very near the
peak of genetic linkage to variations in perfusion recovery
and necrosis in the mouse model of PAD and the potential
role of this receptor in modulating angiogenesis in the set-
ting of PAD has supporting human data.62,63
The IL-21R was identified about 2 decades ago in
the laboratory of Dr Warren J. Leonard at the National
Institutes of Health/National Heart, Lung, and Blood
Institute and has emerged as a key molecule in innate
and adaptive immunity, with IL-21 serving as its sole
ligand.64,65 Early work with IL-21R indicated that it was
angiostatic, but these observations were obtained using
mouse and human ECs cultured in serum supplemented
with bFGF (basic FGF, a potent angiogenic agent) under
normoxic conditions.66 By contrast, skeletal muscle tis-
sue has low levels of VEGF, bFGF, and other cytokine
growth factors.67,68 Accordingly, we altered the in vitro
conditions to be more similar to those seen in isch-
emic skeletal muscle (with ECs subjected to hypoxia
and serum starvation, ie, HSS). Under HSS conditions,
human ECs upregulate IL-21R and exogenous ligand-
induced angiogenesis.69 The differences between the
angiogenic outcomes from the in vitro experiments
conducted under HSS versus the angiostatic outcomes
seen with ECs in growth factor rich, normoxic conditions
June 11, 2021   1947
 Annex and Cooke
were associated with differences in downstream signal-
DISEASE COMPENDIUM
PERIPHERAL VASCULAR
ing. Specifically, under HSS, ligand-mediated IL-21R
activation activated STAT3, not STAT1 nor Akt.62,69
In wild-type C57BL/6 mice, we found that the IL-21R
was significantly upregulated within ischemic muscle fol-
lowing HLI, and the increases were largely within the EC
population. By contrast, the IL-21R showed no increase
in expression in Balb/C mice, a mouse strain that devel-
ops a sustained perfusion deficit and greater tissue loss
following induction of HLI.62 These results suggested
that IL-21R on ECs might play an important role in the
response to ischemia. The IL-21R seemed to have little
role in non-ECs in muscle, as it was not detectable or
present at very low levels in vascular smooth muscle and
skeletal muscle cells in both normoxic and HSS condi-
tions, and IL-21 administration had no effect on vascular
smooth muscle nor skeletal muscle cells survival under
HSS. Furthermore, IL-21 (ligand) levels do not change
following HLI in C57Bl/6 nor Balb/C mice; in human
umbilical vein ECs IL-21 protein was not detectable in
cells before or after HSS.62 Silencing the IL-21R recep-
tor blocked the angiogenic effect of exogenous ligand,
in vitro and in vivo, and when angiogenesis occurred
there was no change in the Akt/eNOS pathway.62 Con-
sistent with its modulation of perfusion changes follow-
ing vascular injury, Lee et al70 later demonstrated that
IL21R−/− mice had greater cerebral infarct volumes
when compared with wild-type C57/BL6 mice in a
stroke model. Furthermore, within that study, a coding
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variant between C57BL/6 and Balb/c mice at amino
acid 200, in the Balb/c allele of IL-21R was linked to
greater neuronal injury and poorer stroke outcomes. At
200 AA, humans express the C57BL/6 sequence, with
only very rare alternative coding variants. Ongoing work
is focused on the IL-21R mediated, nitric oxide-indepen-
dent, angiogenesis in PAD.
Refining Angiogenesis and Modulating
Antiangiogenic Factors
Within the well-studied VEGF receptor-ligand family,
VEGFR2 is widely regarded as the primary receptor
whose activation drives hypoxia-dependent and post-
natal angiogenesis.71 VEGF-A is one of several struc-
turally related ligands for the VEGFR tyrosine kinases:
VEGFR1, R2, and R3.71–73 Within the VEGF-A gene,
there are major alternative splicing sites resulting in
proteins of varying lengths of 121, 165, and 189 amino
acids. There is a 206 splice variant whose expression
is limited to development. The longer transcripts have
a stronger capacity to bind to the extracellular matrix,
which alters their bioavailability. Strategies are avail-
able to activate multiple isoforms, and these include
activating transcription factors and the use of single
plasmid encoding multiple transcripts. Rebar et al74
reported that the administration of a single isoform
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Vascular Regeneration in PAD
of VEGF-A led to the formation of leaky dysfunc-
tional vessels, whereas the coordinated expression of
all of the splice variants led to the formation of more
functional blood vessels in a mouse ear angiogenesis
model. Dai et al75 demonstrated that a VEGF-A acti-
vating transcription factor could increase expression
of multiple VEGF isoforms in ischemic muscle in vivo
and promote angiogenesis following HLI. An additional
and less appreciated splice variant occurs with alter-
nate splicing of the eighth exon of VEGF-A results in
a 6 amino acid switch that changes the pro-angiogenic
VEGFxxxa (xxx =the number of amino acids) to the anti-
angiogenic VEGFxxxb (VEGF165b for 165 amino acids)
isoform.40,76–78 This splicing can occur for any of the
amino acid length transcripts but has been best stud-
ied for the 165 amino acid isoform.78
Kikuchi et al77 showed that peripheral blood mono-
cytes (isolated by gradient centrifugation) from PAD
versus control patients expressed significantly higher
VEGF165b by quantitative polymerase chain reaction and
then in the mouse HLI model showed that intramuscular
delivery of a VEGF165b specific antibody improved per-
fusion recovery in experimental PAD.77 Their conclusion
and indeed the conclusion in similar studies of the VEGF
system focused on the activation the VEGFR2 pathway
as the mechanism of action.77,79,80 Indeed, even studies
performed with VEGFR1 selective ligands in ischemic
heart or PAD models concluded that the mechanism
for the angiogenic effects of these factors was through
occupying VEGFR1, thus allowing VEGF165a and other
ligands to preferentially activate VEGFR2.81–86 This is
often referred to as a decoy effect.87
Using a similar approach as Kikuchi et al,77 in Balb/C
mice, we found therapeutic benefit following HLI with
delivery of the VEGF165b antibody.40 In these in vitro and
in vivo studies, we consistently found that VEGF165b
impaired endothelial VEGFR1 (not VEGFR2) activa-
tion.40 Administration of the VEGF165b antibody reduced
binding of VEGF165b to VEGFR1 and increased the
activation of VEGFR1, as indicated by its level of phos-
phorylation at Y1333, and this occurred with coimmu-
noprecipitation with activated STAT3. In this report, we
concluded that when VEGF165b reduces the activation
of VEGFR1, the antibody against VEGF165b augments
VEGFR1-mediated angiogenesis. This is not meant to
imply that VEGF165b completely silences VEGFR1. Nota-
bly, in wild-type Balb/C and C57/Bl6 mice after HLI,
both VEGFR1 and VEGF165b are increased, and there-
fore, the level ofVEGFR1 activation is dependent upon
the levels of VEGF165b and other VEGFR1 ligands that
are competing for binding.88,89 This is consistent with
published data that kinase-dead knockout mice and
VEGFR1+/− knockout mice have impaired angiogenesis
and perfusion recovery after HLI when compared with
wild type.90 Within macrophages, VEGFR1 activation
promotes an M2-like angiogenic state in macrophages
 Annex and Cooke
which is enhanced by inhibition of VEGF165b.40 Thus, in
the setting of PAD, the VEGFR1 pathway is regulated
differently than VEGFR2 and may activate angiogenic
pathways by themselves or ones that can have therapeu-
tic synergy with the VEGFR2/eNOS pathway.
Role of Noncoding RNAs (miR and Long Noncoding
RNA)
Micro-RNAs are 15 to 23 nucleotide (noncoding)
RNAs that have emerged as key regulators of the
response to hemodynamic forces, vascular injury, and
hypoxia.91,92 A miR is thought to primarily exert its
effects by incorporating into the RNA-induced silenc-
ing complex which then binds to a target mRNA, usu-
ally in the 3′ UTR of its target mRNA.93,94 The binding
of the miR to the mRNA can suppress a single gene
target or the miR can have the capacity to regulate
multiple genes within a common pathway simultane-
ously by promoting degradation of the mRNA or by
inhibiting mRNA translation to protein.94,95
Although a full review of noncoding RNA in angiogen-
esis and arteriogenesis is beyond the scope of this review,
herein, we will provide examples of noncoding RNAs and
their potential application for future vascular therapeutics
in PAD. The first reports of miRs that were identified as
candidates for modulation of neovascularization in PAD
were of miR-92a96 and miR-100.97 Both of these miRs
were identified based upon the response of EC to injury.
Subsequently, the role of each in PAD-related angiogen-
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esis was tested in preclinical models where the inhibition
of the miR, by its antagomirs, enhanced perfusion recov-
ery.96,97 Antagomirs (antisense miRs) have been shown
to have a high degree of specificity and beyond having
little affect in a cell or tissue where the target miR is not
expressed; antagomers even in target tissue can expres-
sion levels of closely related miR’s unchanged.98 Studies
that focused on the ability of miRs to modulate angio-
genesis in PAD conditions appeared.98–103 The response,
that is, the gene or gene pathway, modulated by a miR
can be expected to be different between cells, or target
tissues, based upon the presence of an injury.103 Moving
from angiogenesis to arteriogenesis, Landskroner-Eiger
et al104 focused on miR(s) that regulated arteriogenesis.
In vitro studies under shear stress, identified candidate
miRs that could regulate arteriogenesis, and in murine
PAD models, approaches that targeted miR-199a and
miR-146a demonstrated improved perfusion recovery
following femoral artery ligation.105,106 Using the same
approach described in the identification of the IL-21R,
we used unbiased genetic, computational, and experi-
mental approaches and in vivo HLI models where we
examined the target tissue in ischemic skeletal muscle
and identified miR-93 as a potential therapeutic target
to modulate PAD.98 For miR-93, although overexpres-
sion has beneficial effects on ECs and macrophages in
PAD relevant conditions, the target gene(s) for ECs was
Circulation Research. 2021;128:1944–1957. DOI: 10.1161/CIRCRESAHA.121.318266
Vascular Regeneration in PAD
shown to be p21, p53, and E2F1, while in macrophages,
DISEASE COMPENDIUM
PERIPHERAL VASCULAR
it was immune response factor-9.39,98
Predicting the net physiological effect of miR modu-
lation in PAD conditions is not always straightforward.
Although miR-155 has antiangiogenic effects in target-
ing eNOS, and VEGF levels are inversely correlated with
miR-155 expression, the inhibition of miR-155 attenu-
ates blood flow recovery and leukocyte recruitment in
vivo following HLI.107–109 Indeed, the totality of data sug-
gests a negative role for miR-155 in the response to HLI.
Thus, in considering miRs for therapeutic modulation, rel-
evant disease models can be informative. In this regard,
however, not all miRs, or the gene targets for a given
miR, are conserved through evolution and miRs that have
retained homology and function from rodents to humans
may be preferential for clinical development.
Another class of noncoding RNAs is the long non-
coding RNAs (lncRNAs) which are typically defined
as RNAs of >200 nucleotides.110 Reports are begin-
ning to emerge on the potential role of lncRNAs in
PAD.111 When compared with miRs, lncRNAs are far
more complex. In general, lncRNAs are known to be
far more species-specific and they contain numerous
potential splice variants. A lncRNA can serve numerous
potential regulatory functions from acting as epigenetic
modifiers, to regulating gene expression, to modifying
protein translation, and acting as protein scaffolds.112,113
For example, the lncRNA LEENE facilitates the recruit-
ment of RNA Polymerase II to the eNOS promoter to
enhance eNOS RNA transcription.114
SENCR is a lncRNA that is highly expressed in EC
and is necessary for EC proliferation, migration, and tube
formation in vitro.115,116 Intriguingly, SENCR is expressed
at lower levels in the vascular tissue of patients with
CLI.116 For reasons that include the potential to exam-
ine rodent models, lncRNAs that are conserved from
mouse to human are far easier to study than are those
that are not conserved. Human-specific lncRNA cannot
readily be used in preclinical models of PAD to examine
effects on angiogenesis, arteriogenesis, perfusion recov-
ery, and potentially tissue loss. Studies have sought to
identify candidate lncRNAs based on their association
with angiogenesis and have, for example, characterized
the response of ECs to hypoxia in vitro where lncRNAs,
including LNC00323, MIR503-HG, GATA-AS, have all
been shown to be differentially regulated in human EC
with after hypoxic injury.117–120 Further analysis showed
that a targeted knockdown of LINC00323 impaired EC
tube formation while MIR503-HG knockdown reduced
EC migration, although both were necessary for prolif-
eration.117 Voellenkle et al120 found H19, MIR210HG,
MEG9, MALAT1, and MIR22HG to be elevated by
hypoxia in human umbilical vein ECs in vitro and because
these were evolutionarily conserved, they were able to be
examined in mouse gastrocnemius muscle following HLI.
MALAT1 inhibition impaired angiogenesis in vitro and
June 11, 2021   1949
 Annex and Cooke
blood flow recovery and reduced capillary density fol-
DISEASE COMPENDIUM
PERIPHERAL VASCULAR
lowing HLI in vivo.118 In aged mice, MEG3 inhibition was
shown to improve blood flow recovery following HLI.121
The systematic examination of human tissue will likely
offer the best way to identify lncRNAs that may play a
role in, or serve as therapeutic targets for, PAD.122
Endothelial Metabolism in Angiogenesis.
Research in therapeutic angiogenesis, certainly from the
cardiovascular perspective, has largely focused on the
use of cytokine growth factors to bind to and activate
receptors on the EC surface to drive receptor signal-
ing within in vitro systems to move EC into a prolifera-
tive state. Even when in a quiescent state, energy from
ATP is required for ECs to maintain physiological func-
tion and homeostasis. In ECs, the majority of ATP is pro-
duced via glycolytic metabolism, and this phenomenon
occurs despite the location of ECs being adjacent to
the bloodstream and in contact with the sufficient lev-
els of oxygen.123 In ECs, the 6-phosphofructo-2-kinase/
fructose-2,6-biphosphatase enzymes PFKFBs prod-
uct is an activator of PFK1 (phosphofructokinase 1), a
rate-limiting enzyme of glycolysis.124 Although there are
4 isoforms of PFKFB, PFKFB3 has a kinase/phospha-
tase activity >700, the kinase to phosphatase activity in
PFKFB2 and PFKFB4 are 1.8 and 0.9, respectively, and
the expression of PFKFB1 in ECs is low.124,125 Therefore,
PFKFB3 is critical for converting F6P to F2,6BP, with
F2,6BP being the main allosteric activator of PFK1; the
Downloaded from http://ahajournals.org by on July 26, 2023
most important regulatory enzymes of glycolysis. Taken
together, PFKFB3 is critical for the regulation of endo-
thelial glycolysis.123–126
Beyond homeostasis, ECs need ATP for angiogenesis.
Tumor angiogenesis is perhaps the most extensively studied
angiogenesis systems and one in which the growth/prolifer-
ation of ECs requires an increase in glycolytic metabolism.127
Exposing ECs to hypoxia alone has been used to model
conditions such as tumor angiogenesis and retinopathy.128
The changes in EC metabolism observed under hypoxia are
similar to what is seen with exposure of ECs to VEGF165a,
where EC growth and proliferation is accompanied with
an increase in glycolytic metabolism that occurs with, and
is dependent on, an upregulation of PFKFB3 to increase
the glycolytic flux.129–131 In human umbilical vein ECs chal-
lenged with hypoxia only, PFKFB3 knockdown using siRNA
or pharmacological inhibition has resulted in decreased cell
proliferation.124 Tumors which are known to use enhanced
glycolysis for angiogenesis when embedded in EC-specific
PFKFB3 knockout mice show slower growth rates than
tumors transplanted in control mice.125 Attempts to promote
blood vessel growth in the ischemic muscle bed to improve
leg blood flow with agents such as VEGF165a has been met
with limited therapeutic success in humans.56,132 The angio-
genesis that occurs in ischemic leg muscle, when compared
with angiogenesis in a tumor, is occurring in a tissue that is
relatively low in both cytokine growth factors and, of course,
1950   June 11, 2021 Circulation Research. 2021;128:1944–1957. DOI: 10.1161/CIRCRESAHA.121.318266
Vascular Regeneration in PAD
oxygen tension.68,98 In the state of hypoxia with serum star-
vation, the addition of VEGF165a to ECs induces angiogen-
esis by reducing cell death and increasing EC proliferation
in association with an increase in glycolysis.133,134 However,
in VEGF-mediated tumor angiogenesis, there is vascular
permeability and leaky blood vessels.133,134 Whether all forms
of angiogenesis use the same EC metabolism pathways
as tumors is not completely understood nor are the poten-
tial consequences of using metabolic pathways other than
glycolysis.133–135
Generating Better Autologous Cell Therapies
As described briefly above, large randomized, well-con-
ducted clinical trials of cell therapies for PAD, such as the
National Heart, Lung, and Blood Institute-funded PACE
study, have not shown benefit of adult stem cell therapy.
It is possible that the difference between the preclinical
models (where many cell therapies have efficacy), and the
clinical trials, is related to the number and the quality of the
autologous stem cells derived from individuals with cardio-
vascular disease.136 In this regard, the ability to provide suf-
ficient numbers of high-quality therapeutic cells is likely to
be critical in obtaining a reproducible and significant benefit.
In any event, different approaches are needed. One novel
approach to cell therapy for PAD is to generate therapeutic
cells using autologous skin fibroblasts.137 In this case, a field
of fibroblasts are exposed to retroviral vectors encoding
the master regulators of pluripotency (eg, Oct 4 [octamer-
binding transcription factor 4], Sox2 [SRY (sex-determining
region Y)-box 2], KLF4 [Kruppel-like factor 4], and cMyc
[cellular myelocytomatosis]). These transcriptional factors
direct the activation of a cascade of factors required for
pluripotency, and over a period of several weeks, many of
the fibroblasts will become induced pluripotent stem cells
(iPSCs). Subsequently, the iPSCs can be differentiated into
any of the 3 germ layers, and further differentiated into the
lineage of choice, using growth factors and small molecules
that facilitate the differentiation into a particular lineage,
such as ECs. Rufaihah et al138 have previously shown that
iPSC-derived ECs manifest all of the expected characteris-
tics and functions, expressing endothelial surface markers
(eg, VE-cadherin), generating nitric oxide and angiogenic
cytokines, and forming vascular networks in Matrigel. Fur-
thermore, when injected into the underperfused limb of the
murine HLI model, perfusion was increased. Interestingly,
when delivered systemically the ECs derived from pluripo-
tent stem cells can migrate to the ischemic hindlimb and
increase perfusion as assessed by bioluminescence and
laser doppler spectroscopy.139 These findings suggest that
autologous iPSC-derived ECs might be useful as a cell
therapy in patients with PAD. However, iPSC derivatives will
need to be rigorously tested for the fidelity of reprogram-
ming and the exclusion of pluripotent cells from the thera-
peutic product.
Another approach to generating autologous ECs is to
reprogram fibroblasts directly into ECs.140–142 This form of
 Annex and Cooke
nuclear reprogramming from one somatic cell lineage to
another is also known as direct reprogramming or trans-
differentiation so as to distinguish it from the process
where the generation of an iPSC is an intermediate step.
Transdifferentiation is a change from one somatic cell
type to another. In addition, the term transdifferentiation
may also apply to radical changes in cell form or function
within a cell lineage, such as when a senescent EC is
transformed into a juvenile phenotype, as with the forced
expression of telomerase.143 Whether transdifferentiation
is across somatic lineages, or across time as in the latter
example, the cell must overcome epigenetic barriers that
maintain cell phenotype, and must rearrange chromatin
configuration so as to support the global change in tran-
scriptional profile that underlies a new cell identity.144,145
In the case of direct reprogramming, retroviral vectors
or mRNA encoding the master regulators of endothe-
lial identity (eg, ETV2, FLI1 [Friend leukemia integra-
tion 1 transcription factor], GATA2 [GATA-binding factor
2], and KLF4) can induce fibroblasts to become ECs.
These induced ECs are similar to iPSC-ECs in endothe-
lial functions, as well as the ability to restore perfusion
when injected into the ischemic limb of the mouse.146 In
either case, the generation of the induced pluripotent
stem cell or the direct reprogramming of a fibroblast to
an EC requires the activation of cell-autonomous innate
immune signaling.147 The activation of innate immune
signaling increases DNA accessibility to facilitate the
action of lineage determination factors to change the cell
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phenotype.147–149 The process by which inflammatory sig-
naling increases DNA accessibility to facilitate changes
in cell fate is termed transflammation (Figure 2).
Specific caveats regarding the use of reprogrammed
cells for therapy relates to concerns regarding residual
pluripotent stem cells that could generate abnormal tis-
sues (in the case of iPSC-ECs); and the fidelity of the
reprogramming to functional ECs.150 Other general con-
cerns of cell therapy remain, for example, dosing, duration
of treatment, delivery methods, and cellular persistence
in a hostile ischemic environment and to what extent the
environment can be modulated before therapy.
Generating Autologous Therapeutic Cells In Situ
If autologous therapeutic cells could be generated in situ,
it might solve the issues regarding delivery of the cells
and could potentially reverse processes contributing to
disease, such as endothelial-to-mesenchyme transition,
which may contribute to scarring in the setting of isch-
emic injury. Indeed, there is evidence that fibroblast-to-
endothelial transdifferentiation (MEndoT) contributes to
the recovery from ischemia in vivo, although this concept
remains controversial.137,141,142,147–149 Ubil et al151 have
presented evidence that resident fibroblasts may trans-
differentiate into ECs, to increase capillary density and
to enhance perfusion in the murine myocardial infarction
model which is mediated partially through p53. However,
Circulation Research. 2021;128:1944–1957. DOI: 10.1161/CIRCRESAHA.121.318266
Vascular Regeneration in PAD
a subsequent paper supported by extensive lineage trac-
DISEASE COMPENDIUM
PERIPHERAL VASCULAR
ing found no evidence of MEndoT in the murine myocar-
dial infarction model.152
The contribution and extent of potential modulation of
MEndoT in the response to limb ischemia is still in the pro-
cess of being understood. Because there are significant
differences in peripheral versus myocardial tissues, in their
vascularity and their response to disease, lineage tracing
and single-cell RNA sequencing were used in an HLI model,
to detect evidence of MEndoT. Surprisingly, 8 distinctly dif-
ferent subpopulations of fibroblasts in the mouse hindlimb,
confirmed by their high expression of genes known to be
preferentially expressed in fibroblasts (eg, Tcf4/Tcf7l2,
fibronectin, and collagen IV) yet distinct by their global tran-
scriptional profile. Two subpopulations expressed some EC
genes at baseline and expanded dramatically during isch-
emia. Genetic or pharmacological suppression of inflamma-
tory signaling abrogated the expansion of this population
and impaired vascularity, recovery of perfusion, and preser-
vation of ischemic tissue. Notably, surface markers for these
2 populations permitted their isolation from the ischemic
hindlimb by fluorescence-activated cell sorting. Whereas
most fibroblasts will form clusters of cells in Matrigel, one of
the subpopulations reproducibly formed networks and dis-
played surface markers characteristic of ECs when placed
into Matrigel.153
The expansion or transdifferentiation of resident
fibroblasts to ECs depends upon inflammatory signaling,
an observation consistent with our prior work showing
that inflammatory signaling promotes DNA accessibility
and phenotypic plasticity.148,153 Hypoxic injury (generat-
ing damage-associated molecular patterns or DAMPs)
and inflammatory cytokines released in the setting of
ischemia activate inflammatory signaling (Figure 2). This
triggers global changes in epigenetic modifiers (such as
upregulation of histone acetylases and downregulation
of histone deacetylases) which favor an open chromatin
configuration.154 In addition, activation of iNOS (induc-
ible nitric oxide synthase) facilitates S-nitrosylation
of the polycomb repressive complex 1 and NURD1
(nucleosome remodelling and deacetylase 1), causing
the dissociation of these repressive complexes from the
chromatin.148,149 Finally, inflammatory signaling induces a
glycolytic shift, which supplies the nucleus with metabo-
lites that participate in epigenetic modification. For exam-
ple, the glycolytic shift is associated with citrate export
from the mitochondria to the nucleus, where it is con-
verted to acetylcoA for histone acetylation.155
To summarize, single-cell profiling and lineage trac-
ing has revealed that there may be some contribution of
specific populations of tissue fibroblasts to angiogenesis
in limb ischemia. It may be possible to enhance cellu-
lar plasticity to enhance therapeutic transdifferentiation
of fibroblasts to angiogenic cells. Drugs which favorably
affect the activity of epigenetic modifiers or the glycolytic
state are worthy of further study for PAD.
June 11, 2021   1951
DISEASE COMPENDIUM Annex and Cooke
PERIPHERAL VASCULAR
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EPILOGUE
The past 25 years of research into angiogenic and cell
therapies for PAD have not yet translated into significant
changes in PAD management. The discordance between
what we have learned and what we have accomplished
therapeutically raises important questions. Are the ani-
mal models that we are using misdirecting our therapeu-
tic trials? The classic hindlimb ischemia model in mice is
typically an acute femoral artery ligation, in the absence
of cardiovascular risk factors such as hypercholester-
olemia, hypertension, tobacco exposure, diabetes, or
senescence. It is critical to consider the effects of risk
factors (in particular diabetes and tobacco exposure) in
preclinical models. Better animal models are available
(eg, hypercholesterolemic cynomolgus monkeys) but
are prohibitively expensive and low throughput. A more
feasible approach may be to preferentially use genetic
mouse models that combine cardiovascular risk factors
and use modifications to the classic ligation/excision to
more closely mimic human conditions. An animal model
that develops peripheral arterial lesions would be ideal
but those will need to reach a state of hemodynamically
significant occlusion. Conversely, multiorgan chip models
that combine microfluidics and physiological hemody-
namic forces with human cells could be useful. Finally,
1952   June 11, 2021 Circulation Research. 2021;128:1944–1957. DOI: 10.1161/CIRCRESAHA.121.318266
Vascular Regeneration in PAD
Figure 2. Damage-associated
molecular patterns (DAMPs)
generated by hypoxic injury
stimulate PRRs (pattern recognition
receptors) which activate signaling
pathways that alter transcription or
posttranslational modifications of
epigenetic enzymes to promote DNA
accessibility.
In addition, a glycolytic shift supplies
metabolites to the nucleus for histone
modifications that further facilitate an
open chromatin configuration. Together
with environmental influences that favor
an endothelial phenotype (eg, VEGF
[vascular endothelial growth factor]),
fibroblasts undergo a transdifferentiation
to angiogenic cells that generate vascular
growth factors and incorporate into the
microvasculature.
the application of contemporary molecular technologies
to human samples may be most useful in generating
novel therapeutic avenues. For example, genome-wide
association studies have provided support for antithrom-
botic treatment of PAD,156 a finding that is consistent
with the improved limb outcomes in patients with PAD
with low dose rivaroxaban in the Compass study. Ongoing
studies using single-cell multiomics (eg, single-cell RNA
sequencing, genome sequencing, and ATAC sequencing)
applied to human tissues will also provide novel insights
into disease mechanisms. Such studies may provide use-
ful information such as target receptor expression in sub-
groups of patients or better validate molecular targets
identified in murine models.
It is critical to remember that in patients with PAD,
there is substantial patient heterogeneity in the response
to similar degrees of atherosclerotic disease. Patients
with the same hemodynamic compromise may have dif-
ferent presentations. Why one patient has IC, whereas
another has CLI is not entirely explained by the large
vessel disease, suggesting that there are microvascular
or end-organ differences. In which case, the pathophysi-
ology, and the therapeutic reversal of disease, may be
different in these 2 sets of patients.
Is the failure of angiogenic and cell therapies related
to our tendency to do single-agent studies? Would a
 Annex and Cooke
combinatorial approach be more successful? Rather
than just facilitating angiogenesis, should we also be
considering other processes such as endothelial per-
meability and pericyte association, which may translate
into less permeable, more functional, and more stable
vessels? What about other processes that may contrib-
ute to angiogenesis, such as clonal EC expansion?157
What is the role of the skeletal muscle itself in vasculo-
genesis? Certainly, metabolic products during exercise
affect vasomotion. More studies of how such metabolic
products effect maintenance or repair of the microvas-
culature are needed.
Are iPSC-derived cells a better form of cell therapy?
Can we ensure the fidelity of reprogramming and the sta-
bility of cell fate? Will such cells be more resistant to the
hostile environment into which they are placed? And how
will we deliver such cells, and in what dose and duration?
Is it possibly better to transdifferentiate resident meso-
dermal cells into ECs that can expand the microvascula-
ture? Can we restore tissue plasticity and regeneration
by epigenetic-directed therapies? Or is any of this going
to be useful without first restoring flow through the large
conduit vessels?
We may also need to rethink how we test novel
PAD therapies in clinical trials. For example, in trials of
angiogenic agents, hemodynamic, and anatomic assess-
ments, such as low ankle-brachial blood pressure index
and angiography, are poor surrogate measurements for
perfusion. Treadmill studies to assess function may be
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complicated by many factors including the fibro-fatty
replacement of skeletal muscle that attends chronic limb
ischemia. Thus, improved measures to assess improve-
ments in muscle perfusion and function will be advan-
tageous. For cell therapy trials, do we have the proper
tools, for example, imaging, cell tracking, etc for conduct-
ing more informative early phase human studies? Inflam-
mation plays a major role in coronary artery disease, and
likely plays an important role in PAD. However, emerging
evidence indicates that inflammatory signaling is impor-
tant for vascular regeneration.158 Methods are needed
to assess inflammatory signaling in patient tissue, so as
to optimally modulate this process to enhance vascular
anatomy, perfusion, and skeletal muscle function. For
patients with CLI, the major criteria for Food and Drug
Administration approval are amputation-free survival.
However, for these patients, should not pain relief and
reduction in narcotic use be approvable end points?
CONCLUSIONS
These are just a few of the difficult questions that face
investigators and regulators in the field of vascular
regeneration. However, it is our feeling that the answers
to these questions are within reach. In recent years, there
have been significant advances in our understanding of
the genetic, epigenetic, proteomic, and cellular regulation
Circulation Research. 2021;128:1944–1957. DOI: 10.1161/CIRCRESAHA.121.318266
Vascular Regeneration in PAD
of angiogenesis, arteriogenesis, adult vasculogenesis,
DISEASE COMPENDIUM
PERIPHERAL VASCULAR
and cell fate decisions regulating vascular recovery. The
answers lie in the refinement of our models, in the appli-
cation of new molecular tools to patient tissues, and in
the persistence of our efforts to benefit our patients.
ARTICLE INFORMATION
Affiliations
Vascular Biology Center, Department of Medicine, Medical College of Georgia,
Augusta University (B.H.A.). Department of Cardiovascular Sciences, Houston
Methodist Research Institute, TX (J.P.C.).
Sources of Funding
Dr Annex is supported by R01HL150003, RO1148590, RO1HL141325,
3RO1HL101200 (Popel, Johns Hopkins, PI), and RO1GM129074 (Mac
Gabhann, Johns Hopkins, PI). Dr Cooke is supported by R01s HL133254
and HL148338; and Cancer Prevention Research Institute of Texas CPRIT
RP150611.
Disclosures
Dr Annex is the founder of Merand Pharmaceutical a start-up company estab-
lished at the University of Virginia to commercialize miR-93 for peripheral arte-
rial disease (PAD) but the work for this article was supported by the National
Institutes of Health (NIH) grants to Dr Annex before the formation of the com-
pany and no financial or time support from Merand Pharmaceutical was used.
Dr Cooke is on the Scientific Advisory Boards for Humann, Fibralign, and Ja-
nOne and is the majority owner of Cooke Consulting; none of which companies
provided financial support for this work. No products of these companies were
discussed in this review.
REFERENCES
1. Benjamin EJ, Virani SS, Callaway CW, Chamberlain AM, Chang AR, Cheng
S, Chiuve SE, Cushman M, Delling FN, Deo R, et al; American Heart Asso-
ciation Council on Epidemiology and Prevention Statistics Committee and
Stroke Statistics Subcommittee. Heart Disease and Stroke Statistics-2018
Update: a Report From the American Heart Association. Circulation.
2018;137:e67–e492. doi: 10.1161/CIR.0000000000000558
2. Fowkes FG, Rudan D, Rudan I, Aboyans V, Denenberg JO, McDermott
MM, Norman PE, Sampson UKA, Williams LJ, Mensah GE, et al. Com-
parison of global estimates of prevalence and risk factors for peripheral
artery disease in 2000 and 2010: a systematic review and analysis. Lancet.
2013;382:1329–40. doi: 10.1016/S0140-6736(13)61249-0
3. Hirsch AT, Duval S. The global pandemic of peripheral artery disease. Lan-
cet. 2013;382:1312–1314. doi: 10.1016/S0140-6736(13)61576-7
4. Kullo IJ, Rooke TW. CLINICAL PRACTICE. Peripheral artery disease. N Engl
J Med. 2016;374:861–871. doi: 10.1056/NEJMcp1507631
5. Allison MA, Ho E, Denenberg JO, Langer RD, Newman AB, Fabsitz RR,
Criqui MH. Ethnic-specific prevalence of peripheral arterial disease in
the United States. Am J Prev Med. 2007;32:328–333. doi: 10.1016/j.
amepre.2006.12.010
6. Nead KT, Cooke JP, Olin JW, Leeper NJ. Alternative ankle-brachial
index method identifies additional at-risk individuals. J Am Coll Cardiol.
2013;62:553–559. doi: 10.1016/j.jacc.2013.04.061
7. Selvin E, Erlinger TP. Prevalence of and risk factors for peripheral arterial
disease in the United States: results from the National Health and Nutri-
tion Examination Survey, 1999-2000. Circulation. 2004;110:738–43. doi:
10.1161/01.CIR.0000137913.26087.F0
8. Olin JW, Sealove BA. Peripheral artery disease: current insight into the dis-
ease and its diagnosis and management. Mayo Clin Proc. 2010;85:678–
692. doi: 10.4065/mcp.2010.0133
9. Bonaca MP, Bauersachs RM, Anand SS, Debus ES, Nehler MR, Patel MR,
Fanelli F, Capell WH, Diao L, Jaeger N, et al. Rivaroxaban in Peripheral Artery
Disease after Revascularization. N Engl J Med. 2020;382:1994–2004. doi:
10.1056/NEJMoa2000052
10. Gerhard-Herman MD, Gornik HL, Barrett C, Barshes NR, Corriere MA,
Drachman DE, Fleisher LA, Fowkes FGR, Hamburg NM, Kinlay S, et al.
2016 AHA/ACC Guideline on the Management of Patients With Lower
Extremity Peripheral Artery Disease: executive Summary: a Report of the
June 11, 2021   1953
 Annex and Cooke
American College of Cardiology/American Heart Association Task Force on
DISEASE COMPENDIUM
PERIPHERAL VASCULAR
Clinical Practice Guidelines. J Am Coll Cardiol. 2017;69:1465–1508. doi:
10.1016/j.jacc.2016.11.008
11. Baubeta Fridh E, Andersson M, Thuresson M, Sigvant B, Kragsterman
B, Johansson S, Hasvold P, Falkenberg M, Nordanstig J. Amputation
rates, mortality, and pre-operative comorbidities in patients revascula-
rised for intermittent claudication or critical limb ischaemia: a popula-
tion based study. Eur J Vasc Endovasc Surg. 2017;54:480–486. doi:
10.1016/j.ejvs.2017.07.005
12. Criqui MH, Aboyans V. Epidemiology of peripheral artery disease. Circ Res.
2015;116:1509–1526. doi: 10.1161/CIRCRESAHA.116.303849
13. Vartanian SM, Conte MS. Surgical intervention for peripheral arterial disease.
Circ Res. 2015;116:1614–1628. doi: 10.1161/CIRCRESAHA.116.303504
14. McDermott MM, Ades P, Guralnik JM, Dyer A, Ferrucci L, Liu K, Nelson
M, Lloyd-Jones D, Van Horn L, Garside D, et al. Treadmill exercise and
resistance training in patients with peripheral arterial disease with and
without intermittent claudication: a randomized controlled trial. JAMA.
2009;301:165–74. doi: 10.1001/jama.2008.962. Erratum in: JAMA.
2012;307:1694.
15. Garg PK, Liu K, Tian L, Guralnik JM, Ferrucci L, Criqui MH, Tan J,
McDermott MM. Physical activity during daily life and functional decline
in peripheral arterial disease. Circulation. 2009;119:251–260. doi:
10.1161/CIRCULATIONAHA.108.791491
16. Annex BH. Therapeutic angiogenesis for critical limb ischaemia. Nat Rev
Cardiol. 2013;10:387–396. doi: 10.1038/nrcardio.2013.70
17. Adams RH, Alitalo K. Molecular regulation of angiogenesis and lymphangio-
genesis. Nat Rev Mol Cell Biol. 2007;8:464–478. doi: 10.1038/nrm2183
18. Mentzer SJ, Konerding MA. Intussusceptive angiogenesis: expansion and
remodeling of microvascular networks. Angiogenesis. 2014;17:499–509.
doi: 10.1007/s10456-014-9428-3
19. Aicher A, Rentsch M, Sasaki K, Ellwart JW, Fändrich F, Siebert R, Cooke JP,
Dimmeler S, Heeschen C. Nonbone marrow-derived circulating progenitor
cells contribute to postnatal neovascularization following tissue ischemia. Circ
Res. 2007;100:581–589. doi: 10.1161/01.RES.0000259562.63718.35
20. Rehman J, Li J, Parvathaneni L, Karlsson G, Panchal VR, Temm CJ,
Mahenthiran J, March KL. Exercise acutely increases circulating endothelial
progenitor cells and monocyte-/macrophage-derived angiogenic cells. J Am
Coll Cardiol. 2004;43:2314–2318. doi: 10.1016/j.jacc.2004.02.049
Downloaded from http://ahajournals.org by on July 26, 2023
21. Rehman J, Li J, Orschell CM, March KL. Peripheral blood “endothelial pro-
genitor cells” are derived from monocyte/macrophages and secrete angio-
genic growth factors. Circulation. 2003;107:1164–1169. doi: 10.1161/01.
cir.0000058702.69484.a0
22. Heeschen C, Jang JJ, Weis M, Pathak A, Kaji S, Hu RS, Tsao PS, Johnson
FL, Cooke JP. Nicotine stimulates angiogenesis and promotes tumor growth
and atherosclerosis. Nat Med. 2001;7:833–839. doi: 10.1038/89961
23. Kiuchi K, Matsuoka M, Wu JC, Lima e Silva R, Kengatharan M, Verghese M,
Ueno S, Yokoi K, Khu NH, Cooke JP, et al. Mecamylamine suppresses Basal
and nicotine-stimulated choroidal neovascularization. Invest Ophthalmol Vis
Sci. 2008;49:1705–1711. doi: 10.1167/iovs.07-0089
24. Semenza GL. Oxygen sensing, hypoxia-inducible factors, and disease
pathophysiology. Annu Rev Pathol. 2014;9:47–71. doi: 10.1146/annurev-
pathol-012513-104720
25. Qian HS, Liu P, Huw LY, Orme A, Halks-Miller M, Hill SM, Jin F, Kretschmer P,
Blasko E, Cashion L, et al. Effective treatment of vascular endothelial growth
factor refractory hindlimb ischemia by a mutant endothelial nitric oxide syn-
thase gene. Gene Ther. 2006;13:1342–1350. doi: 10.1038/sj.gt.3302781
26. Ferraro B, Cruz YL, Baldwin M, Coppola D, Heller R. Increased perfusion
and angiogenesis in a hindlimb ischemia model with plasmid FGF-2 deliv-
ered by noninvasive electroporation. Gene Ther. 2010;17:763–769. doi:
10.1038/gt.2010.43
27. Morishita R, Nakamura S, Hayashi S, Taniyama Y, Moriguchi A, Nagano T,
Taiji M, Noguchi H, Takeshita S, Matsumoto K, et al. Therapeutic angiogen-
esis induced by human recombinant hepatocyte growth factor in rabbit
hind limb ischemia model as cytokine supplement therapy. Hypertension.
1999;33:1379–1384. doi: 10.1161/01.hyp.33.6.1379
28. Banfi A, von Degenfeld G, Gianni-Barrera R, Reginato S, Merchant MJ,
McDonald DM, Blau HM. Therapeutic angiogenesis due to balanced single-
vector delivery of VEGF and PDGF-BB. FASEB J. 2012;26:2486–2497.
doi: 10.1096/fj.11-197400
29. Lekas M, Lekas P, Mei SH, Deng Y, Dumont DJ, Stewart DJ. Tie2-depen-
dent neovascularization of the ischemic hindlimb is mediated by angiopoi-
etin-2. PLoS One. 2012;7:e43568. doi: 10.1371/journal.pone.0043568
30. Ho HK, Jang JJ, Kaji S, Spektor G, Fong A, Yang P, Hu BS, Schatzman
R, Quertermous T, Cooke JP. Developmental endothelial locus-1 (Del-1), a
1954   June 11, 2021 Circulation Research. 2021;128:1944–1957. DOI: 10.1161/CIRCRESAHA.121.318266
Vascular Regeneration in PAD
novel angiogenic protein: its role in ischemia. Circulation. 2004;109:1314–
1319. doi: 10.1161/01.CIR.0000118465.36018.2D
31. Wolf C, Cai WJ, Vosschulte R, Koltai S, Mousavipour D, Scholz D,
Afsah-Hedjri A, Schaper W, Schaper J. Vascular remodeling and altered
protein expression during growth of coronary collateral arteries. J Mol Cell
Cardiol. 1998;30:2291–2305. doi: 10.1006/jmcc.1998.0790
32. Cai WJ, Koltai S, Kocsis E, Scholz D, Kostin S, Luo X, Schaper W, Schaper J.
Remodeling of the adventitia during coronary arteriogenesis. Am J Physiol
Heart Circ Physiol. 2003;284:H31–H40. doi: 10.1152/ajpheart.00478.2002
33. Fung E, Helisch A. Macrophages in collateral arteriogenesis. Front Physiol.
2012;3:353. doi: 10.3389/fphys.2012.00353
34. Heuslein JL, Meisner JK, Li X, Song J, Vincentelli H, Leiphart RJ, Ames
EG, Blackman BR, Blackman BR, Price RJ. Mechanisms of ampli-
fied arteriogenesis in collateral artery segments exposed to reversed
flow direction. Arterioscler Thromb Vasc Biol. 2015;35:2354–2365. doi:
10.1161/ATVBAHA.115.305775
35. Heil M, Ziegelhoeffer T, Wagner S, Fernández B, Helisch A, Martin S, Tribulova
S, Kuziel WA, Bachmann G, Schaper W. Collateral artery growth (arterio-
genesis) after experimental arterial occlusion is impaired in mice lacking
CC-chemokine receptor-2. Circ Res. 2004;94:671–7. doi: 10.1161/01.
RES.0000122041.73808.B5
36. Krishnasamy K, Limbourg A, Kapanadze T, Gamrekelashvili J, Beger C,
Häger C, Lozanovski VJ, Falk CS, Napp LC, Bauersachs J, et al. Blood ves-
sel control of macrophage maturation promotes arteriogenesis in ischemia.
Nat Commun. 2017;8:952. doi: 10.1038/s41467-017-00953-2
37. Zbinden S, Zbinden R, Meier P, Windecker S, Seiler C. Safety and effi-
cacy of subcutaneous-only granulocyte-macrophage colony-stimulating
factor for collateral growth promotion in patients with coronary artery
disease. J Am Coll Cardiol. 2005;46:1636–1642. doi: 10.1016/j.
jacc.2005.01.068
38. Caradu C, Couffinhal T, Chapouly C, Guimbal S, Hollier PL, Ducasse E,
Bura-Rivière A, Dubois M, Gadeau AP, Renault MA. Restoring endothe-
lial function by targeting desert Hedgehog downstream of Klf2 improves
critical limb Ischemia in adults. Circ Res. 2018;123:1053–1065. doi:
10.1161/CIRCRESAHA.118.313177
39. Ganta VC, Choi MH, Kutateladze A, Fox TE, Farber CR, Annex BH. A
MicroRNA93-interferon regulatory factor-9-immunoresponsive gene-
1-itaconic acid pathway modulates M2-like macrophage polarization to
revascularize Ischemic muscle. Circulation. 2017;135:2403–2425. doi:
10.1161/CIRCULATIONAHA.116.025490
40. Ganta VC, Choi M, Farber CR, Annex BH. Antiangiogenic VEGF165b
regulates macrophage polarization via S100A8/S100A9 in periph-
eral artery disease. Circulation. 2019;139:226–242. doi: 10.1161/
CIRCULATIONAHA.118.034165
41. Libby P. Inflammation in atherosclerosis-No Longer a theory. Clin Chem.
2021;67:131–142. doi: 10.1093/clinchem/hvaa275
42. Meier P, Gloekler S, Oezdemir B, Indermuehle A, Traupe T, Vogel R, de Marchi
S, Seiler C. G-CSF induced arteriogenesis in humans: molecular insights
into a randomized controlled trial. Curr Vasc Pharmacol. 2013;11:38–46.
43. Grundmann S, Hoefer I, Ulusans S, Bode C, Oesterle S, Tijssen JG, Piek
JJ, Buschmann I, van Royen N. Granulocyte-macrophage colony-stimulating
factor stimulates arteriogenesis in a pig model of peripheral artery disease
using clinically applicable infusion pumps. J Vasc Surg. 2006;43:1263–
1269. doi: 10.1016/j.jvs.2006.02.049
44. Meier P, Gloekler S, de Marchi SF, Indermuehle A, Rutz T, Traupe T, Steck
H, Vogel R, Seiler C. Myocardial salvage through coronary collateral growth
by granulocyte colony-stimulating factor in chronic coronary artery dis-
ease: a controlled randomized trial. Circulation. 2009;120:1355–1363. doi:
10.1161/CIRCULATIONAHA.109.866269
45. Asahara T, Murohara T, Sullivan A, Silver M, van der Zee R, Li T, Witzenbichler
B, Schatteman G, Isner JM. Isolation of putative progenitor endothe-
lial cells for angiogenesis. Science. 1997;275:964–967. doi: 10.1126/
science.275.5302.964
46. Ceradini DJ, Kulkarni AR, Callaghan MJ, Tepper OM, Bastidas N, Kleinman
ME, Capla JM, Galiano RD, Levine JP, Gurtner GC. Progenitor cell traffick-
ing is regulated by hypoxic gradients through HIF-1 induction of SDF-1. Nat
Med. 2004;10:858–864. doi: 10.1038/nm1075
47. Grunewald M, Avraham I, Dor Y, Bachar-Lustig E, Itin A, Jung S, Yung S,
Chimenti S, Landsman L, Abramovitch R, et al. VEGF-induced adult neo-
vascularization: recruitment, retention, and role of accessory cells. Cell.
2006;124:175–189. doi: 10.1016/j.cell.2005.10.036
48. Yoon CH, Hur J, Park KW, Kim JH, Lee CS, Oh IY, Kim TY, Cho HJ, Kang
HJ, Chae IH, et al. Synergistic neovascularization by mixed transplantation
of early endothelial progenitor cells and late outgrowth endothelial cells:
 Annex and Cooke
the role of angiogenic cytokines and matrix metalloproteinases. Circulation.
2005;112:1618–1627. doi: 10.1161/CIRCULATIONAHA.104.503433
49. Yoder MC. Defining human endothelial progenitor cells. J Thromb Haemost.
2009;7(suppl 1):49–52. doi: 10.1111/j.1538-7836.2009.03407.x
50. van Royen N, Piek JJ, Buschmann I, Hoefer I, Voskuil M, Schaper W.
Stimulation of arteriogenesis; a new concept for the treatment of arterial
occlusive disease. Cardiovasc Res. 2001;49:543–553. doi: 10.1016/s0008-
6363(00)00206-6
51. Heil M, Eitenmüller I, Schmitz-Rixen T, Schaper W. Arteriogenesis versus
angiogenesis: similarities and differences. J Cell Mol Med. 2006;10:45–55.
doi: 10.1111/j.1582-4934.2006.tb00290.x
52. Robbins JL, Jones WS, Duscha BD, Allen JD, Kraus WE, Regensteiner
JG, Hiatt WR, Annex BH. Relationship between leg muscle capillary den-
sity and peak hyperemic blood flow with endurance capacity in peripheral
artery disease. J Appl Physiol (1985). 2011;111:81–6. doi: 10.1152/
japplphysiol.00141.2011
53. Duscha BD, Robbins JL, Jones WS, Kraus WE, Lye RJ, Sanders JM, Allen
JD, Regensteiner JG, Hiatt WR, Annex BH. Angiogenesis in skeletal
muscle precede improvements in peak oxygen uptake in peripheral artery
disease patients. Arterioscler Thromb Vasc Biol. 2011;31:2742–2748. doi:
10.1161/ATVBAHA.111.230441
54. Isner JM, Pieczek A, Schainfeld R, Blair R, Haley L, Asahara T, Rosenfield
K, Razvi S, Walsh K, Symes JF. Clinical evidence of angiogenesis after arte-
rial gene transfer of phVEGF165 in patient with ischaemic limb. Lancet.
1996;348:370–374. doi: 10.1016/s0140-6736(96)03361-2
55. Cooke JP, Losordo DW. Modulating the vascular response to limb isch-
emia: angiogenic and cell therapies. Circ Res. 2015;116:1561–1578. doi:
10.1161/CIRCRESAHA.115.303565
56. Iyer SR, Annex BH. Therapeutic angiogenesis for peripheral artery dis-
ease: lessons learned in translational science. JACC Basic Transl Sci.
2017;2:503–512. doi: 10.1016/j.jacbts.2017.07.012
57. Cooke JP, Meng S. Vascular regeneration in peripheral artery disease. Arte-
rioscler Thromb Vasc Biol. 2020;40:1627–1634. doi: 10.1161/ATVBAHA.
120.312862
58. Schleicher M, Yu J, Murata T, Derakhshan B, Atochin D, Qian L, Kashiwagi
S, Di Lorenzo A, Harrison KD, Huang PL, et al. The Akt1-eNOS axis illus-
trates the specificity of kinase-substrate relationships in vivo. Sci Signal.
2009;2:ra41. doi: 10.1126/scisignal.2000343
Downloaded from http://ahajournals.org by on July 26, 2023
59. Ackah E, Yu J, Zoellner S, Iwakiri Y, Skurk C, Shibata R, Ouchi N, Easton
RM, Galasso G, Birnbaum MJ, et al. Akt1/protein kinase B alpha is
critical for ischemic and VEGF-mediated angiogenesis. J Clin Invest.
2005;115:2119–2127.
60. Murohara T, Asahara T, Silver M, Bauters C, Masuda H, Kalka C, Kearney
M, Chen D, Symes JF, Fishman MC, et al. Nitric oxide synthase modulates
angiogenesis in response to tissue ischemia. J Clin Invest. 1998;101:2567–
2578. doi: 10.1172/JCI1560
61. Dokun AO, Keum S, Hazarika S, Li Y, Lamonte GM, Wheeler F,
Marchuk DA, Annex BH. A quantitative trait locus (LSq-1) on mouse
chromosome 7 is linked to the absence of tissue loss after surgical
hindlimb ischemia. Circulation. 2008;117:1207–1215. doi: 10.1161/
CIRCULATIONAHA.107.736447
62. Wang T, Cunningham A, Dokun AO, Hazarika S, Houston K, Chen L, Lye
RJ, Spolski R, Leonard WJ, Annex BH. Loss of interleukin-21 recep-
tor activation in hypoxic endothelial cells impairs perfusion recovery after
hindlimb ischemia. Arterioscler Thromb Vasc Biol. 2015;35:1218–1225. doi:
10.1161/ATVBAHA.115.305476
63. Wang T, Cunningham A, Houston K, Sharma AM, Chen L, Dokun AO, Lye
RJ, Spolski R, Leonard WJ, Annex BH. Endothelial interleukin-21 receptor
up-regulation in peripheral artery disease. Vasc Med. 2016;21:99–104. doi:
10.1177/1358863X15621798
64. Leonard WJ. The Yin and Yang of Interleukin-21 in allergy, autoimmunity
and cancer. Blood. 2011;118:1809–1810.
65. Spolski R, Leonard WJ. Interleukin-21: a double-edged sword with thera-
peutic potential. Nat Rev Drug Discov. 2014;13:381–393.
66. Castermans K, Tabruyn SP, Zeng R, van Beijnum JR, Eppolito C, Leonard
WJ, Shrikant PA, Griffioen AW. Angiostatic activity of the antitumor cyto-
kine interleukin-21. Blood. 2008;112:4940–4947. doi: 10.1182/blood-
2007-09-113878
67. Palmer-Kazen U, Wariaro D, Luo F, Wahlberg E. Vascular endothelial cell
growth factor and fibroblast growth factor 2 expression in patients with
critical limb ischemia. J Vasc Surg. 2004;39:621–628. doi: 10.1016/j.
jvs.2003.07.006
68. Annex BH, Torgan CE, Lin P, Taylor DA, Thompson MA, Peters KG, Kraus
WE. Induction and maintenance of increased VEGF protein by chronic motor
Circulation Research. 2021;128:1944–1957. DOI: 10.1161/CIRCRESAHA.121.318266
Vascular Regeneration in PAD
nerve stimulation in skeletal muscle. Am J Physiol. 1998;274:H860–H867.
DISEASE COMPENDIUM
PERIPHERAL VASCULAR
doi: 10.1152/ajpheart.1998.274.3.H860
69. Wang T, Cunningham A, Dokun A, Hazarika S, Chen L, Lye R, Leonard W,
Annex B. Angiogenic properties of interleukin-21 under hypoxic conditions.
Blood. 2014: (e-letter to Castermans et al.)
70. Lee HK, Keum S, Sheng H, Warner DS, Lo DC, Marchuk DA. Natural allelic
variation of the IL-21 receptor modulates ischemic stroke infarct volume. J
Clin Invest. 2016;126:2827–2838. doi: 10.1172/JCI84491
71. Olsson AK, Dimberg A, Kreuger J, Claesson-Welsh L. VEGF receptor sig-
nalling - in control of vascular function. Nat Rev Mol Cell Biol. 2006;7:359–
371. doi: 10.1038/nrm1911
72. Cébe-Suarez S, Zehnder-Fjällman A, Ballmer-Hofer K. The role of VEGF
receptors in angiogenesis; complex partnerships. Cell Mol Life Sci.
2006;63:601–615. doi: 10.1007/s00018-005-5426-3
73. Shibuya M. Differential roles of vascular endothelial growth factor recep-
tor-1 and receptor-2 in angiogenesis. J Biochem Mol Biol. 2006;39:469–
478. doi: 10.5483/bmbrep.2006.39.5.469
74. Rebar EJ, Huang Y, Hickey R, Nath AK, Meoli D, Nath S, Chen B, Xu L,
Liang Y, Jamieson AC, et al. Induction of angiogenesis in a mouse model
using engineered transcription factors. Nat Med. 2002;8:1427–1432. doi:
10.1038/nm1202-795
75. Dai Q, Huang J, Klitzman B, Dong C, Goldschmidt-Clermont PJ,
March KL, Rokovich J, Johnstone B, Rebar EJ, Spratt SK, et al. Engi-
neered zinc finger-activating vascular endothelial growth factor tran-
scription factor plasmid DNA induces therapeutic angiogenesis in
rabbits with hindlimb ischemia. Circulation. 2004;110:2467–2475. doi:
10.1161/01.CIR.0000145139.53840.49
76. Oltean S, Bates DO. Hallmarks of alternative splicing in cancer. Oncogene.
2014;33:5311–5318. doi: 10.1038/onc.2013.533
77. Kikuchi R, Nakamura K, MacLauchlan S, Ngo DT, Shimizu I, Fuster JJ,
Katanasaka Y, Yoshida S, Qiu Y, Yamaguchi TP, et al. An antiangiogenic iso-
form of VEGF-A contributes to impaired vascularization in peripheral artery
disease. Nat Med. 2014;20:1464–1471. doi: 10.1038/nm.3703
78. Harper SJ, Bates DO. VEGF-A splicing: the key to anti-angiogenic thera-
peutics? Nat Rev Cancer. 2008;8:880–887. doi: 10.1038/nrc2505
79. Roberts DM, Kearney JB, Johnson JH, Rosenberg MP, Kumar R,
Bautch VL. The vascular endothelial growth factor (VEGF) recep-
tor Flt-1 (VEGFR-1) modulates Flk-1 (VEGFR-2) signaling dur-
ing blood vessel formation. Am J Pathol. 2004;164:1531–1535. doi:
10.1016/S0002-9440(10)63711-X
80. Shibuya M, Claesson-Welsh L. Signal transduction by VEGF recep-
tors in regulation of angiogenesis and lymphangiogenesis. Exp Cell Res.
2006;312:549–560. doi: 10.1016/j.yexcr.2005.11.012
81. Silvestre JS, Tamarat R, Ebrahimian TG, Le-Roux A, Clergue M, Emmanuel
F, Duriez M, Schwartz B, Branellec D, Lévy BI. Vascular endothelial growth
factor-B promotes in vivo angiogenesis. Circ Res. 2003;93:114–123. doi:
10.1161/01.RES.0000081594.21764.44
82. Luttun A, Tjwa M, Carmeliet P. Placental growth factor (PlGF) and its receptor
Flt-1 (VEGFR-1): novel therapeutic targets for angiogenic disorders. Ann N
Y Acad Sci. 2002;979:80–93. doi: 10.1111/j.1749-6632.2002.tb04870.x
83. Luttun A, Tjwa M, Moons L, Wu Y, Angelillo-Scherrer A, Liao F, Nagy
JA, Hooper A, Priller J, De Klerck B, et al. Revascularization of isch-
emic tissues by PlGF treatment, and inhibition of tumor angiogenesis,
arthritis and atherosclerosis by anti-Flt1. Nat Med. 2002;8:831–840.
doi: 10.1038/nm731
84. Carmeliet P, Moons L, Luttun A, Vincenti V, Compernolle V, De Mol M, Wu
Y, Bono F, Devy L, Beck H, et al. Synergism between vascular endothelial
growth factor and placental growth factor contributes to angiogenesis and
plasma extravasation in pathological conditions. Nat Med. 2001;7:575–
583. doi: 10.1038/87904
85. Bry M, Kivelä R, Holopainen T, Anisimov A, Tammela T, Soronen J, Silvola J,
Saraste A, Jeltsch M, Korpisalo P, et al. Vascular endothelial growth factor-B
acts as a coronary growth factor in transgenic rats without inducing angio-
genesis, vascular leak, or inflammation. Circulation. 2010;122:1725–1733.
doi: 10.1161/CIRCULATIONAHA.110.957332
86. Lähteenvuo JE, Lähteenvuo MT, Kivelä A, Rosenlew C, Falkevall A, Klar J,
Heikura T, Rissanen TT, Vähäkangas E, Korpisalo P, et al. Vascular endothe-
lial growth factor-B induces myocardium-specific angiogenesis and arterio-
genesis via vascular endothelial growth factor receptor-1- and neuropilin
receptor-1-dependent mechanisms. Circulation. 2009;119:845–856. doi:
10.1161/CIRCULATIONAHA.108.816454
87. Boucher JM, Clark RP, Chong DC, Citrin KM, Wylie LA, Bautch VL. Dynamic
alterations in decoy VEGF receptor-1 stability regulate angiogenesis. Nat
Commun. 2017;8:15699. doi: 10.1038/ncomms15699
June 11, 2021   1955
 Annex and Cooke
88. Chu LH, Ganta VC, Choi MH, Chen G, Finley SD, Annex BH, Popel AS.
DISEASE COMPENDIUM
PERIPHERAL VASCULAR
A multiscale computational model predicts distribution of anti-angiogenic
isoform VEGF165b in peripheral arterial disease in human and mouse. Sci
Rep. 2016;6:37030. doi: 10.1038/srep37030
89. Clegg LE, Ganta VC, Annex BH, Mac Gabhann F. Systems Pharmacology
of VEGF165b in peripheral artery disease. CPT Pharmacometrics Syst
Pharmacol. 2017;6:833–844. doi: 10.1002/psp4.12261
90. Amano H, Kato S, Ito Y, Eshima K, Ogawa F, Takahashi R, Sekiguchi K,
Tamaki H, Sakagami H, Shibuya M, et al. The role of vascular endothelial
growth factor receptor-1 signaling in the recovery from Ischemia. PLoS
One. 2015;10:e0131445. doi: 10.1371/journal.pone.0131445
91. Neth P, Nazari-Jahantigh M, Schober A, Weber C. MicroRNAs in flow-
dependent vascular remodelling. Cardiovasc Res. 2013;99:294–303. doi:
10.1093/cvr/cvt096
92. Urbich C, Kuehbacher A, Dimmeler S. Role of microRNAs in vascular dis-
eases, inflammation, and angiogenesis. Cardiovasc Res. 2008;79:581–
588. doi: 10.1093/cvr/cvn156
93. Ha M, Kim VN. Regulation of microRNA biogenesis. Nat Rev Mol Cell Biol.
2014;15:509–524. doi: 10.1038/nrm3838
94. Jonas S, Izaurralde E. Towards a molecular understanding of microR-
NA-mediated gene silencing. Nat Rev Genet. 2015;16:421–433. doi:
10.1038/nrg3965
95. van Rooij E. The art of microRNA research. Circ Res. 2011;108:219–234.
doi: 10.1161/CIRCRESAHA.110.227496
96. Bonauer A, Carmona G, Iwasaki M, Mione M, Koyanagi M, Fischer A,
Burchfield J, Fox H, Doebele C, Ohtani K, et al. MicroRNA-92a controls
angiogenesis and functional recovery of ischemic tissues in mice. Science.
2009;324:1710–1713. doi: 10.1126/science.1174381
97. Grundmann S, Hans FP, Kinniry S, Heinke J, Helbing T, Bluhm F, Sluijter JP,
Hoefer I, Pasterkamp G, Bode C, et al. MicroRNA-100 regulates neovascu-
larization by suppression of mammalian target of rapamycin in endothelial
and vascular smooth muscle cells. Circulation. 2011;123:999–1009. doi:
10.1161/CIRCULATIONAHA.110.000323
98. Hazarika S, Farber CR, Dokun AO, Pitsillides AN, Wang T, Lye RJ, Annex BH.
MicroRNA-93 controls perfusion recovery after hindlimb ischemia by mod-
ulating expression of multiple genes in the cell cycle pathway. Circulation.
2013;127:1818–1828. doi: 10.1161/CIRCULATIONAHA.112.000860
99. Caporali A, Meloni M, Völlenkle C, Bonci D, Sala-Newby GB, Addis R,
Downloaded from http://ahajournals.org by on July 26, 2023
Spinetti G, Losa S, Masson R, Baker AH, et al. Deregulation of microR-
NA-503 contributes to diabetes mellitus-induced impairment of endothe-
lial function and reparative angiogenesis after limb ischemia. Circulation.
2011;123:282–291. doi: 10.1161/CIRCULATIONAHA.110.952325
100. Yin KJ, Olsen K, Hamblin M, Zhang J, Schwendeman SP, Chen YE.
Vascular endothelial cell-specific microRNA-15a inhibits angiogen-
esis in hindlimb ischemia. J Biol Chem. 2012;287:27055–27064. doi:
10.1074/jbc.M112.364414
101. Welten SM, Bastiaansen AJ, de Jong RC, de Vries MR, Peters EA, Boonstra
MC, Sheikh SP, La Monica N, Kandimalla ER, Quax PH, et al. Inhibition of
14q32 MicroRNAs miR-329, miR-487b, miR-494, and miR-495 increas-
es neovascularization and blood flow recovery after ischemia. Circ Res.
2014;115:696–708. doi: 10.1161/CIRCRESAHA.114.304747
102. Icli B, Li H, Pérez-Cremades D, Wu W, Ozdemir D, Haemmig S, Guimaraes
RB, Manica A, Marchini JF, Orgill DP, et al. MiR-4674 regulates angiogenesis
in tissue injury by targeting p38K signaling in endothelial cells. Am J Physiol
Cell Physiol. 2020;318:C524–C535. doi: 10.1152/ajpcell.00542.2019
103. Feinberg MW. Healing the injured vessel wall using microRNA-facilitated
gene delivery. J Clin Invest. 2014;124:3694–3697. doi: 10.1172/JCI77509
104. Landskroner-Eiger S, Qiu C, Perrotta P, Siragusa M, Lee MY, Ulrich V,
Luciano AK, Zhuang ZW, Corti F, Simons M, et al. Endothelial miR-17~92
cluster negatively regulates arteriogenesis via miRNA-19 repression of
WNT signaling. Proc Natl Acad Sci U S A. 2015;112:12812–12817. doi:
10.1073/pnas.1507094112
105. Heuslein JL, McDonnell SP, Song J, Annex BH, Price RJ. MicroRNA-146a
regulates perfusion recovery in response to arterial occlusion via arteriogen-
esis. Front Bioeng Biotechnol. 2018;6:1. doi: 10.3389/fbioe.2018.00001
106. Heuslein JL, Gorick CM, McDonnell SP, Song J, Annex BH, Price RJ.
Exposure of endothelium to biomimetic flow waveforms yields identifi-
cation of miR-199a-5p as a potent regulator of arteriogenesis. Mol Ther
Nucleic Acids. 2018;12:829–844. doi: 10.1016/j.omtn.2018.08.001
107. Sun HX, Zeng DY, Li RT, Pang RP, Yang H, Hu YL, Zhang Q, Jiang Y,
Huang LY, Tang YB, et al. Essential role of microRNA-155 in regulat-
ing endothelium-dependent vasorelaxation by targeting endothelial ni-
tric oxide synthase. Hypertension. 2012;60:1407–1414. doi: 10.1161/
HYPERTENSIONAHA.112.197301
1956   June 11, 2021 Circulation Research. 2021;128:1944–1957. DOI: 10.1161/CIRCRESAHA.121.318266
Vascular Regeneration in PAD
108. Liu Q, Yang J. Expression and significance of miR155 and vascular endo-
thelial growth factor in placenta of rats with preeclampsia. Int J Clin Exp
Med. 2015;8:15731–7.
109. Pankratz F, Bemtgen X, Zeiser R, Leonhardt F, Kreuzaler S, Hilgendorf
I, Smolka C, Helbing T, Hoefer I, Esser JS, et al. MicroRNA-155 exerts
cell-specific antiangiogenic but proarteriogenic effects during adaptive
neovascularization. Circulation. 2015;131:1575–1589. doi: 10.1161/
CIRCULATIONAHA.114.014579
110. Statello L, Guo CJ, Chen LL, Huarte M. Gene regulation by long non-coding
RNAs and its biological functions. Nat Rev Mol Cell Biol. 2021;22:96–118.
doi: 10.1038/s41580-020-00315-9
111. Monteiro JP, Bennett M, Rodor J, Caudrillier A, Ulitsky I, Baker AH.
Endothelial function and dysfunction in the cardiovascular system: the long
non-coding road. Cardiovasc Res. 2019;115:1692–1704. doi: 10.1093/
cvr/cvz154
112. Yu B, Wang S. Angio-LncRs: LncRNAs that regulate angiogenesis and
vascular disease. Theranostics. 2018;8:3654–3675. doi: 10.7150/
thno.26024
113. Hung J, Miscianinov V, Sluimer JC, Newby DE, Baker AH. Targeting non-
coding RNA in vascular biology and disease. Front Physiol. 2018;9:1655.
doi: 10.3389/fphys.2018.01655
114. Miao Y, Ajami NE, Huang TS, Lin FM, Lou CH, Wang YT, Li S, Kang J,
Munkacsi H, Maurya MR, et al. Enhancer-associated long non-coding RNA
LEENE regulates endothelial nitric oxide synthase and endothelial func-
tion. Nat Commun. 2018;9:292. doi: 10.1038/s41467-017-02113-y
115. Lyu Q, Xu S, Lyu Y, Choi M, Christie CK, Slivano OJ, Rahman A, Jin ZG,
Long X, Xu Y, et al. SENCR stabilizes vascular endothelial cell adherens
junctions through interaction with CKAP4. Proc Natl Acad Sci U S A.
2019;116:546–555. doi: 10.1073/pnas.1810729116
116. Boulberdaa M, Scott E, Ballantyne M, Garcia R, Descamps B, Angelini GD,
Brittan M, Hunter A, McBride M, McClure J, et al. A role for the long non-
coding RNA SENCR in commitment and function of endothelial cells. Mol
Ther. 2016;24:978–990. doi: 10.1038/mt.2016.41
117. Fiedler J, Breckwoldt K, Remmele CW, Hartmann D, Dittrich M, Pfanne
A, Just A, Xiao K, Kunz M, Müller T, et al. Development of long noncod-
ing RNA-based strategies to modulate tissue vascularization. J Am Coll
Cardiol. 2015;66:2005–2015. doi: 10.1016/j.jacc.2015.07.081
118. Michalik KM, You X, Manavski Y, Doddaballapur A, Zörnig M, Braun T,
John D, Ponomareva Y, Chen W, Uchida S, et al. Long noncoding RNA
MALAT1 regulates endothelial cell function and vessel growth. Circ Res.
2014;114:1389–1397. doi: 10.1161/CIRCRESAHA.114.303265
119. Neumann P, Jaé N, Knau A, Glaser SF, Fouani Y, Rossbach O, Krüger M,
John D, Bindereif A, Grote P, et al. The lncRNA GATA6-AS epigenetically
regulates endothelial gene expression via interaction with LOXL2. Nat
Commun. 2018;9:237. doi: 10.1038/s41467-017-02431-1
120. Voellenkle C, Garcia-Manteiga JM, Pedrotti S, Perfetti A, De Toma I,
Da Silva D, Maimone B, Greco S, Fasanaro P, Creo P, et al. Implication
of Long noncoding RNAs in the endothelial cell response to hy-
poxia revealed by RNA-sequencing. Sci Rep. 2016;6:24141. doi:
10.1038/srep24141
121. Boon RA, Hofmann P, Michalik KM, Lozano-Vidal N, Berghäuser D,
Fischer A, Knau A, Jaé N, Schürmann C, Dimmeler S. Long noncoding
RNA Meg3 controls endothelial cell aging and function: implications for
regenerative angiogenesis. J Am Coll Cardiol. 2016;68:2589–2591. doi:
10.1016/j.jacc.2016.09.949
122. Terwilliger ZS, Ryan TE, Goldberg EJ, Schmidt CA, Yamaguchi DJ, Karnekar
R, Brophy P, Green TD, Zeczycki TN, Mac Gabhann F, et al. Racial dif-
ferences in the limb skeletal muscle transcriptional programs of patients
with critical limb ischemia. Vasc Med. 2021;6:1358863X20983918. doi:
10.1177/1358863X20983918. Epub ahead of print.
123. De Bock K, Georgiadou M, Schoors S, Kuchnio A, Wong BW, Cantelmo
AR, Quaegebeur A, Ghesquière B, Cauwenberghs S, Eelen G, et al. Role of
PFKFB3-driven glycolysis in vessel sprouting. Cell. 2013;154:651–663.
doi: 10.1016/j.cell.2013.06.037
124. Ros S, Schulze A. Balancing glycolytic flux: the role of 6-phosphofructo-
2-kinase/fructose 2,6-bisphosphatases in cancer metabolism. Cancer
Metab. 2013;1:8. doi: 10.1186/2049-3002-1-8
125. Xu Y, An X, Guo X, Habtetsion TG, Wang Y, Xu X, Kandala S, Li Q, Li
H, Zhang C, et al. Endothelial PFKFB3 plays a critical role in angiogen-
esis. Arterioscler Thromb Vasc Biol. 2014;34:1231–9. doi: 10.1161/
ATVBAHA.113.303041
126. Doddaballapur A, Michalik KM, Manavski Y, Lucas T, Houtkooper RH, You
X, Chen W, Zeiher AM, Potente M, Dimmeler S, et al. Laminar shear stress
inhibits endothelial cell metabolism via KLF2-mediated repression of
 Annex and Cooke
PFKFB3. Arterioscler Thromb Vasc Biol. 2015;35:137–145. doi: 10.1161/
ATVBAHA.114.304277
127. Zecchin A, Kalucka J, Dubois C, Carmeliet P. How endothelial cells adapt
their metabolism to form vessels in tumors. Front Immunol. 2017;8:1750.
doi: 10.3389/fimmu.2017.01750
128. Wong BW, Marsch E, Treps L, Baes M, Carmeliet P. Endothelial cell metab-
olism in health and disease: impact of hypoxia. EMBO J. 2017;36:2187–
2203. doi: 10.15252/embj.201696150
129. Cao Y, Zhang X, Wang L, Yang Q, Ma Q, Xu J, Wang J, Kovacs L, Ayon RJ,
Liu Z, et al. PFKFB3-mediated endothelial glycolysis promotes pulmonary
hypertension. Proc Natl Acad Sci U S A. 2019;116:13394–13403. doi:
10.1073/pnas.1821401116
130. Wu J, Ke X, Fu W, Gao X, Zhang H, Wang W, Ma N, Zhao M, Hao X, Zhang
Z. Inhibition of hypoxia-induced retinal angiogenesis by specnuezhenide,
an effective constituent of ligustrum lucidum Ait., through suppression of
the HIF-1α/VEGF signaling pathway. Molecules. 2016;21:E1756. doi:
10.3390/molecules21121756
131. Cao R, Jensen LD, Söll I, Hauptmann G, Cao Y. Hypoxia-induced retinal
angiogenesis in zebrafish as a model to study retinopathy. PLoS One.
2008;3:e2748. doi: 10.1371/journal.pone.0002748
132. Rajagopalan S, Mohler ER III, Lederman RJ, Mendelsohn FO, Saucedo
JF, Goldman CK, Blebea J, Macko J, Kessler PD, Rasmussen HS, et al.
Regional angiogenesis with vascular endothelial growth factor in periph-
eral arterial disease: a phase II randomized, double-blind, controlled study
of adenoviral delivery of vascular endothelial growth factor 121 in patients
with disabling intermittent claudication. Circulation. 2003;108:1933–8.
doi: 10.1161/01.CIR.0000093398.16124.29
133. Domigan CK, Warren CM, Antanesian V, Happel K, Ziyad S, Lee S, Krall A,
Duan L, Torres-Collado AX, Castellani LW, et al. Autocrine VEGF maintains
endothelial survival through regulation of metabolism and autophagy. J Cell
Sci. 2015;128:2236–48. doi: 10.1242/jcs.163774
134. McDonald DM, Baluk P. Significance of blood vessel leakiness in cancer.
Cancer Res. 2002;62:5381–5.
135. Lugano R, Ramachandran M, Dimberg A. Tumor angiogenesis: causes, con-
sequences, challenges and opportunities. Cell Mol Life Sci. 2020;77:1745–
1770. doi: 10.1007/s00018-019-03351-7
136. Leeper NJ, Hunter AL, Cooke JP. Stem cell therapy for vascular regen-
eration: adult, embryonic, and induced pluripotent stem cells. Circulation.
Downloaded from http://ahajournals.org by on July 26, 2023
2010;122:517–26. doi: 10.1161/CIRCULATIONAHA.109.881441
137. Han JK, Chang SH, Cho HJ, Choi SB, Ahn HS, Lee J, Jeong H, Youn SW,
Lee HJ, Kwon YW, et al. Direct conversion of adult skin fibroblasts to en-
dothelial cells by defined factors. Circulation. 2014;130:1168–1178. doi:
10.1161/CIRCULATIONAHA.113.007727
138. Rufaihah AJ, Huang NF, Jamé S, Lee JC, Nguyen HN, Byers B, De A,
Okogbaa J, Rollins M, Reijo-Pera R, et al. Endothelial cells derived from
human iPSCS increase capillary density and improve perfusion in a
mouse model of peripheral arterial disease. Arterioscler Thromb Vasc Biol.
2011;31:e72–9. doi: 10.1161/ATVBAHA.111.230938
139. Huang NF, Niiyama H, Peter C, De A, Natkunam Y, Fleissner F, Li Z, Rollins
MD, Wu JC, Gambhir SS, et al. Embryonic stem cell-derived endothelial
cells engraft into the ischemic hindlimb and restore perfusion. Arterioscler
Thromb Vasc Biol. 2010;30:984–91. doi: 10.1161/ATVBAHA.110.202796
140. Ginsberg M, James D, Ding BS, Nolan D, Geng F, Butler JM, Schachterle
W, Pulijaal VR, Mathew S, Chasen ST, et al. Efficient direct reprogram-
ming of mature amniotic cells into endothelial cells by ETS factors
and TGFβ suppression. Cell. 2012;151:559–575. doi: 10.1016/j.
cell.2012.09.032
141. Lee S, Park C, Han JW, Kim JY, Cho K, Kim EJ, Kim S, Lee SJ, Oh SY,
Tanaka Y, et al. Direct reprogramming of human dermal fibroblasts into
Circulation Research. 2021;128:1944–1957. DOI: 10.1161/CIRCRESAHA.121.318266
Vascular Regeneration in PAD
endothelial cells using ER71/ETV2. Circ Res. 2017;120:848–861. doi:
DISEASE COMPENDIUM
PERIPHERAL VASCULAR
10.1161/CIRCRESAHA.116.309833
142. Li Y, Zhou G, Bruno IG, Cooke JP. Telomerase mRNA reverses senes-
cence in progeria cells. J Am Coll Cardiol. 2017;70:804–805. doi:
10.1016/j.jacc.2017.06.017
143. Matsushita H, Chang E, Glassford AJ, Cooke JP, Chiu CP, Tsao PS.
eNOS activity is reduced in senescent human endothelial cells: pres-
ervation by hTERT immortalization. Circ Res. 2001;89:793–798. doi:
10.1161/hh2101.098443
144. Xia B, Zhao D, Wang G, Zhang M, Lv J, Tomoiaga AS, Li Y, Wang X, Meng S,
Cooke JP, et al. Machine learning uncovers cell identity regulator by histone
code. Nat Commun. 2020;11:2696. doi: 10.1038/s41467-020-16539-4
145. Wang G, Xia B, Zhou M, Lv J, Zhao D, Li Y, Bu Y, Wang X, Cooke JP, Cao
Q, et al. MACMIC reveals a dual role of CTCF in epigenetic regulation of
cell identity genes. Genomics Proteomics Bioinformatics. 2021;21:S1672-
0229(21)00057-7. doi: 10.1016/j.gpb.2020.10.008
146. Clayton ZE, Yuen GS, Sadeghipour S, Hywood JD, Wong JW, Huang NF,
Ng MK, Cooke JP, Patel S. A comparison of the pro-angiogenic potential
of human induced pluripotent stem cell derived endothelial cells and in-
duced endothelial cells in a murine model of peripheral arterial disease. Int
J Cardiol. 2017;234:81–89. doi: 10.1016/j.ijcard.2017.01.125
147. Sayed N, Wong WT, Ospino F, Meng S, Lee J, Jha A, Dexheimer P, Aronow
BJ Cooke JP. Transdifferentiation of human fibroblasts to endothelial cells:
role of innate immunity. Circulation. 2015;131:300–309.
148. Meng S, Zhou G, Gu Q, Chanda PK, Ospino F, Cooke JP. Transdifferentiation
requires iNOS activation: role of RING1A S-nitrosylation. Circ Res.
2016;119:e129–e138.
149. Chanda PK, Meng S, Lee J, Leung HE, Chen K, Cooke JP. Nuclear
S-nitrosylation defines an optimal zone for inducing pluripotency.
Circulation. 2019;140:1081–1099.
150. Cooke JP. Therapeutic transdifferentiation: a novel approach for vascu-
lar disease. Circ Res. 2013;112:748–50. doi: 10.1161/CIRCRESAHA.
113.301053
151. Ubil E, Duan J, Pillai IC, Rosa-Garrido M, Wu Y, Bargiacchi F, Lu Y,
Stanbouly S, Huang J, Rojas M, et al. Mesenchymal-endothelial transition
contributes to cardiac neovascularization. Nature. 2014;514:585–590.
doi: 10.1038/nature13839
152. He L, Huang X, Kanisicak O, Li Y, Wang Y, Li Y, Pu W, Liu Q, Zhang H, Tian
X, et al. Preexisting endothelial cells mediate cardiac neovascularization after
injury. J Clin Invest. 2017;127:2968–2981. doi: 10.1172/JCI93868
153. Meng S, Lv J, Chanda PK, Owusu I, Chen K, Cooke JP. Reservoir of fibro-
blasts promotes recovery from limb Ischemia. Circulation. 2020;142:1647–
1662. doi: 10.1161/CIRCULATIONAHA.120.046872
154. Lee J, Sayed N, Hunter A, Au KF, Wong WH, Mocarski ES, Pera RR,
Yakubov E, Cooke JP. Activation of innate immunity is required for efficient
nuclear reprogramming. Cell. 2012;151:547–558.
155. Lai L, Reineke E, Hamilton DJ, Cooke JP. Glycolytic switch is required for
transdifferentiation to endothelial lineage. Circulation. 2019;139:119–
133. doi: 10.1161/CIRCULATIONAHA.118.035741
156. Klarin D, Lynch J, Aragam K, Chaffin M, Assimes TL, Huang J, Lee
KM, Shao Q, Huffman JE, Natarajan P, et al; VA Million Veteran
Program. Genome-wide association study of peripheral artery disease
in the Million Veteran Program. Nat Med. 2019;25:1274–1279. doi:
10.1038/s41591-019-0492-5
157. Yoder MC, Annex BH. Does multicolor lineage tracing of endothelial cells
provide a black and white answer on clonal expansion in post-natal angio-
genesis? Circ Res. 2018;122:643–645.
158. Cooke JP. Inflammation and its role in regeneration and repair. Circ Res.
2019;124:1166–1168.
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