Peripheral Artery Disease Compendium

Circulation Research Compendium on Peripheral Artery Disease

Epidemiology of Peripheral Artery Disease
Pathogenesis of the Limb Manifestations and Exercise Limitations in Peripheral Artery Disease
Lower Extremity Manifestations of Peripheral Artery Disease: The Pathophysiologic and Functional Implications of Leg Ischemia
The Genetic Basis of Peripheral Arterial Disease: Current Knowledge, Challenges and Future Directions
Modulating the Vascular Response to Limb Ischemia: Angiogenic and Cell Therapies
Pharmacological Treatment and Current Management of Peripheral Artery Disease
Endovascular Intervention for Peripheral Artery Disease
Surgical Intervention for Peripheral Arterial Disease
John Cooke, Guest Editor

Pathogenesis of the Limb Manifestations and Exercise

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Limitations in Peripheral Artery Disease

William R. Hiatt, Ehrin J. Armstrong, Christopher J. Larson, Eric P. Brass

Abstract: Patients with peripheral artery disease have a marked reduction in exercise performance and daily
ambulatory activity irrespective of their limb symptoms of classic or atypical claudication. This review will evaluate
the multiple pathophysiologic mechanisms underlying the exercise impairment in peripheral artery disease based
on an evaluation of the current literature and research performed by the authors. Peripheral artery disease results
in atherosclerotic obstructions in the major conduit arteries supplying the lower extremities. This arterial disease
process impairs the supply of oxygen and metabolic substrates needed to match the metabolic demand generated
by active skeletal muscle during walking exercise. However, the hemodynamic impairment associated with the
occlusive disease process does not fully account for the reduced exercise impairment, indicating that additional
pathophysiologic mechanisms contribute to the limb manifestations. These mechanisms include a cascade of
pathophysiological responses during exercise-induced ischemia and reperfusion at rest that are associated with
endothelial dysfunction, oxidant stress, inflammation, and muscle metabolic abnormalities that provide opportunities
for targeted therapeutic interventions to address the complex pathophysiology of the exercise impairment in
peripheral artery disease.   (Circ Res. 2015;116:1527-1539. DOI: 10.1161/CIRCRESAHA.116.303566.)
Key Words: exercise ■ metabolism ■ peripheral artery disease ■ peripheral vascular diseases
■ physiopathology ■ therapeutics

P eripheral artery disease (PAD) is initiated by athero-

genic mechanisms common to all major vascular terri-
tories but encompasses several distinctly important clinical
sequelae.1 The global burden of PAD is large and rising,
with substantial morbidity and mortality found in high-,
middle-, and low-income countries.2 The atherosclerotic
occlusive disease underlying PAD is associated with an
exercise limitation in the majority of patients, whereas the
classic symptoms of intermittent claudication, ischemic
rest pain, and ischemic ulceration are less common mani-
festations.3 Patients with PAD are also at increased risk of
myocardial infarction, ischemic stroke, heart failure, re-
novascular hypertension, and vascular death reflecting the
systemic atherosclerotic burden.4–6 The epidemiology of

Original received September 18, 2014; revision received December 8, 2014; accepted December 17, 2014. In February 2015, the average time from
submission to first decision for all original research papers submitted to Circulation Research was 13.9 days.
From the Division of Cardiology, Department of Medicine (W.R.H., E.J.A.), CPC Clinical Research (W.R.H.), University of Colorado School of
Medicine, Aurora; Cardiovascular & Metabolic Diseases Drug Discovery Unit, Takeda Pharmaceuticals, San Diego, CA (C.J.L.); and Department of
Medicine, Harbor-UCLA Center for Clinical Pharmacology, Torrance, CA (E.P.B.).
Correspondence to William R. Hiatt, MD, Division of Cardiology, Department of Medicine, University of Colorado School of Medicine, C/O CPC
Clinical Research, 13199 E Montview Blvd, Suite 200, Aurora, CO 80045. E-mail Will.Hiatt@UCDenver.edu
© 2015 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org DOI: 10.1161/CIRCRESAHA.116.303566

1527
 1528  Circulation Research  April 24, 2015
Nonstandard Abbreviations and Acronyms
ABI ankle-brachial index
ERR estrogen related receptor
ETA endothelin receptor A
ETB endothelin receptor B
FFR fractional flow reserve
PAD peripheral artery disease
PPAR peroxisome proliferator–activated receptor
PAD is linked to that of the other common atherosclerotic
processes, such as coronary and cerebral artery diseases,
and affects 15% of the adult population aged >45 years.7
Predominant risk factors for PAD include advancing age,
smoking, and diabetes mellitus; hypertension and lipid
disorders contribute less risk when compared with their
association with ischemic stroke and myocardial infarc-
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tion.2,8 Patients with PAD have cardiovascular outcomes
similar to patients with coronary artery disease or isch-
emic stroke, with the risk of myocardial infarction, stroke,
or vascular death >5% per year.9–15
The diagnosis of PAD can be ascertained by simple hemo-
dynamic measurements, in particular, the ankle-brachial index
(ABI), a test performed by measuring the systolic blood pres-
sure in each arm and the dorsalis pedis and posterior tibial
arteries of each ankle. The highest of the dorsalis pedis or
posterior tibial artery pressures is the numerator of the ABI
specific to each leg, whereas the highest arm pressure on ei-
ther side is the denominator. A ratio of <0.90 in either leg is
considered hemodynamic evidence of PAD.16 When imaged,
PAD can be characterized as one or more arterial stenoses or
occlusions involving the distal aorta, iliac, femoral, popliteal,
or tibial arteries.17
The underlying mechanisms responsible for the functional
limitations in PAD are hemodynamic in origin, which affects
the supply of oxygen and substrates to metabolically active
tissue: skeletal muscle in the case of intermittent claudication
and skin and subcutaneous tissues in the case of critical leg
ischemia. However, additional pathophysiologic mechanisms
contribute to the limb manifestations, where a reduction in
blood flow alone does not fully account for the exercise limita-
tion. Recent research into the pathophysiology of this exercise
limitation has identified several important sequelae resultant
from the chronic hemodynamic deficit. These changes affect-
ing the vasculature and skeletal muscle distal to the primary
hemodynamic lesions likely contribute to the mechanisms of
exercise limitation in PAD. These advances are reviewed here
with an additional focus on identifying possible future thera-
peutic strategies.
Symptomatic Manifestations and Exercise
Limitations in PAD
In the coronary circulation, plaque rupture leading to acute
ischemic events is a common clinical presentation; in PAD,
progressive atherosclerosis leading to chronic stenosis and
occlusion, often in series, in the arteries supplying the lower
extremities dominates the symptomatic manifestations.6
Limb Symptoms
Patients with PAD may present with a range of limb symp-
toms. Most patients are either asymptomatic or have atypical
limb symptoms during exercise, whereas only a third have
typical symptoms of claudication.18 The clinical character-
istics of PAD have been characterized in a longitudinal co-
hort study that defined typical and atypical limb symptoms.
Typical claudication is defined by exercise-induced calf dis-
comfort that is relieved by rest.19 Atypical limb symptoms
can be characterized as exertional calf symptoms that do not
begin at rest but are otherwise not consistent with classical
claudication, such as muscle symptoms that do not include
the calves, and pain at rest and pain with exertion.18 These
symptoms should not be confused with other causes of atypi-
cal limb pain, including statin-induced myalgias, spinal ste-
nosis, and other orthopedic conditions. The pathogenesis can
usually be differentiated with a careful history and physical
examination, although some patients may have PAD concom-
itant orthopedic disease.
Atypical exertional leg symptoms are more common than
classic claudication and are also associated with decreased
overall functional exercise capacity.20 Patients with hemo-
dynamic evidence of PAD in the peripheral circulation (eg,
low ABI) consistently demonstrate a marked reduction in
peak exercise performance and daily ambulatory activity.21–23
Thus, the pathophysiology, which results in reduced exercise
performance in PAD, affects a broad spectrum of patients ir-
respective of their limb symptoms. In this context, although
patients with typical intermittent claudication have been the
best studied and the term is in common usage, the mecha-
nisms underlying the exercise limitation are likely common
to all patients regardless of the nature of their limb symptoms.
Therefore, using the term claudication to describe all symp-
tomatic patients with PAD is inappropriate, and in this review,
the functional deficit associated with PAD will be referred to
as an exercise limitation and includes otherwise asymptomatic
patients who also have exercise impairment.
Severity of the Exercise Impairment
The severity of the functional limitation in PAD has been
well characterized. On a population basis, patients with PAD
have an ≈50% reduction in peak exercise performance when
compared with an age-matched healthy cohort.21 This exer-
cise limitation is associated with marked impairments in daily
physical activity as assessed by questionnaire and daily ener-
gy expenditure.24,25 Patients with PAD also have an accelerated
functional decline over time that is related to the underlying
hemodynamic severity of the disease in the leg.26 The primary
treatment goal for all patients with PAD is cardiovascular risk
reduction, whereas on the basis of the above considerations,
an additional goal is to improve exercise performance, daily
functional activity, and quality of life.
Arterial Hemodynamics in PAD
The reduction in blood flow to the lower extremity as a conse-
quence of the arterial occlusive disease process is a contribu-
tor to the exercise impairment in PAD. Ischemia with exercise
followed by reperfusion is also an important initiator of the
subsequent pathophysiology in skeletal muscle.
 Hiatt et al   Exercise Limitation in Peripheral Artery Disease   1529
Resting Hemodynamics
Volumetric blood flow in the major conduit arteries delivering
oxygen to the lower extremities is determined by the driving
pressure, geometry of the vessel and associated stenosis, and
blood viscosity.27 Poiseuille first described these fundamental
relationships by showing that flow was inversely proportional
to the length of a tube, directly proportional to the pressure
gradient, and directly proportional to the fourth power of the
tube diameter.28 Under normal conditions, flow is laminar and
there is minimal pressure drop from the heart to the distal ar-
terial circulation. However, with an arterial stenosis, there is
a drop in pressure and flow across the stenosis. This pressure
drop is accentuated by a loss of kinetic energy because of tur-
bulent flow induced by the stenosis (Figure 1). In the lower
extremity, a series of arterial stenoses, for example, a stenosis
or occlusion in the external iliac, superficial femoral arteries,
and popliteal arteries, will be hemodynamically additive with
the overall flow limitation of the sum of the individual compo-
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nents.29,30 These hemodynamic manifestations of PAD can be
assessed with the ABI, which reflects the total hemodynamic
burden of stenoses and occlusions from the central circulation
to the ankle. Except for patients with noncompressible tibial
vessels where the ABI does not reflect the intra-arterial pres-
sure, patients with PAD will typically have an ABI <0.90 at
rest. However, these patients have no limb symptoms at rest in
the absence of more severe hemodynamic compromise caus-
ing critical leg ischemia. Under conditions of rest, skeletal
muscle oxygen demand is low and limb oxygen delivery is ad-
equate despite the drop in systolic pressure from the arm to the
ankle. Resting calf muscle blood flow in patients with claudi-
cation is similar to that in an age-matched control population,
Figure 1. Hemodynamic alterations in peripheral artery disease. Top, A healthy arterial bed with normal large vessel and
microcirculatory flow characteristics. Bottom, A peripheral artery with atherosclerotic occlusive disease. ABI indicates ankle-brachial
index. Reprinted from Hiatt and Brass.32
indicating that under the conditions of low muscle metabolic
demand at rest, flow can be maintained by a corresponding
decrease in peripheral resistance to compensate for the drop in
arterial pressure across the stenosis.31
Exercise Hemodynamics
In normal subjects during exercise, the increase in limb oxy-
gen uptake is driven by an increase in metabolic demand.33 In
healthy adults, the system is dynamic in the rest-to-exercise
transition, with blood flow increasing 10-fold at low levels of
exercise and 40-fold at high levels.34 At steady-state exercise,
this leads to a close coupling of ventilatory oxygen update in
the lungs, delivery of oxygen by the heart and arterial circula-
tion to active skeletal muscle, and muscle mitochondrial oxy-
gen uptake at the tissue level.35 In patients with PAD, there is a
markedly blunted flow response in the rest-to-exercise transi-
tion that plateaus at flows well below that obtained in healthy
subjects. This flow limitation is the result of the fixed stenoses
becoming flow limiting (a concept termed critical arterial ste-
nosis). Furthermore, the vasodilator response in the peripheral
circulation is also abnormal and is unable to fully compensate
for the fixed resistance lesions.36,37
A related approach to understanding the change in arterial
hemodynamics with exercise is the fractional flow reserve
(FFR), which is the ratio of the flow distal to a stenosis di-
vided by the pressure proximal to the stenosis under condi-
tions of maximal vasodilation. For example, an FFR of 0.80
indicates that the stenotic vessel can only deliver 80% of the
flow of a normal vessel when the distal arteriolar bed is maxi-
mally vasodilated. The vasodilator response in PAD can also
be impaired by endothelial dysfunction, as discussed below. In
humans with coronary artery disease, these relationships have
 1530  Circulation Research  April 24, 2015
been confirmed to have physiological significance for quanti-
fying coronary ischemia.38 In the coronary circulation, clinical
studies have shown that an FFR of <0.80 is physiologically
significant and that this FFR value identifies a clinically sig-
nificant cutoff point where symptomatic patients benefit from
coronary revascularization.39 In patients with claudication,
initial studies have shown that FFR measurements correlate
with the lesion peak systolic velocity, a noninvasive measure-
ment of lesion severity.40 Further studies will be necessary to
correlate the clinical use of FFR measurements to potentially
guide revascularization or other treatment strategies in PAD.
A complementary approach to determining the physiologi-
cal significance of flow limitations in patients with PAD is
the assessment of tissue oxygenation. Blood-oxygen-level–
dependent MRI imaging techniques can measure the extent
of tissue deoxygenation during postocclusive reactive hyper-
emia.41 Near-infrared spectroscopy has also been used to make
dynamic assessments of tissue oxygenation in patients with
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PAD. For example, the phosphodiestersase-5 inhibitor silde-
nafil improved muscle oxygenation as assessed by near-infra-
red spectroscopy without improving walking performance in a
small study of patients with PAD.42 These approaches have the
potential to differentiate the effect of oxygen delivery versus
oxygen use on muscle dysfunction.
Endothelial and Microcirculatory Dysfunction
As described above, optimizing muscle blood flow during ex-
ercise is dependent on vasodilation in the limb to minimize
the overall resistance to flow delivery to active muscle. This
vasodilation is an active process with several important vaso-
active mediators, including nitric oxide.43 Patients with PAD
have significant abnormalities in endothelium-dependent va-
sodilation.44,45 Oxidant stress leads to generation of superox-
ide anion and other mediators of endothelial dysfunction.10,46
Exercise-induced release of endothelin-1, a potent vasocon-
strictor, can also antagonize exercise-induced skeletal muscle
vasodilation.47,48 Through these mechanisms, impaired endo-
thelial function as assessed by flow-mediated dilation has been
correlated with the clinical severity of PAD.49,50 Furthermore,
in studies where leg blood flow was measured by using a
thermodilution catheter, there was a correlation (r=0.71) be-
tween maximal leg blood flow and performance on a bicycle
ergometer.31
Interestingly, endothelial dysfunction was recently associat-
ed with walking impairment independent of the ABI, suggest-
ing that endothelial dysfunction may contribute to the exercise
impairment in PAD.49 Other studies have suggested a correla-
tion between ABI and endothelial dysfunction as assessed by
flow-mediated dilation.51 Exercise training, which improves
functional status in patients with PAD, also improves flow-
mediated dilation.52 A recent study of patients undergoing a
12-week exercise training program demonstrated an improve-
ment in flow-mediated dilation compared with baseline, sug-
gesting that improvements in endothelial function may be one
mechanism mediating reduced symptoms of claudication after
exercise training.53 In addition, in an animal model of chronic
atherosclerosis that simulated some features of PAD, a phar-
macological intervention that improved exercise-induced
blood flow also improved the animals’ functional status.54 The
impaired vasodilation in patients with PAD is likely a sequela
of the obstruction, and improvement in physiological vasodi-
lation with exercise offers a potential therapeutic target.
Microcirculatory dysfunction and local skeletal muscle
flow can also be assessed by novel noninvasive imaging tech-
niques, including arterial spin–labeling MRI and contrast-en-
hanced ultrasound. In a pilot study, arterial spin–labeling MRI
demonstrated that microvascular flow was relatively preserved
among patients with mild PAD (ABI>0.50), but more severe
PAD (ABI<0.50) was associated with a significant decrement
in microvascular blood flow.55 This technique can also be used
to image calf muscle flow at peak exercise and may be a useful
tool to assess changes in calf blood flow after therapeutic in-
terventions.56 In another small study, perfusion imaging of the
skeletal muscle microcirculation was performed by contrast-
enhanced ultrasound at rest and with exercise. In a multivari-
ate model, the treadmill time to the onset of claudication was
correlated with microcirculatory exercise flow and the differ-
ence between resting and exercise microcirculatory flow.57
Contrast-enhanced MRI measures of peak exercise blood flow
have also been shown to correlate with exercise performance
and 6-minute walk times.58
Rheological Factors
Alterations in blood hemorheology can also impair arte-
rial flow. Changes in the blood concentration of fibrinogen
and von Willebrand factor can increase blood viscosity, par-
ticularly at the microcirculatory level, which according to
Pousseille law could contribute to reduced arterial flow—but
less so than a change of equal magnitude in the percent steno-
sis.59,60 Activated leukocytes may also promote microvascular
plugging and thrombosis, thereby further increasing the re-
sistance to arterial flow. In a large cohort of patients from the
Edinburgh Artery Study, blood viscosity and fibrinogen were
independently associated with hemodynamic disease severity
of PAD, and fibrin degradation products were also associated
with progression of PAD.61,62 These findings suggest that in-
creased blood viscosity may be a component of the functional
limitation or simply secondary to the systemic atherosclerotic
disease process.
Existing Medical Therapy for the Exercise
Limitation in PAD
Current medical therapy for the exercise limitation in PAD is
limited. Reasonable evidence exists for the use of phospho-
diesterase type 3 inhibition, particularly cilostazol. Several
meta-analyses have demonstrated a consistent clinical benefit
of this medication despite a lack of detailed understanding of
its mechanism of disease benefit.63 Specifically, it is not clear
why either the inhibition of platelet aggregation or the stimu-
lation of vasodilation—both mediated by phosphodiesterase
type 3 inhibition—would translate into functional benefit in
patients with PAD. As noted above, a nonspecific vasodilator,
such as cilostazol, has the potential for steal of flow by health-
ier, more responsive vessels. Also, no convincing explanation
exists for why the clinical benefits of phosphodiesterase type
3 inhibition continue to increase during 24 weeks of treat-
ment when the direct pharmacological effects—vasodilation
and inhibition of platelet aggregation—are immediate.64 This
 Hiatt et al   Exercise Limitation in Peripheral Artery Disease   1531
time-dependent improvement suggests a complex physiologi-
cal response to treatment. Although modest improvements in
ABI have been observed with phosphodiesterase inhibition, it
is unlikely that this effect alone accounts for the mechanism of
benefit.65 The efficacy signals from 2 additional phosphodies-
terase type 3 inhibitors support a disease benefit for the class,
albeit one that is not completely understood.66,67
Older medications, such as pentoxifylline and naftidrofuryl,
are approved for use in patients with PAD within and outside
of the United States, respectively. The overall data supporting
any functional benefit for treatment of patients with PAD with
pentoxifylline are weak.68,69 The limited benefit of naftidro-
furyl in patients with PAD also cannot be linked to any well-
defined mechanism of action.69–71
Exercise training has been a mainstay of treatment for PAD,
with a well-established benefit during a typical 12-week train-
ing program.72 Exercise training may affect several pathways
associated with clinical benefit, including improved skeletal
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muscle metabolism, endothelial function, and the biomechan-
ics of gait.73,74 Given the established efficacy of exercise train-
ing, it would be logical to use pharmacotherapy as an adjunct
to structured exercise rehabilitation in patients with PAD.
Candidates for pharmacotherapy might include drugs with
mechanisms that augment or complement the beneficial ef-
fects of training. In part to due to lack of good candidates and
the complexity of studying drug therapy against a background
of exercise training, there is a paucity of data addressing this
hypothesis. A randomized trial of exercise training added to
optimal medical therapy, including cilostazol, demonstrated
a clear additive benefit of training, but the design did not
address if cilostazol added benefit compared with exercise
training without the drug.75 In addition, a study combining
treatment with propionyl-l-carnitine and exercise training
failed to show significantly better improvement in walking
performance when compared with exercise training alone.76
Endovascular revascularization is a commonly used inter-
vention to improve skeletal muscle blood flow. When provided
on a background of best medical therapy, revascularization of
aortoiliac disease results in improved peak walking time and
associated parameters of quality of life, and the magnitude of
benefit is similar to that achieved with supervised exercise.75
Cellular and Molecular Pathophysiology of the
Exercise Impairment in PAD
Although the primary pathophysiology of the exercise limita-
tion in PAD is initiated by the underlying arterial occlusive
disease and associated reduction in exercise blood flow and
oxygen delivery, the correlation is not strong between the flow
limitation versus the observed decrease in exercise perfor-
mance. Studies examining the resting ABI showed a weak to
no correlation with peak treadmill exercise performance.22,77,78
In addition, interventions, such as exercise training, that lead
to a large increase in peak exercise performance have little ef-
fect on skeletal muscle blood flow or ABI.79 Thus, although the
occlusive atherosclerotic disease process is associated with a
well-defined limitation of blood flow and oxygen delivery to
exercising skeletal muscle, the absence of a strong correla-
tion between the arterial obstruction and exercise performance
indicates that factors other than changes in limb vascular re-
sistance contribute to the exercise impairment in patients with
PAD. Underlying mechanisms that may induce factors that
are linked to the functional impairment include inflammation
and oxidant stress with subsequent endothelial and microcir-
culatory dysfunction, skeletal muscle structural abnormalities,
altered oxygen coupling and mitochondrial respiration, and
skeletal muscle metabolic abnormalities. A better understand-
ing of these pathways may help guide novel medical therapies
for the treatment of PAD and improve functional outcomes in
patients with claudication.
Endothelial and Microcirculatory Dysfunction
As described above, patients with PAD have significant ab-
normalities in endothelium-dependent vasodilation.44,45 A
potential therapeutic strategy with respect to endothelial and
microcirculatory dysfunction is antagonism of the release or
action of endothelin. Endothelin can bind to either endothelin
receptor A (ETA) or endothelin receptor B (ETB) subtypes
1 and 2. ETA receptors are located on the smooth muscle
cells of vascular tissue, and endothelin binding causes vaso-
constriction through agonist-induced receptor-mediated sig-
naling.80 ETB receptor action is more complex, with ETB1
mediating vasodilation through nitric oxide release, whereas
ETB2 causes vasoconstriction. Thus, antagonism of vasocon-
striction mediated by ETA represents a potential therapeutic
strategy to improve microcirculatory flow in PAD. However,
the resultant vasodilator effect will be of benefit only if it im-
proves microvascular flow to ischemic muscle and does not
induce a steal phenomenon. Several selective ETA receptor
antagonists are available for clinical testing. Ambrisentan is
an ETA selective antagonist approved in the United States and
Europe for the treatment of pulmonary arterial hypertension.81
Two other candidates for testing would be the ETA and ETB
dual antagonists macitentan and bosentan, which are both also
approved for the treatment of pulmonary artery hypertension.
Macitentan represents an intermediate pharmacological pro-
file between bosentan and ambrisentan, with a 50-fold selec-
tivity for the ETA subtype compared with the ETB subtype.82,83
Given the high prevalence of diabetes mellitus in patients
with PAD, it is interesting to note that studies have also sug-
gested that pioglitazone could improve vascular function.84–86
The relevance of these changes to functional status in patients
with PAD remains unknown.
Inflammation and Oxidant Stress
Inflammation is a key mediator of the atherosclerotic process
and likely contributes to the limb manifestations of PAD as
well.9,10 In addition, the mismatch between oxygen demand
and oxygen delivery during exercise in patients with PAD
induces an inflammatory response.10 In PAD, exercise is as-
sociated with an increase in plasma levels of numerous in-
flammatory mediators, including thiobarbituric acid–reactive
substances (formed as a byproduct of lipid peroxidation),
thromboxane, interleukin-8, tumor necrosis factor-α, soluble
intercellular adhesion molecule-1, vascular cell adhesion mol-
ecule-1, von Willebrand factor, E-selectin, and thrombomodu-
lin.87–93 Inflammatory mediators can aggravate endothelial
dysfunction, and markers, such as interleukin-6, are inversely
 1532  Circulation Research  April 24, 2015
correlated with maximum treadmill performance.94 When ex-
ercise impairment was evaluated by a series of walking tests,
higher serum C-reactive protein and serum amyloid A concen-
trations were associated with reductions in 6-minute-walk dis-
tance, 4-meter walking velocity, and a summary performance
score that combined performance in walking speed, stand-
ing balance, and time for 5 repeated chair rises.95 Elevated
C-reactive protein levels were also associated with a greater
annual decline in 6-minute walk performance for a period of 3
years and peak walking time.96,97
During ischemia, skeletal muscle mitochondria release free
radicals, including superoxide and other reactive oxygen spe-
cies, that are derived from the oxidation–reduction cascade.98
Reperfusion of ischemic muscle after exercise may also lead
to an increase in oxidant stress.99,100 These reactive oxygen
species have the potential to trigger numerous pathophysi-
ologic pathways, including endothelial dysfunction and cova-
lent modification of muscle macromolecules. Over time, this
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oxidant stress can also lead to mitochondrial DNA injury as
demonstrated in patients with PAD.101,102 Mitochondrial DNA
injury can also be detected in the less affected limbs of pa-
tients with unilateral PAD, suggesting that the mitochondrial
injury may reflect systemic inflammation and not simply local
skeletal muscle ischemia. Although exercise acutely induces
oxidant stress in patients with PAD, exercise training has con-
sistently been shown to improve symptoms among patients
with PAD. Although this may seem to be contradictory, it is
important to differentiate the effects of acute versus training
exercise paradigms.103 For example, in model systems, exercise
training has been shown to upregulate modulators of oxidative
stress, including superoxide dismutase, inducible nitric oxide
synthase, and thioredoxin.104–106 This modulation of oxidative
stress defense mechanisms may be an important mechanism
for the benefit of exercise training in patients with PAD.
Inflammatory mediators may also have proangiogenic and
antiangiogenic effects, potentially modulating the endogenous
response to ischemia.107 For example, circulating concentra-
tions of vascular endothelial growth factor are decreased in
patients with PAD when compared with controls with other
comorbid conditions.93
Innovative anti-inflammatory therapies developed for other
indications may have relevance for PAD. For example, inter-
cellular adhesion molecules and vascular cell adhesion mol-
ecules play important roles in intracellular signaling and the
immune and inflammatory responses and are elevated in pa-
tients with PAD.108 Antagonists of vascular cell adhesion mol-
ecule and intercellular adhesion molecule function have been
developed that could be tested for efficacy in PAD. In particu-
lar, antagonists of very late antigen-4 have shown promise in
treating inflammatory disorders in several animal models and
could be advanced into clinical trials for this indication.109–112
Selectins, also elevated in PAD populations, are a set of
cell adhesion molecules consisting of 3 subsets: E-selectins
expressed on endothelial cells in skin microvessels after ex-
posure to cytokines, L-selectins basally expressed on leuko-
cytes, such as granulocytes and monocytes, and P-selectins
basally expressed on platelets and endothelial cells. Recently,
inclacumab, a potent and selective P-selectin–neutralizing
antibody, has completed safety, tolerability, pharmacokinetic,
and pharmacodynamic studies showing reduction in myo-
cardial damage after percutaneous coronary intervention for
non–ST-segment–elevation myocardial infarction.113 Whether
modulating P-selectin in PAD has longer term clinical benefit
has not yet been studied. In addition, inclacumab has been
demonstrated to reduce elevated circulating platelet–leuko-
cyte aggregate levels in patients with PAD, thus providing
pharmacodynamic data useful for hypothesis generation.114
The angiotensin-converting enzyme inhibitor ramipril re-
cently has been reported to improve placebo-corrected walk-
ing distance.115 However, the placebo group in this study
did not show the usual walking performance improvements
typically seen in these trials, which may have inflated the
net benefit of the drug over placebo. Potential mechanisms
of ramipril-mediated improvement in walking performance
include associations with an increase in angiogenic biomark-
ers and reduction in the markers of thrombosis, inflammation,
and leukocyte adhesion.116 These biomarker changes may re-
flect modulation of more fundamental processes. As a meta-
analysis of angiotensin-converting inhibition did not find any
benefit on exercise performance recently,117 it seems unlikely
that renin–angiotensin–aldosterone system pathway interven-
tion provides consistent functional benefit.
Metabolic agents, such as propionyl-l-carnitine, have also
shown a signal of benefit but have not been fully developed
as an approved pharmacological therapy.118 Although several
large studies have demonstrated the atherosclerotic disease–
modifying benefit of statin treatment, which may include
actions through anti-inflammatory pathways, inconsistent
benefits on exercise performance have been observed in
PAD.119–121
Skeletal Muscle Structural Abnormalities
Numerous pathological changes have been identified in the
skeletal muscle of patients with PAD, including muscle apop-
tosis and atrophy, increased fiber type switching, altered
myosin heavy-chain expression, and muscle fiber denerva-
tion.122–125 These structural changes may be mediated, in part,
by higher levels of inflammatory mediators in PAD.126 On the
basis of biopsy specimens from the gastrocnemius muscles of
patients with PAD, ≈4% of gastrocnemius cells are apoptotic,
and caspase-3 levels are twice as high as in patients without
PAD.125 Selective fiber type switching from type I (aerobic)
to type II (glycolytic) fibers may impair skeletal muscle per-
formance and has been associated with decreased exercise
tolerance.123 Decreased expression of myosin heavy-chain iso-
form II in patients with PAD may also result in altered muscle
contraction kinetics and decreased cellular efficiency.127 The
observation of muscle fiber denervation among patients with
PAD has also led to the hypothesis that arterial insufficiency
coexists with a distal motor neuron neuropathy that worsens
muscle weakness independent of arterial flow.128 Consistent
with this hypothesis, impaired peroneal nerve conduction
velocity was associated with decreased calf muscle area and
lower 6-minute walk distance among patients with PAD.129
Numerous studies have correlated changes in calf muscle
ultrastructure and overall muscle strength with subsequent car-
diovascular outcomes in patients with claudication. Computed
 Hiatt et al   Exercise Limitation in Peripheral Artery Disease   1533
tomographic measurements of the content of muscle and fat
in the calf have shown that calf muscle content is inversely
associated with loss of mobility, even after adjusting for other
risk factors and comorbidities.130 During long-term follow-up,
a reduced calf muscle content was associated with increased
all-cause and cardiovascular mortality independent of the
ABI.131 Overall leg strength has also been shown to associate
with mortality in men, but not women, with PAD.132 Taken to-
gether, these findings suggest that the morphological changes
in calf muscle predispose to significant functional decline and
may be an additional marker for increased risk among patients
with symptomatic PAD.
One approach for modulating muscle apoptosis and atrophy
is through exploitation of the myostatin pathway. Multiple
new pharmacological agents that target this pathway are al-
ready in clinical development, including the soluble ActRIIB-
Fc fusion decoy receptor ramatercept,133,134 antimyostatin
antibody MYO-029, and the ActRIIB antibody BYM338.135–137
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However, previous studies have shown that although endur-
ance training improves function in patients with PAD, strength
training does not result in the same degree of improvement.
This discordance between the strong predictive value of en-
dogenous muscle mass and the poor response to interven-
tion may mean that endogenous muscle mass is an integrated
marker of the complex limb changes occurring with chronic
PAD. Thus, the likelihood of success of a muscle mass–based
strategy alone may be low.74 Furthermore, this strategy is like-
ly to be associated with systemic adverse events as exempli-
fied by the epistaxis, gum bleeding, and skin vessel dilation
observed in patients treated with ramatercept.133
Alterations in Oxygen Coupling and Mitochondrial
Respiration
There is a tight coupling between pulmonary oxygen uptake,
circulatory delivery of oxygen, and muscle mitochondrial
respiration in healthy subjects during steady-state exercise.
The transition from rest-to-exercise causes a rapid increase
in the cellular degradation of ATP to ADP to support mus-
cle contraction. Regeneration of ATP is initially supported
by nonoxidative mechanisms, including a rapid depletion of
phosphocreatine. Oxidative resynthesis of ATP occurs more
slowly and requires delivery of oxygen to exercising skeletal
muscle. The delay in oxidative adjustment to a new metabolic
demand has been termed metabolic inertia.138
The transition to increased oxygen consumption can be
characterized by several noninvasive measurements, including
the kinetics of pulmonary oxygen consumption at the onset of
exercise and the rate of hemoglobin desaturation in exercising
skeletal muscle. In healthy subjects at the onset of exercise,
these kinetic assessments are an attempt to characterize rates
of mitochondrial oxidative metabolism rather than overall
oxygen delivery.138 In patients with PAD, a profound prolon-
gation of the kinetic rates of pulmonary oxygen consumption
and tissue hemoglobin desaturation have been described at
the onset of exercise relative to healthy, age-matched con-
trols.139,140 This prolonged kinetic response associated with
the reduced exercise performance in subjects with PAD sug-
gests that alterations in skeletal muscle mitochondrial respira-
tion may contribute to the decreased exercise performance in
PAD.141 Studies have also been performed using 31P magnetic
resonance spectroscopy in PAD. When assessed at the onset
of exercise, changes in muscle pH and phosphocreatine con-
centration suggested inefficient oxidative metabolism neces-
sitating a higher rate of ATP turnover for given power output
as observed in control muscle.142 Taken together, these obser-
vations demonstrate increased metabolic inertia in patients
with PAD and provide evidence of impaired muscle metabolic
function in PAD.
Other evidence supports impaired mitochondrial function
as an important factor in functional impairment in PAD. The
electron transport chain in mitochondria oxidizes nicotinamide
adenine dinucleotide in a series of enzymatic steps that trans-
fer electrons to oxygen (Figure 2). Key mitochondrial path-
ways, including the electron transport chain, are vulnerable to
free-radical injury.143 Skeletal muscle from the affected leg in
patients with PAD has reduced the activities of mitochondrial
nicotinamide adenine dinucleotide dehydrogenase of complex
I and ubiquinol-cytochrome c oxidoreductase (complex III).144
The impairment in complex III of the electron transport chain
may result in a higher redox potential to maintain electron
flux, which may in turn lead to free-radical leakage in the mi-
tochondria. Catalase and superoxide dismutase minimize the
deleterious effects of hydrogen peroxide and superoxide, re-
spectively, by catalyzing the conversion of these species into
oxygen and hydrogen peroxide, which is subsequently con-
verted to water. Damage ensues when reactive oxygen species
levels exceed the reductive capacity of the cell.145 These ob-
servations suggest that electron transport chain activity is im-
paired, likely due to the ischemia-reperfusion injury in PAD,
which may contribute to propagation of oxidative injury and
metabolic dysfunction. Future strategies to modulate mito-
chondrial respiratory function and efficiency could represent
novel therapeutic targets for patients with PAD.
Skeletal Muscle Metabolic Abnormalities
Numerous alterations in metabolic pathways have been stud-
ied in the skeletal muscle of patients with PAD. A commonly
used experimental paradigm has been used to identify patients
with predominant evidence of hemodynamic abnormalities in
one leg with no ABI abnormality or claudication symptoms in
the contralateral leg. This allowed comparisons between the
affected leg, an apparently unaffected leg in the same patient,
and an age-matched control population. These studies have
identified many key metabolic abnormalities in PAD.
Early observations focused on alterations in muscle car-
nitine metabolism. Under normal metabolic conditions, fuel
substrates, such as fatty acids, proteins, and carbohydrates,
are converted into acyl-CoAs as intermediates in their com-
plete oxidation. These coenzyme A–coupled intermediates
are linked to the cellular carnitine pool through the reversible
transfer of acyl groups between carnitine and coenzyme A.146
One of the functions of carnitine is to serve as a buffer for the
acyl-CoA pool through the formation of acylcarnitines. Under
conditions of metabolic stress, incomplete oxidation or incom-
plete use of acyl-CoAs leads to their accumulation. Transfer
of the acyl group to carnitine results in the accumulation of the
corresponding acylcarnitine, which can be measured.147 Thus,
an increase in the concentration of acylcarnitines in plasma or
 1534  Circulation Research  April 24, 2015
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muscle in PAD is consistent with abnormal, incomplete oxida-
tive metabolism of fuel substrates.
In patients with PAD, initial observations demonstrated ac-
cumulation of short-chain acylcarnitines in plasma that was
inversely correlated with exercise performance.148 Subsequent
studies evaluated changes in muscle carnitine metabolism us-
ing gastrocnemius biopsy specimens. These studies confirmed
the accumulation of acylcarnitines in the affected skeletal
muscle with no evidence of accumulation in the unaffected
muscle.149 Acylcarnitine accumulation was also associated
with reduced peak treadmill exercise performance and was
a stronger predictor of exercise performance than the ABI.149
Muscle lactate levels are also significantly increased in the
skeletal muscle of patients with PAD as a result of incomplete
oxidation of glucose. Lactate accumulation likely reflects high
nicotinamide adenine dinucleotide content (because of im-
paired entry into the electron transport chain) and decreased
pyruvate dehydrogenase activity.150
Pharmacological approaches to restoring mitochondrial
function have been reviewed recently.151 These approaches
could include modifying mitochondrial biogenesis, mito-
chondrial dynamics, mitophagy, or the mitochondrial unfold-
ed protein response. Many therapeutic hypotheses focused
on mitochondrial function can be formulated and tested in
PAD based on the established efficacy of exercise training,
as training likely affects mitochondrial function in PAD.
Mitochondria from subjects with PAD who undergo exercise
training demonstrate improved oxidation of pyruvate,152 and
exercise training results in a redistribution of the carnitine
Figure 2. Metabolic and mitochondrial
abnormalities in peripheral artery
disease. FAD indicates flavin adenine
dinucleotide; and NAD, nicotine adenine
dinucleotide. Reprinted from Hiatt and
Brass.32
pool from acylcarnitine to carnitine, indicating an improve-
ment in complete substrate oxidation.73
Peroxisome proliferator–activated receptor (PPAR)-α and
PPAR-δ both modulate mitochondrial expression, and their
levels correlate well with type I fiber levels and endurance
training and capacity.153,154 Although agonists of PPAR-δ, such
as L165041 and GW501516, have not advanced substantially
through clinical development because of concerns of neoplas-
tic risk,155 the PPAR-α agonist fibrates have been marketed
for many years. Fibrates could be used to test the therapeutic
hypothesis that a PPAR-α agonist in patients with PAD would
improve skeletal muscle in type I oxidative capacity and that
this enrichment will translate into improved walking perfor-
mance in PAD.
Several molecular interventions that modulate a subset of
targets for mitochondrial biogenesis beyond the PPAR family
are in preclinical development. These include the sirtuins and
5′ AMP-activated protein kinase pathway,156 estrogen related
receptor (ERR)-α,157 estrogen related receptor (ERR)-γ,158
and nuclear respiratory factor 1.159 Although the use of the 5′
AMP-activated protein kinase activator 5-aminoimidazole-
4-carboxamide ribonucleotide for doping in cycling and other
professional sports is suspect, clinical trial results assessing
its effect on performance have been generally disappoint-
ing.160 However, lack of benefit in healthy subjects may not
exclude effectiveness in patients with PAD. The indirect 5′
AMP-activated protein kinase activator R118 has improved
exercise performance and vascular insufficiency in high-fat
fed mice.54 Although the overlapping metabolic functions of
 Hiatt et al   Exercise Limitation in Peripheral Artery Disease   1535

Table 1.  Mechanisms of Exercise Impairment in PAD
Healthy Physiology
Arterial flow
Endothelial and microvasculature
dysfunction
Inflammation
Reactive oxygen species and
oxidative/reductive stress
Muscle structural abnormalities
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PAD Pathophysiology
Normal at rest, inadequate increment
with exercise to meet metabolic
demand
Impaired endothelium-dependent
vasodilation on exercise challenge
Increase in plasma levels of numerous
inflammatory mediators; inflammation-
impaired actions of progenitor/satellite
cell differentiation and responses and
growth factors
During ischemia, skeletal muscle
mitochondria release free radicals,
including superoxide and other reactive
oxygen species that are derived from
the oxidation–reduction cascade
Muscle apoptosis and atrophy; fiber
type switching; altered myosin heavy
underlying the reduced exercise performance in these patients
and potential new targets for therapeutic interventions.
Disclosures
Dr Hiatt reports grant awards in the past 2 years to CPC Clinical
Research (a nonprofit academic research organization and affiliated
to the University of Colorado) from the following sponsors: Aastrom,
AstraZeneca, Bayer, National Institutes of Health, CSI, Cytokinetics,
DNAVEC, Kowa, Kyushu University, Merck, Pluristem, Regeneron,
Rigel, and Takeda. Dr Armstrong reports being a consultant in the
past 2 years to Pfizer, Abbott Vascular, and Spectranetics. Dr Larson
is an employee of Takeda Pharmaceuticals Company, Inc. Dr Brass
reports being a consultant in the past 2 years to GlaxoSmithKline,
Novartis, McNeil Consumer Pharmaceuticals, Novo Nordisk,
3D Communications, Catabasis Pharmaceuticals, Allergan,
NovaDigm Therapeutics, Bayer, Endo Pharmaceuticals, Amgen,
Boston Scientific, World Self-Medication Institute, Merck, NPS
Pharmaceuticals, HeartWare International, Takeda Pharmaceuticals,
Genzyme, Consumer Healthcare Products Association, DepoMed,
Cangene, Aveo Oncology, BioMarin, Galderma, QRx Pharma,
University of Washington, Amarin, EnteroMedics, Acerta, Cerexa,

chain expression; fiber denervation
Muscle metabolic abnormalities Altered oxygen coupling and
mitochondrial respiration: in patients
with PAD, prolongation of the kinetic
rates of oxygen consumption, and
tissue hemoglobin desaturation has
been described at the onset of exercise
PAD indicates peripheral artery disease.
ERRα and ERRγ suggest potential benefit for patients with
PAD, breast cancer association studies suggest that ERRα
and ERRγ have differing effects on cancer progression, with
high levels of ERRα associated with expression of ErbB2, in-
creased risk of recurrence, and adverse clinical outcomes.161,162
Thus, careful choices of pharmacological agents will need to
be made if mitochondrial biogenesis in patients with PAD will
be attempted through ERR modulation. At this time, there is
minimal literature reporting ERRγ modulators likely to be
useful for experimental medicine hypothesis testing, leaving
only small molecules associated with the unclear benefit/risk
ratio of ERRα modulation available for use in the near future.
Likewise, no facile methods to clinically modulate the impor-
tant regulator of mitochondrial expression nuclear respiratory
factor 1 exist at this time. Thus, both basic research and drug
discovery in the area of mitochondrial biogenesis are newly
needed to generate hypotheses and new targets for clinical
testing.
Conclusions
PAD represents a complex pathophysiology that results in
significant functional limitations in exercise performance. A
dominant mechanism in the initiation of the exercise limita-
tion is a reduction in exercise blood flow and oxygen delivery
to exercising skeletal muscle in the lower extremity. This flow
limitation is also associated with skeletal muscle ischemia with
walking exercise and reperfusion with rest that set up a cas-
cade of pathophysiological responses leading to further tissue
injury, endothelial dysfunction, muscle metabolic abnormali-
ties, and other changes that further reduce exercise capacity.
Understanding these sequelae offers insights into mechanisms
Kythera Biopharmaceuticals, and Trius Therapeutics. Dr Brass has
equity in Calistoga Pharmaceuticals and Catabasis Pharmaceuticals.
None of the authors received financial support for this article.
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 Pathogenesis of the Limb Manifestations and Exercise Limitations in Peripheral Artery
Disease
William R. Hiatt, Ehrin J. Armstrong, Christopher J. Larson and Eric P. Brass
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Circ Res. 2015;116:1527-1539

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