Peripheral Artery Disease Compendiumcircres.ahajournals.org/content/circresaha/116/9/1527.full.pdf · 1528 Circulation Research April 24, 2015 PAD is linked to that of the other common

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Peripheral 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

Peripheral Artery Disease Compendium

© 2015 American Heart Association, Inc.

Circulation Research is available at http://circres.ahajournals.org DOI: 10.1161/CIRCRESAHA.116.303566

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

Pathogenesis of the Limb Manifestations and Exercise Limitations in Peripheral Artery Disease

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

Circulation Research Compendium on Peripheral Artery Disease

Epidemiology of Peripheral Artery DiseasePathogenesis of the Limb Manifestations and Exercise Limitations in Peripheral Artery DiseaseLower Extremity Manifestations of Peripheral Artery Disease: The Pathophysiologic and Functional Implications of Leg IschemiaThe Genetic Basis of Peripheral Arterial Disease: Current Knowledge, Challenges and Future DirectionsModulating the Vascular Response to Limb Ischemia: Angiogenic and Cell TherapiesPharmacological Treatment and Current Management of Peripheral Artery DiseaseEndovascular Intervention for Peripheral Artery DiseaseSurgical Intervention for Peripheral Arterial Disease

John Cooke, Guest Editor

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 [email protected]

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1528 Circulation Research April 24, 2015

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-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 SymptomsPatients 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 ImpairmentThe 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 PADThe 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.

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


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Resting HemodynamicsVolumetric 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-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,

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 HemodynamicsIn 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

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


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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 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 DysfunctionAs 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 FactorsAlterations 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


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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 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 DysfunctionAs 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 StressInflammation 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


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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 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 AbnormalitiesNumerous 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


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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 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 RespirationThere 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 AbnormalitiesNumerous 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


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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

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

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


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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.

ConclusionsPAD 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

underlying the reduced exercise performance in these patients and potential new targets for therapeutic interventions.

DisclosuresDr 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, 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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Table 1. Mechanisms of Exercise Impairment in PAD

Healthy Physiology PAD Pathophysiology

Arterial flow Normal at rest, inadequate increment with exercise to meet metabolic demand

Endothelial and microvasculature dysfunction

Impaired endothelium-dependent vasodilation on exercise challenge

Inflammation Increase in plasma levels of numerous inflammatory mediators; inflammation-impaired actions of progenitor/satellite cell differentiation and responses and growth factors

Reactive oxygen species and oxidative/reductive stress

During ischemia, skeletal muscle mitochondria release free radicals, including superoxide and other reactive oxygen species that are derived from the oxidation–reduction cascade

Muscle structural abnormalities Muscle apoptosis and atrophy; fiber type switching; altered myosin heavy 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.


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

Pathogenesis of the Limb Manifestations and Exercise Limitations in Peripheral Artery

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Peripheral Artery Disease Compendiumcircres.ahajournals.org/content/circresaha/116/9/1527.full.pdf · 1528 Circulation Research April 24, 2015 PAD is linked to that of the other common