In addition to this, plasma markers of endothelial and microvascular function are often measured, although up to date this has not been extensively studied in the PAD population. Evaluation of certain pathways and new management strategies in PAD may adopt assessment of endothelial function as a surrogate end-point. This suggests that there is a requirement for a standardized method of assessing endothelial function, specific for PAD, which is clinically and cost-effective.
Assessment of endothelial function in PAD has employed a variety of other methods which may provide indirect information. Migration and adhesion of cells have been studied by measuring plasma and cell adhesion molecule concentrations, whereas platelet function has been assessed by aggregometry and monocyte activation by measuring cytokine levels. These methods may provide an insight into the mechanisms of atherothrombosis management in PAD, but are of little relevance to vasomotion and claudication symptoms.
Endothelial function is traditionally assessed by the responsiveness of a conduit artery to intra-arterial infusion of endothelium-dependent vasodilators. This method has the advantage of directly measuring changes in vessel calibre, and can be used to study large and small arteries. Ultrasound in conjunction with local drug delivery, plethysmography, and iontophoresis have been used to investigate peripheral vasomotor responsiveness, but the clinical utility of these techniques is limited by cost and availability.
Microvascular function could significantly contribute to exercise intolerance in patients with PAD. While endothelial function mainly determines wall shear stress and hence vasodilation, it is not the sole determinant of the latter. Impaired myogenic constriction and structural alterations of resistance arteries may affect vasodilator reserve and contribute to symptoms of intermittent claudication.
Overview of Peripheral Arterial Disease (PAD)
In addition to this huge impact, it is known that the cardiovascular events in patients with PAD are equivalent to those with coronary artery disease, and these patients have a higher risk of death compared to patients without CAD or PAD.
This is a very significant disease state given the large number of people affected. It affects 8-12 million people in the United States, possibly more as many patients with the condition have not been properly diagnosed. This includes 5% of the population aged 55 to 64 and 20% of individuals over 70 years old. Healthcare costs for this condition are very burdensome, with estimated direct and indirect costs as high as $4.69 billion. It is a major cause of disability and reduced quality of life in the elderly. In some cases, it can also be limb-threatening and can possibly lead to amputation. The worst-case scenario for patients with critical limb ischemia is a major amputation. More than 100,000 amputation procedures are performed yearly.
There are two types of patients with PAD: those with intermittent claudication and those with critical limb ischemia. The latter have severe ischemic pain at rest or a non-healing wound or gangrene. Symptoms of PAD also include color changes in the skin, temperature changes, decreased hair growth on the affected limb, and the development of sores that do not heal. Infection of those sores is also a common symptom.
Peripheral arterial disease (PAD) is a condition marked by atherosclerotic occlusive disease of the lower extremities. It is caused by the narrowing of the arteries in the lower extremities. During walking or exercise, the muscles do not get enough blood and the flow is inadequate to meet the increased oxygen demand. The most common symptom of the condition is “intermittent claudication,” which is ischemic muscle pain in the lower extremities during exercise. This is a chronic condition that generally continues to worsen over time.
Importance of Endothelial Dysfunction in PAD
Endothelium, a single layer of squamous endothelial cells that line the inner surface of blood vessels, and the continuous system of cells has been reported to have a total weight of 1 kg. It is now well recognized that endothelial cells have multiple critical functions beyond the classical role in regulating vasomotor tone, myocardium, and monocytes. These include active participation in the regulation of inflammation, blood coagulation, fibrinolysis, and platelet and leukocytes (PMN) adhesion. The vascular endothelium serves a critical role in the maintenance of vascular homeostasis and is a key determinant of whether atherosclerosis and thrombosis will be initiated and progressed. Primarily, it is the location at which the blood directly contacts the vessel wall. Functional disorders of the endothelium are implicated in the pathogenesis of diverse vascular diseases. L-arginine is initially converted to N-hydroxyl-arginine. As with other risk factors, the relative contribution of the various EDRFs and underlying mechanisms to the pathogenesis of specific cardiovascular diseases is difficult to assess. However, once the specific EDRF(s) and its beneficial effects on the vessel wall have been identified, it may be possible to prevent or reverse the associated vascular disease by augmenting the production or activity of the EDRF(s) concerned. A clear example of this concept is the elucidation of the role of NO in the regulation of vascular tone and platelet activity. Demonstration of the presence of endothelial vasodilator dysfunction in patients with coronary artery disease, hypertension, and congestive heart failure has led to research aimed at increasing the release or activity of NO in these patients using both pharmacological and non-pharmacological approaches.
Understanding Endothelial Dysfunction
The endothelium provides a dynamic, multifunctional interface between circulating blood and underlying tissues. The endothelium plays a critical role in maintaining vascular health and its functions include regulation of vascular tone, haemostasis, platelet and leukocyte interactions with the vessel wall, inflammatory and immune reactions, and vascular growth and remodelling. Endothelial function can be assessed by determining the balance between the endothelium-derived vasodilators and vasoconstrictors. Nitric oxide is the primary vasodilator released by the endothelium and has been shown to be a key factor in the regulation of vascular tone. In addition, prostacyclin, bradykinin, endothelium-derived hyperpolarising factor, and adenosine are potent vasodilators. The vasoconstrictor function of the endothelium is primarily regulated by endothelin-1. The dynamic equilibrium between vasodilators and vasoconstrictors helps to maintain blood flow and adequate perfusion of organs under various physiological conditions. An imbalance favouring vasoconstriction or reduced vasodilator bioactivity leads to a decrease in blood flow to organs and tissues and is a primary event in the pathogenesis of atherosclerosis and its clinical manifestations including PAD. A reduction in the bioactivity of NO occurs early in the development of atherosclerosis and has profound effects on the vasculature. Measures of NO bioactivity have been shown to be a predictor of future cardiovascular events.
Role of Endothelium in Vascular Health
NO has several other anti-atherosclerotic effects. NO inhibits platelet aggregation and adhesion to vascular endothelium. NO also has an anti-inflammatory action, as it prevents expression of leukocyte adhesion molecules and the migration of leukocytes into the vessel wall. Finally, NO inhibits the proliferation of vascular smooth muscle cells and production of extracellular matrix. These combined effects prevent the development of atherosclerotic lesions and maintain the integrity of the vascular wall. This is important for prevention of plaque rupture and thrombosis which are the cause of ischemia in PAD. Although eNOS knockout mice studies have shown the importance of NO in the prevention of atherosclerosis and thrombus formation, the specific role of NO in PAD has not been well defined.
NO Figure 1. Effects of nitric oxide on vascular smooth muscle cells. Created with BioRender.com
The endothelium consists of a single layer of squamous cells, which lines the inner surface of blood vessels. Endothelial cells perform a crucial role in the regulation of vascular homeostasis through production of vasoactive substances. Nitric oxide, the most potent endogenous vasodilator, is produced by endothelial nitric oxide synthase (eNOS) from L-arginine. NO diffuses into the underlying vascular smooth muscle cells, where it promotes relaxation through an increase in cyclic GMP. Endothelin, a potent vasoconstrictor, acts in an autocrine and paracrine manner on endothelin A and B receptors located on endothelial and vascular smooth muscle cells. Prostacyclin, another potent vasodilator and inhibitor of platelet aggregation, is also produced by the endothelium. In contrast, prostacyclin is quickly metabolized to less active compounds, thus its effects are mainly mediated in a paracrine manner. Prostacyclin acts on vascular smooth muscle cells to cause vasorelaxation through an increase in cyclic AMP. These substances function to maintain vascular tone by either promoting vasorelaxation or vasoconstriction.
Causes and Mechanisms of Endothelial Dysfunction
Several pathways and processes lead to endothelial dysfunction. Endothelial cells are typically in a quiescent, anti-inflammatory state. An essential aspect of endothelial dysfunction is a pro-inflammatory state, characterized by the expression of pro-inflammatory mediators. Increased oxidative stress is a common trigger of this change in the endothelium, as it reduces the bioavailability of nitric oxide. In several instances of risk factors for PAD, the high levels of oxidative stress are due to increased activity of NADPH oxidase. As oxidative stress increases, there is also an increase in the production of asymmetric dimethylarginine (ADMA) which directly inhibits nitric oxide synthase, further reducing nitric oxide production. An additional mechanism for decreased nitric oxide bioactivity is the reaction of nitric oxide with superoxide to produce peroxynitrite. High levels of cholesterol, specifically LDL, which is a significant risk factor for PAD, have several detrimental effects on the endothelium. Cholesterol accumulates in macrophages to form foam cells, which are a characteristic of early atherosclerotic lesions. Foam cells and LDL can induce endothelial cell apoptosis and necrosis, as well as increasing inflammatory mediators and decreasing nitric oxide bioavailability. High cholesterol levels also upregulate the expression of adhesion molecules.
Impact of Endothelial Dysfunction on PAD Progression
A dysfunctional endothelium can have a significant impact on the progression of PAD. The impaired endothelial cells lose their anti-thrombotic and anti-atherogenic properties. This can lead to a decrease in production of nitric oxide (NO) and an increase in release of von Willebrand factor, endothelin-1, and adhesion molecules. NO is a major regulator of vasodilatation, inflammation, platelet aggregation, and proliferation of vascular smooth muscle cells. In the presence of PAD, NO helps to maintain blood flow and prevent thrombosis in arteries by inhibiting platelet aggregation and promoting vasodilatation. A decrease in NO bioavailability greatly reduces this protective effect. NO also has an inhibitory effect on expression of adhesion molecules and cytokines, thus helping to prevent inflammation and the propagation of atherosclerotic plaque. Inhibition of NO activity promotes the expression of these harmful substances. This results in an increase in leukocyte adhesion, a key event in occurrence of atherogenesis and progression of PAD. Circulating endothelin-1 is a potent vasoconstrictor and mitogen which promotes proliferation of vascular smooth muscle cells. Increased production of this substance would promote occurrence and progression of atherosclerosis as well as restenosis following revascularisation. Von Willebrand factor plays an important role in thrombosis by aiding platelet adhesion to sites of vascular injury. An increase in this substance in the presence of PAD would greatly increase risk of thrombotic events in the affected arteries. All these harmful effects of increased release of such substances contribute to propagation of atherosclerotic plaque and occlusion of affected arteries in PAD. Endothelial dysfunction can also lead to the progression of PAD through promotion of inflammation and atherogenesis. Inflammatory changes in the vessel wall are a key feature in all stages of PAD. The aforementioned increase in adhesion of leukocytes to the vessel wall promotes their migration into the intima. Here they are transformed into macrophages which are responsible for uptake of LDL and foam cell formation. Foam cells are a key component of the fatty streak which is the first visible lesion of atherosclerosis. Formation and progression of atherosclerotic plaque leads to occlusion of the artery and stenotic changes in the vessel. This further reduces blood flow below the affected area, setting up a cyclical pattern which will lead to ischemia and further damage to the affected limb. The adverse effects of adhesion molecules on progression of PAD were discussed previously in the context of their inhibitory effects on NO and the beneficial effects of NO in preventing atherosclerosis.
Targeting Endothelial Dysfunction for PAD Treatment
3.1. Current Treatment Approaches for PAD Given that PAD is essentially atherosclerosis of the peripheral vasculature, the primary management for PAD is modification of cardiovascular risk factors and antiplatelet medication, which are beneficial in preventing myocardial infarction, stroke, and death rather than improving symptoms of intermittent claudication. In trying to improve symptoms, the use of cilostazol is advocated as it both inhibits platelet aggregation and is a vasodilator, which may increase walking performance. However, the most effective treatment for intermittent claudication is a formal supervised exercise program. Unfortunately, patients with severe PAD who are most likely to benefit are often unable to complete an exercise program due to their disease. For those with critical limb ischemia, revascularization is the most effective means of relieving symptoms and avoiding amputation. This is usually achieved through percutaneous intervention or bypass surgery. In severe cases, limb amputation may be necessary for symptom relief and to improve quality of life.
Peripheral artery disease (PAD) occurs secondary to atherosclerosis and is one of the most common cardiovascular diseases, affecting over ten million people in the United States alone. The prevalence of PAD is rapidly increasing due to an older population demographic and an alarming rise in the incidence of diabetes, hypertension, and obesity, all of which are risk factors for PAD. Moreover, the incidence of PAD in women appears to be increasing, particularly among minority populations. Consequent to atherosclerosis, the obstruction of blood flow can cause a range of symptoms, including intermittent claudication, ischemic rest pain, and finally critical limb ischemia that may require limb amputation. With a substantial impact on quality of life and a prognosis comparable to that of patients with coronary artery disease, PAD is a major health concern.
Current Treatment Approaches for PAD
Several clinical trials have examined the effects of lipid-lowering agents on cardiovascular events and progression of PAD. Although there are no specific trials that have used the regression of PAD as an endpoint, the evidence would suggest that lipid lowering would be beneficial based on the clinical outcomes. The efficacy of lipid-lowering agents and their effect on the proatherogenic state of PAD can vary from drug to drug. However, clear benefits can be seen in reducing the rate of myocardial infarction and stroke and improving walking distance.
PAD treatment options can be divided into relief of symptoms and attempts to produce a cure. They may vary from controlling risk factors such as diabetes and hypertension with medication, through implementing exercise programs or dietary modification to promote a healthier lifestyle, to an invasive intervention aimed at resolving the disease. The choice of therapeutic approach in a patient with PAD is highly dependent upon the individual patient’s symptom status and the location and severity of the arterial occlusive disease. Although current therapies can be effective in the relief of symptoms, the high rate of cardiovascular events and lack of disease regression in PAD patients suggests that the methods are not truly beneficial in altering the course of the disease.
Potential Therapeutic Strategies to Improve Endothelial Function
Potential therapeutic strategies to improve endothelial function in PAD can be categorized into several approaches. All of these approaches aim to increase blood flow to the affected limb. Several commonly used pharmacological agents aim to improve the risk factor profile in these patients by addressing hypertension, hyperlipidemia, and diabetes. The overall goal is to improve endothelial function. Examples of these agents include ACE inhibitors, statins, and antioxidants. While the use of these agents is the cornerstone of medical management of PAD, there is little direct evidence to suggest that they will improve endothelial function in the affected limb. A recent study using ramipril, an ACE inhibitor, demonstrated improved walking times in patients with intermittent claudication. However, this improvement was not specifically attributed to improved blood flow to the limb and had a relatively modest effect. Similarly, in the recent CLAIR study using atorvastatin, there was no significant improvement in maximal walking distance in patients with intermittent claudication, despite a significant increase in ankle-brachial pressure indices. These negative findings suggest that addressing risk factors and improving systemic endothelial function is beneficial to PAD patients, but may have limited effects on the diseased arterial wall.
Clinical Trials and Research in Endothelial Dysfunction for PAD
The final part of endothelial research lies in clinical outcome studies. As yet, no study has directly tested whether PAD patients with improved endothelial function have better clinical outcome. That confirmation would require the planning and executing of a longer multicentre study of a chosen therapeutic intervention in which the aim would be not just to evaluate claudication symptoms or treadmill walking performance, but to find a therapy which actually alters the natural history of the disease. This might be done by studying whether regression of atherosclerosis occurs at a slower rate in those receiving the intervention or whether the rate of clinical events such as myocardial infarction, stroke or need for limb revascularisation is lower in the treated group. All options have scientifically solid rationales, the choice between them being a question of availability of funding and the appetite of those involved to embark on studies of different degrees of complexity and the provision of adequate protection for the patients’ interests.
Conclusion
Approximately 10 million people in the United States are estimated to suffer from peripheral arterial disease (PAD) at an annual cost greater than 4 billion dollars. These numbers are expected to increase in the upcoming years as the population continues to grow older and fatter. The underlying cause of PAD is atherosclerosis. Atherosclerosis is not a single disease, rather it is a degenerative process that results from injury to the endothelial lining of arteries. Progressive narrowing and occlusion of the arterial lumen by atherosclerotic plaque limits blood flow to the limbs, causing the classic symptom of claudication. In addition to muscle ischemia and necrosis, which can lead to limb amputation, PAD is also associated with a marked increase in cardiovascular events and mortality. It is clear that preventing and treating atherosclerosis is of utmost importance in PAD. A large body of evidence now exists which demonstrates that inflammation plays a critical role in the development of atherosclerosis. This evidence comes from epidemiologic studies demonstrating that levels of various inflammatory markers correlate with the incidence of cardiovascular events and from pathological studies which show that inflammation is present at all stages of atherosclerotic lesion development. Nevertheless, to date no specific therapies for treating the inflammation of atherosclerosis have been tested in a PAD clinical trial, much less approved for use in PAD. This stands in marked contrast to the treatment of coronary artery disease in which several anti-inflammatory therapies have been tested and are currently used. Given the well known efficacy of antithrombotic and lipid lowering therapies in preventing cardiovascular events in PAD, it can be expected that anti-inflammatory therapies would also be effective. Furthermore, since inflammation is also implicated in other forms of atherosclerosis-related ischemic vascular disease such as stroke and acute coronary syndromes, successful anti-inflammatory therapy for PAD would have further-reaching implications.
Summary of the Importance of Targeting Endothelial Dysfunction in PAD
The role of the endothelium in the pathophysiology of PAD appears to be extremely important and potentially crucial for the progression of atherosclerosis and the ischemic symptoms seen in affected patients. It is clear that endothelial damage and dysfunction from the various risk factors for atherosclerosis and PAD potentiate the abnormal muscle proliferation and promote a pro-thrombotic state. Also, given that risk factor modification is often seen as the cornerstone of treatment for atherosclerosis, the unique ability of the endothelium to “integrate” and respond to the biochemical changes from these risk factors make it an attractive target for intervention.
Future Directions and Implications for PAD Treatment
With an enhanced understanding of the molecular mechanisms that underlie the development of endothelial dysfunction in PAD, several strategies for treatment can be envisioned. Among the most intriguing are those that attempt to restore the balance of pro and anti-inflammatory mediators in the endothelium and systemic circulation. Inflammatory pathways are now understood to prominently involve oxidative stress reactions, with superoxide inactivating NO and also reacting with it to produce the potent oxidant peroxynitrite. Superoxide is increased in the aortocaval fistula model and in human PAD and is likely responsible for much of the NO degradation in these settings. Superoxide dismutase, which scavenges superoxide, has shown promise to prevent the development of endothelial dysfunction after acute limb ischemia in rats by preserving NO bioactivity. Similarly, statin drugs, which have known pleiotropic effects to improve endothelial function and reduce cardiovascular events, were recently found to increase NO bioactivity in the setting of PAD in association with reduction of an oxidative stress marker. Other experimental measures to suppress oxidative stress or its detrimental effects on NO may therefore hold great potential to prevent or treat PAD.
