Peripheral arterial disease (PAD) is a manifestation of systemic atherosclerosis that affects the arteries supplying the legs and leads to a diverse range of clinical presentations. Most patients with PAD are asymptomatic or have atypical leg symptoms. A smaller group of patients have critical leg ischemia (CLI), defined as ischemic rest pain or tissue loss, that is most often due to chronic total occlusion of an infrapopliteal artery. While many patients with intermittent claudication have a benign course with little morbidity other than impaired walking capacity, those with CLI have a high rate of major amputation and cardiovascular event, and their mortality rate approaches 50% at 5 years. It is essential that patients with PAD are identified early and risk stratified accurately. This will allow interventions to be targeted to those who are most likely to benefit and the effects of new treatments to be evaluated more precisely. Current diagnostic methods have various limitations and the ideal test for PAD would provide not only an early diagnosis of disease but also information about severity and outcome. An exciting possibility is that the advent of a range of promising new therapies for PAD will stimulate interest in the development of such a test. The purpose of this article is to review emerging biomarkers and diagnostic tests for PAD and to assess the potential impact of these developments on patient care and future research. Although there are many similarities between disease in the lower limb and that affecting the aorto-iliac bed, this review will focus on the former since epidemiology, presentation, and prognosis are often quite different.
Biomarkers for Peripheral Arterial Disease
In over half a century, there has been little change in the conventional non-invasive diagnostic algorithm for PAD, which includes comparing blood pressures between limbs and segmental assessment of pressures and waveforms. A clear goal would be to improve the identification of PAD, particularly in its asymptomatic stages, in such a way that earlier intervention can prevent progression to symptomatic disease. This may, in part, be achieved by identifying novel circulating biomarkers of PAD not only for diagnostic purposes but also to understand disease mechanisms and to monitor the effectiveness of ongoing treatments. Early steps to date in biomarker exploration have been multifactorial and have varying methods and results.
Biomarkers for peripheral artery disease (PAD) have emerged from research on cardiovascular disease or other metabolic conditions. These potential markers of severity or activity of the underlying PAD process may be derived from serum, plasma, or urine samples and reflect levels of inflammation, hemostatic activation, or tissue ischemia. Simultaneously, novel technologies have enabled seeking abnormal gene expression in affected limbs with PAD, in due course providing a large selection of potential gene-based biomarkers for this condition.
Novel Biomarkers
Cytokines are small secreted proteins that are either inflammatory or anti-inflammatory mediators. These act in an auto and paracrine manner when binding to their specific receptors. There is evidence to show that increased levels of some pro-inflammatory cytokines, such as tumor necrosis factor, interleukin-6, and interleukin-18, are associated with cardiovascular disease. High sensitivity C-reactive protein (hs-CRP), a strong acute phase reactant and downstream marker of inflammatory cytokine activity, has been shown to be a powerful independent predictor of PAD. It is also well-documented and easily measured. Inflammatory markers and mediators have also been implicated in the pathophysiology of claudication and may affect muscle metabolism. It has been postulated that inflammation is a possible reason for the variability in symptoms, and a specific marker could help identify and give more targeted therapy to these patients. Recent work has uncovered the link between statins and their anti-inflammatory effects. This gives promise to other drugs with similar effects, which could be tailored to treat PAD. This is an area of much ongoing research. Identification of an exact marker of a specific pathway may give rise to new targeted treatments for claudication and intermittent claudication.
Osteoprotegerin is a member of the TNF receptor superfamily. Its role as a receptor activator of NF-kB (RANK) decoy receptor is well understood. OPG appears to have an inhibitory effect on RANK/RANK-L mediated inflammation, promoting osteoclastogenesis. The OPG/RANK/RANK-L system has been strongly implicated as a mediator of vascular calcification in ESKD patients and diabetes. High levels of OPG could be a biomarker of coronary artery calcification in PAD. In a recent study in the US, the Cardiovascular Health Study, investigated a population-based cohort to ascertain if there was an association between PAD and higher prevalence and greater burden of extracoronary arterial calcification. They concluded that there is a strong independent association between PAD and extracoronary calcification. OPG has a well-defined ELISA testing, so it could be measured relatively inexpensively. OPG could be a significant marker in PAD and other peripheral vascular diseases in the future. Upcoming research is centered on an attempt to identify inflammatory mediators and markers in order to find more tailored treatment for symptomatic PAD. There are various ways in which inflammation can be measured. Results of investigations may also give leads to other potential markers.
Novel usefulness as a diagnostic tool is the key element of defining a biomarker. Potential biomarkers are now being discovered due to advances in the understanding of the biology of PAD. New candidates include molecules or classes of molecules as yet unmeasured in PAD, such as cytokines or osteopontin, that could further elucidate the pathophysiology of PAD.
Established Biomarkers
The measurement of ankle brachial pressure index (ABPI) could be considered as a biomarker in itself and as a reference standard for the performance of functional tests. ABPI is a highly reproducible method of comparing blood pressures and is associated with a decrease in morbidity and mortality in patients presenting with typical leg symptoms. Low ABPI values have been shown to correlate with future cardiovascular events and mortality, making ABPI a robust prognostic indicator. Dynamic measures of ABPI before and after exercise are termed ABPI response and categorisation of a >20% drop in ABPI following exercise as a positive result can diagnose peripheral artery disease and is associated with poorer lower extremity functional performance and faster rates of functional decline.
An ideal biomarker would reflect biological processes in the arterial wall in health or disease and would correlate with the disease stage. It would be reproducible and representative of the patient’s clinical condition, easily and non-invasively assayed, cost-effective, and predictive of the progression of disease or response to therapy. A number of established biomarkers have been proposed in PAD that reflect different aspects of disease pathophysiology, severity, and prognosis.
Diagnostic Tools for Peripheral Arterial Disease
The most validated and widely used anatomic test for PAD is the ankle-brachial index (ABI). This simple, noninvasive test is the ratio of systolic blood pressure in the ankle to that in the arm. According to a recent meta-analysis, a low ABI has a high overall accuracy for the diagnosis of PAD, with a summary sensitivity and specificity of 0.77 and 0.92, compared to the criterion of angiographic stenosis of 50% or greater. The main advantage of the ABI is that it also provides a measure of disease severity. An ABI between 0.91 and 0.70 indicates mild disease, while an ABI between 0.40 and 0.70 is associated with moderate disease. An ABI less than 0.40 indicates severe disease, and is often seen in patients with critical limb ischemia.
The remainder of this chapter will focus on diagnostic tools that provide direct or indirect anatomic information about atherosclerotic lesions. This is in contrast to functional tests, which mainly assess the severity of ischemia and functional impairment.
Thus, diagnosis of PAD can be considered from two different angles. First, there are diagnostic tools that provide an indirect but accurate measure of the functional impairment that brought the patient to seek medical attention. An example would be the treadmill exercise test, in which patients with claudication walk on a treadmill while monitoring blood pressure in the arms and ankles. More direct information can be obtained by measuring the relative increase in blood pressure after exercise, which should be at least 20 mm Hg. On the other hand, there are diagnostic tools aimed at detecting atherosclerotic lesions themselves, often in patients who do not yet have symptoms. These are often termed “screening” tools, and are becoming increasingly important as the disease burden shifts to an asymptomatic.
Essentially, the diagnosis of peripheral arterial disease (PAD) is not complex – but at the same time, it is often quite elusive. The classic symptom of intermittent claudication is commonly used as the starting point for diagnosis. And indeed, for research purposes, this can be useful. However, when the goal is to actually visualize and characterize the atherosclerotic process, more objective measures are needed.
Non-Invasive Diagnostic Tools
The emergence of endovascular therapies and the recurrent failure of best medical therapy to alleviate symptoms in many PAD patients has increased the demand for accurate lesion assessment to determine the most appropriate treatment strategy. Intravascular ultrasound is the most accurate means of determining lesion severity and morphology, guiding changes in procedural strategy in 40% of cases by demonstrating severe calcium, neointimal hyperplasia, dissection or inadequate lesion preparation. The value of lesion assessment with IVUS has been confirmed in coronary disease and the development of IVUS technology specifically for use in the peripheral arteries has increased its feasibility and ease of use. High frequency miniaturized ultrasound catheters provide an alternative means of lesion assessment and hold promise in the future direction of PAD therapy. Measures of lesion morphology and vessel dimensions obtained from IVUS and IVUS-catheter based techniques guide subsequent treatment with plain balloon angioplasty, the optimal choice and sizing of stents and plaque modification devices.
Non-invasive diagnosis of PAD has traditionally been focused on measuring ankle and brachial systolic blood pressure indices. An ankle-brachial index (ABI) less than 0.9 is suggestive of PAD, with a sensitivity of 95% and a specificity of 100%. Pulsed waveform analysis and segmental pressure measurement are extensions of the ABI, offering information as to the level and extent of disease. Photoplethysmography detects changes in blood volume in the foot at different heart beat phases and is widely employed, despite its poor sensitivity and specificity for detecting PAD. Duplex ultrasound is another widely used non-invasive tool, with a sensitivity of 92% and a specificity of 96% compared to CTA for detecting stenosis ≥50%. Magnetic resonance angiography and CT angiography are equivalent and highly accurate non-invasive tests for the detection and quantification of PAD. Their high negative predictive value makes them useful tools for excluding PAD when the diagnosis is unclear. Benefit, or harm, from revascularization procedures or medical treatment is often erroneously assumed through the presence and severity of stenosis. Invasive angiography is associated with a significant risk of complications in PAD patients, including death, MI, stroke, and access site complications, and is often a poor predictor of lesion severity, is of limited benefit and should be discouraged.
Invasive Diagnostic Tools
Invasive diagnostic tools have improved the accuracy of PAD diagnosis and assessment over the years. Contrast angiography is generally considered the gold standard of lower extremity arterial imaging. However, invasive angiography is infrequently used as a primary diagnostic tool compared with its historical use of defining anatomy in patients with both peripheral and coronary artery disease. Limitations in the information provided by angiography have led to the development of other more frequently utilized invasive diagnostic tests. The severity of ischemia can be defined using various techniques. Catheter-derived information on the direct (ankle/brachial/PK/DP pressures) and indirect (toe pressure) measurement of distal blood pressure is useful and predictive of wound healing potential and amputation risk. Digital subtraction angiography provides a more detailed anatomic assessment of the arteries and has been used to define endpoints in minimally invasive endovascular therapies. Although not yet a widely available bedside tool.
Future Directions and Conclusion
The measurement of end organ ischaemia and intermittent claudication in PAD has already been revolutionised by the use of patient questionnaires. New methods, including skin perfusion pressure and transcutaneous oximetry, have provided an objective measure of limb ischaemia, essential for the assessment of a disease which is so often minimised by patients and practitioners. More technical and expensive diagnostic methods, such as magnetic resonance angiography, computed tomographic angiography, and intravascular ultrasound, provide high-resolution images of the arterial lumen and have been considered effective alternatives to traditional angiography. The ability of these tests to image peripheral arteries remains limited, and as the authors point out, these tests only assess anatomy and not the severity of ischaemia incurred, hence the reluctance to move away from traditional angiography. Newer techniques, such as contrast-enhanced magnetic resonance angiography and fusion imaging modalities, promise to overcome these limitations but are still very much in the research and development stage.
The obvious future direction in the assessment of peripheral arterial disease is the better standardisation of the existing tests, such as the ankle brachial pressure index (ABPI). The next generation of tests must undergo rigorous clinical validation to ensure their effectiveness. Better characterisation of the disease itself is essential. Is the determination of severity based on a percentage stenosis really the best approach, or would a system which correlates different levels of claudication to angiographic findings be more useful? Would any new test for PAD be more useful if it could also predict a response to therapy, be it surgical, radiological, or medical?