Infectious Contributions to Atherosclerosis and Vascular Disease

Do We Really Know the Answer?

Clinical Trials Investigating the Utility of Antibiotics in Cardiovascular Disease

Chlamydia pneumoniae in atherosclerotic lesions

When we became aware of the presence of Chlamydia pneumoniae (C. pneumoniae) in atherosclerotic lesions in 1991, we undertook a series of 9 studies to solidify and expand this surprising finding. The tissues of the 362 persons we studied came from autopsy, surgery and atherectomy, and included atheromatous lesions of the coronary, carotid and femoral/popliteal arteries as well as the aorta. The populations studied came from South Africa and throughout the United States, including Alaska. Persons studied included Caucasians, Blacks, Asians and Alaska Natives. C. pneumoniae was present in atheroma in all these populations. Regardless of age or extent of disease, the organism was found in lesions in young persons as well as in extensive disease in older persons. In these studies, the organism was not found in normal artery specimens. The youngest person with an atheromatous lesion positive for C. pneumoniae was 19 years old. In 2000, Kuo and Campbell reviewed the results of our studies of atheroma and those of others.¹ While the original observation of C. pneumoniae in atheroma was made by electron microscopy (EM), most subsequent studies used immunocytochemistry (ICC) and polymerase chain reaction (PCR) because they could be done more easily. The ICC used C. pneumoniae-specific monoclonal antibodies (MA). Isolation is difficult but it has been done and has further proved the presence of C. pneumoniae in atheroma. Association does not mean cause, but the organism was in the plaques, atheroma, and aorta as well as in the coronary, carotid, and peripheral arteries.

Trial Design

A decade ago, when the tissue studies provided definitive evidence of an association of C. pneumoniae and atherosclerosis, we began planning a trial to see if antibiotic treatment would affect coronary heart disease (CHD).² A human antibiotic treatment trial would be speculative and would only be worth considering if it was safe for the participants. The enormous public health importance of CHD gave urgency to such a trial. Because of the speculative nature of the study hypothesis, we chose to design the trial on a relatively large reduction (25%) in events. The endpoint events were cardiac death, myocardial infarction (MI), coronary bypass surgery, a percutaneous revascularization procedure and hospitalization for unstable angina.

Based on studies using similar endpoints, a 6.5% annual event rate was estimated. We planned to use participants with stable CHD. Considerations of robustness and power to test secondary hypotheses led to a sample size of 4,000, observed for 4 years. If participants at the time of recruitment had an acute coronary syndrome (ACS), the estimated annual event rate would be higher and the size or length of the trial could be reduced. The antibiotic used for these trials should be effective against C. pneumoniae. Long-term treatment is essential to have an effect on a chronic C. pneumoniae infection.²

Preliminary or Pilot Trials

Eight small trials using macrolide antibiotics for secondary prevention of CHD events were reported before the large, adequately-powered trials could be funded and completed. All 8 of these trials were underpowered.³ Six of the 8 recruited participants at the time of an ACS. There were 60 to 1400 participants, observed from 3 months to 2 years. In addition to the limitations of small numbers of participants and short observation periods, most of these trials had very limited courses of antibiotic treatment. All used a macrolide antibiotic known to be effective against C. pneumoniae. The treatment period varied from 3 days to 3 months. While it is unknown if 3 months of treatment might be long enough to modify a chronic C. pneumoniae infection, treatment of one month or less would be unlikely to do so.

Four of the trials reported a reduction in cardiac events and four found no reduction. Because they lacked adequate statistical power, these trials have little usefulness.³ A ninth trial has recently been reported in which 4,373 patients were randomized and followed for 3 years. There was no reduction in CHD events, but an increase in cardiac deaths in the treated group was reported. This trial is considered inadequate because the macrolide treatment was given for only two weeks.⁴

Results of Large Clinical Trials

There are three clinical trials of antibiotic therapy in CHD that are robustly powered to give a secure result.^5-7 Two trials enrolled participants with stable, known CHD, and one enrolled patients hospitalized with an acute coronary syndrome. Treatment of two was with Azithromycin, a macrolide/azalide antibiotic. It was given once a week for 3 months in the Weekly Intervention with Zithromax for Atherosclerosis and its Related Disorders (WIZARD) trial and for one year in the Azithromycin and Coronary Events (ACES) trial. Gatifloxin, a fluoroquinolone antibiotic, was given the first 10 days of each month for 2 years in the Pravastatin or Atorvastatin Evaluation and Infection Therapy (PROVE IT) trial.

ACES and PROVE IT had from 22–25% of the participants suffering a primary endpoint event over the period of observation. WIZARD had fewer events due to a shorter observation period. Antibiotic treatment was not associated with any clinically significant benefit in the secondary prevention of coronary events.

Discussion

The limitations of these antibiotic treatment trials include the possibility that the antibiotic does not reach the microorganism in the chronic lesions of atherosclerosis; that the treatment was not continued long enough; that the dose of antibiotic given was too small; or the antibiotic was too weak. These limitations do not, for practical purposes, weaken the basic conclusion of the studies that antibiotics cannot be recommended for treatment of chronic CHD. The most important limitation in these trials is the advanced stage of the disease in the participants. Review of the ACES study population revealed that 89% had had at least one coronary revascularization procedure prior to the study and that over half were suffering from angina.

Most of the subjects were receiving vigorous standard treatment for CHD. Events occurring in this trial, as well as in WIZARD and PROVE IT, could reasonably be attributed to destabilization and rupture of plaques, rather than extension of the atherosclerotic process with vessel narrowing. Assuming that the treatment was adequate, these findings suggest that neither C. pneumoniae nor another organism susceptible to the antibiotics plays an important role in the late-stage disease events of CHD. However, since the trials were not designed to study the possible role of C. pneumoniae in the pathogenesis of atherosclerosis, the trial results do not tell us anything about a possible pathogenic role of C. pneumoniae in the initiation or development of atherosclerosis. Atherosclerosis begins in childhood and continues to develop in adults. Complications occur mostly in older adults. In the trials, treatment was given after at least one CHD event had occurred, late in the long-term development of the disease.

Clinical Trials of Other Atherosclerotic Diseases

In addition to the trials in CHD, there have been clinical trials of antibiotic therapy directed at atherosclerotic changes in carotid artery, peripheral arteries and the aorta.³ These trials were based on non-invasive measurements of changes in the artery walls. The trials did not rely on events, but each subject contributed to the endpoint. Four trials had antibiotic treatment for one month and one for only 3 days. Each trial reported a statistically significant favorable effect of antibiotic on progression of the disease, except for the one with 3-day treatment. These results should be considered preliminary. If the positive results of these trials can be repeated with larger numbers, they will offer more evidence suggesting that C. pneumoniae is involved in the pathogenesis of atherosclerotic occlusive disease.

Chlamydia pneumoniae in vascular disease — pathogen or passerby?

The first report of an association of C. pneumoniae infection and atherosclerosis was by Saikku and colleagues⁸ who found that Finnish men with IgG antibody against C. pneumoniae were at increased risk for MI and CHD in comparison to population controls. Over 100 papers have been published to address the association of C. pneumoniae antibody and atherosclerotic disease and many have demonstrated an association while a few well designed studies have not.⁹

The first report of C. pneumoniae in atherosclerotic tissue was based on finding these ultrastructures in electron micrographs of atheromas and by immunoctyochemical stain using C. pneumoniae-specific monoclonal antibodies.¹⁰ There have been over sixty studies conducted by investigators world-wide that have found the organism in atherosclerotic lesions throughout the arterial tree using various methodologies including immunocytochemical staining, PCR, in situ hybridization, and culture, although isolation of Chlamydia from chronic infections is rare.

When C. pneumoniae was identified in atherosclerotic lesions, it was hypothesized that following respiratory tract infections, lung macrophages became infected and disseminated infections to the vasculature. Thus, two possibilities were considered. First, that the organism established persistent infection in the normal artery, and in combination with other risk factors of atherosclerosis, induced atherogenesis. The other possibility was that the organism established persistent infection in an artery with atheroma and accelerated the process. The in vitro evidence represents 15 years of work by many investigators.

First, the organism is able to infect critical cells of the atherosclerotic lesion, including endothelial cells, smooth muscle cells and macrophages. Importantly, the organism can also infect foam cells that are derived from macrophages and smooth muscle cells. In the laboratory, we have shown chlamydial inclusions form in infected foam cells, resulting in the production of infectious particles. In humans, the organism is found in foam cells within the atherosclerotic lesion. In co-culture experiments, we found that macrophages transmitted infection to endothelial cells and smooth muscle cells and also enhanced the infectivity of C. pneumoniae for these cells. In addition, infected monocytes bind better to endothelial cells. This finding supports the hypothesis that following endothelial activation, C. pneumoniae-infected macrophages are home to the site of endothelial activation and can therefore transmit infection to endothelial cells.

C. pneumoniae also up-regulates the expression of adhesion molecules on endothelial cells and the cognate receptors on macrophages. Infection then induces smooth muscle cell migration and proliferation. A key role of C. pneumoniae in atherosclerosis is that C. pneumoniae can live in macrophages, and can induce various pro-atherogenic factors, including pro-inflammatory cytokine and inducible nitric oxide synthase. In Dr. Gerry Byrne’s laboratory, it was revealed that in the presence of low-density lipoprotein (LDL), C. pneumoniae induces foam cell formation, and the oxidation of LDL occurs through chlamydial heat shock protein 60.^11,12C. pneumoniae can induce foam cell death, matrix metalloproteinases and tissue factor. The induction of tissue factor is through Egr-1. Egr-1 is pro-atherogenic and is elevated in human atherosclerotic lesions. Egr-1 also contributes to the expression of a variety of proteins that play a role in atherosclerosis, including growth factors, cytokines and adhesion molecules.

Our laboratories focused on mouse models of C. pneumoniae infection and atherosclerosis. C57BL/6J mice that were fed a normal chow diet did not develop atherosclerosis. In contrast, those fed an atherogenic diet developed fatty streak lesions. The other model is ApoE-/-mice on a C57BL/6J background, which is hypercholesterolemic and develops lesions spontaneously in a time-dependent manner. The first group of experiments was designed to determine whether or not hyperlipidemic mice infected with C. pneumoniae developed accelerated atherosclerosis. We either used ApoE knockout mice or simultaneously infected and fed C57BL/6J mice a high-fat/high-cholesterol diet. The idea behind these experiments was to simulate infection in adults.

C. pneumoniae infection accelerated lesion development in ApoE knockout mice, in comparison to sham inoculated mice, as indicated by increased plaque size. C. pneumoniae infection also accelerated lesion development in C57BL/6J mice fed a high-fat/high-cholesterol diet. The conclusion was that in conjunction with hyperlipidemia, C. pneumoniae accelerates atherosclerosis in those mouse models.

Another two experiments were conducted to determine what would happen if the mice were injected before they developed hyperlipidemia. One experiment was to infect normolipidemice mice, and in the other experiment, mice were infected prior to administration of a high-fat/high-cholesterol diet. The idea was that this would simulate infection in childhood. If mice were fed an atherogenic diet simultaneously with repeated infection and maintained on the diet, there was a statistically significant increase in lesion development in infected mice in comparison to uninfected mice. In contrast, if mice were infected three times prior to initiation of the atherogenic diet (administered at five and seven weeks after the first inoculation), there was no acceleration of atherosclerosis in infected mice.There are various other models that have been used, including hyperlipidemic mouse, rat and rabbit models. All of these have demonstrated enhanced lesion development when infected with C. pneumoniae.^13–19

Thus, the conclusions from these studies are that infection accelerates atherosclerotic lesion development in hyperlipidemic animals, and C. pneumoniae is a co-risk factor with hyperlipidemia for atherosclerosis. There are other effects of C. pneumoniae that have been demonstrated in different animal models. Specifically, C. pneumoniae has been shown to induce endothelial dysfunction, and increase fibrinogen levels and matrix metalloproteinase production with a reduced fibrous cap area.^20–22

In conclusion, there is evidence for the biological plausibility that C. pneumoniae contributes to atherosclerotic processes. Specifically, C. pneumoniae infects lung macrophages, which disseminate infection. Infected macrophages are home to the activated endothelium and have increased adherence due to up-regulation of adhesions. The infected macrophages can migrate into the intima. Alternatively, infection can be transmitted to endothelial cells, which results in up-regulation of adhesion molecules. The endothelial cells can release factors that have not yet been identified that result in oxidation of LDL and production of tissue factor. Infected cells can transmit and establish persistent infection. C. pneumoniae infection of macrophages can promote uptake and the oxidation of LDL. C. pneumoniae induces smooth muscle cell proliferation, leading to fibrosis. Infection of macrophages and smooth muscle cells induces up-regulation of cytokines. Collectively, these factors can contribute to acceleration of plaque development. Additionally, infection of macrophages induces matrix metalloproteinase production and foam cell death, which can lead to plaque destabilization. There is also pro-coagulant stimulation induced by C. pneumoniae, which can lead to thrombosis and acute MI.

Atherosclerosis

Atherosclerosis is a heterogenous process that occurs in all of the arteries of the body, but with multiple clinical manifestations related to different means of expression in different vascular beds, resulting in cerebrovascular and peripheral vascular as well as coronary artery disease (CAD). Disease manifestations in the various vascular beds are associated with one another. Of patients with cerebrovascular disease, 37% have another form of vascular disease, and of patients with coronary disease, about 40%. Symptomatic coronary or cerebrovascular disease is found in 50% of patients with peripheral vascular disease (PVD). All the major cardiovascular risk factors relate to atherosclerosis in all of the vascular beds; however, some are more important in one bed than another.

LDL appears to be more strongly related than other risk factors to coronary disease, whereas smoking and diabetes seem to be more strongly related than other risk factors to clinical manifestations of PVD. Hypertension is most strongly related to the disease of the cerebral vessels. In the early 1960s, McGill and his colleagues, using data obtained at autopsy, focused attention on the prolonged pre-clinical phase of symptom development: plaques developed over 30 years before clinical sequelae were manifest (the “clinical threshold”). In the early period of understanding this phenomenon, the only way to gain any insight into the pre-clinical phase of atherosclerosis development was through examination of autopsy specimens, which are biased, or through coronary angiography studies, which also focus on a biased sample of symptomatic individuals.

More recently we have come to understand more about the pathogenesis of atherosclerosis, and particularly about the relationship of atherosclerosis to the development of clinical events. We now understand that progression of the atherosclerotic plaque only defines the substrates within which further changes may occur that result in symptom development. Further understanding of the pathogenetic process necessitates study of plaque dynamics that lead to changes in composition and structure (the plaque with a large lipid core and thin fibrous cap is more unstable and prone to rupture). The final step in the pathway to event development relates to the consequences of plaque destabilization, since not all unstable plaques that rupture lead to events. Dynamic factors within the arterial wall as well as within the lumen of the artery additionally determine the consequences of plaque rupture; the vast majority of plaques that rupture no doubt do so and heal with no clinical sequelae whatsoever. In the carotid system, plaque instability can have other consequences, such as unstable plaques, which can lead to emboli that frequently cause symptom development.

Thus, there is a long, slow pre-clinical phase of atherosclerosis development that has not been possible to study until recently. It was not until the mid-1980s that investigators began to use non-invasive methods to study populations and to ask questions about the determinants and consequences of pre-clinical disease. The non-invasive method that has been used most extensively is B-mode ultrasound of the extracranial carotid arteries. The advantage of this technology is that use of non-invasive imaging provides unique advantages for the study of pathogenesis. The method can be used to study asymptomatic individuals and measure atherosclerosis progression. B-mode ultrasound reliably measures the walls of arteries, and can be related to risk factors and clinical outcome.

Essentially all of the traditional cardiovascular disease risk factors (age, male gender, smoking, cholesterol, hypertension, diabetes) have been shown to be related to carotid atherosclerosis using B-mode ultrasound. Many non-traditional risk factors for cardiovascular disease have also been related to wall thickening of the carotid arteries, including passive smoking, increased homocysteine, dietary saturated fat, psychosocial factors, and Chlamydia infection. These same risk factors have also been related to carotid atherosclerosis progression, and there appears to be an interaction between coronary status and influence of risk factors on carotid disease progression. Patients with coronary disease progress atherosclerosis of the carotid arteries 4–5 times faster than those free of coronary disease, and risk factors are more strongly related to progression of carotid atherosclerosis in patients with coronary disease than in patients free of coronary disease. High-density lipoprotein (HDL) cholesterol, for instance, is strongly related to carotid atherosclerosis progression in patients with coronary disease but not in patients free of CAD.²⁴

Several interventions (most notably, lipid-lowering therapy) have been tested for their influence on both clinical events and progression of carotid atherosclerosis quantified by B-mode ultrasound. In these studies, reduction of clinical events has been correlated with the retardation of the progression of atherosclerosis measured by B-mode ultrasound. The correlation is not strong, which is likely due to the fact that other factors, alluded to previously and not measured in these trials (plaque instability, for example), contribute to event development along with atherosclerosis progression. There have been a number of trials testing the effect of C. pneumoniae treatment on clinical cardiovascular events.²⁵ One of these studies used B-mode ultrasound to evaluate the effects of intervention with roxithromycin on the progression of atherosclerosis in the carotid arteries. Individuals who had had a prior ischemic stroke were evaluated, and 62/272 (25%) were chlamydia positive at IgG titers of 1:64 or greater.26 The results showed that patients who did not have C. pneumoniae antibody titers progressed their carotid atherosclerosis at a slow rate, whereas those with C. pneumoniae antibody titers progressed almost twice as fast. Those with positive titers treated with the antibiotic had about a 60% retardation of progression compared to those treated with placebo, whereas in those with negative antibody titers the antibiotic had no effect on the (slower) B-mode progression rate. Antibiotic treatment had no effect on clinical events in that trial. In conclusion, atherosclerosis is a diffuse disease with unique regional characteristics. The development of events represents a late manifestation of the overall process.

We believe that epidemiologic and observational studies of pre-clinical disease with non-invasive methods may provide some insight into mechanisms involved in the development of clinical events. Clinical trials with atherosclerosis outcome may be informative about pathogenesis in the pre-clinical state. New developments in imaging methods such as MRI may afford the opportunity to quantify changes in plaque characteristics that will provide further information on the pathogenesis of clinical events.

Trial Design and Endpoints in Peripheral Arterial Disease

The pathological consequences of vascular disease can be divided into sequelae of its systemic and limb manifestations. Patients with peripheral arterial disease (PAD) carry a high risk of systemic cardiovascular events, including MI, stroke, or vascular death. Epidemiologic studies reveal that the risks for these events are increased as much as six-fold compared with age-matched healthy individuals. The annual rate of non-fatal MI, stroke and vascular death averages 5% in PAD. The natural history of limb manifestations is quite stable. Most claudicants have stable symptoms for approximately a five-year period. Claudication is disabling, however, and limits daily living activities to the degree that symptomatic treatment is warranted in most patients. With that background, PAD trials and cardiovascular disease trials in general can study either events driven by systemic disease manifestations, or symptoms associated with disease. Event-based trials are largely designed to demonstrate reduced risk of fatal and non-fatal cardiovascular events. Secondary endpoints in these trials typically address the need for revascularization and hospitalization.

Because of the relative infrequency of these events, occurring in the range of 6–8% per year in a patient cohort, large numbers of patients (4,000–10,000) must be followed for many years, depending on the anticipated treatment effect. In contrast, symptomatic trials for PAD are designed to demonstrate improvement in claudication symptoms. Each patient provides baseline and post treatment measures of symptoms, and trials can be powered with far fewer patients than in event-based trials, even for relatively modest anticipated treatment benefits. Other endpoints studied include quality of life, treadmill performance, and exercise capacity. The treadmill endpoint is the primary endpoint for regulatory approval in PAD studies in patients with claudication.

Event-Based Trials in PAD

There are several examples of different pharmacologic targets and therapies that have been assessed in the PAD population. The Heart Protection Study examined the effects of Simvastatin versus placebo on cardiovascular events and the need for revascularization. A group of patients with PAD were included, and experienced a significant reduction in the risk of subsequent cardiovascular events associated with treatment. Antiplatelet agents demonstrate benefit in PAD patients, specifically clopidogrel in a trial versus aspirin. and the angiotensin-converting enzyme inhibitor ramipril has demonstrated reduced cardiovascular event rates associated with its use.

All of these trials studied thousands of patients to determine the reduced risk associated with therapy. In symptom trials in PAD, the primary endpoint is peak walking time on a graded treadmill. In critical limb ischemia (CLI) studies, the primary endpoint is amputation-free survival. CLI trials tend to be larger, with 300–500 patients to achieve this endpoint, simply because of the decreased frequency of amputation compared to symptom measurement, whereas claudication trials can be done with 75–100 patients per group. Secondary endpoints in symptom trials typically include time of onset for claudication, quality of life, and changes in ankle brachial index.

Non-Revascularization PAD Therapy Trials and Development

Exercise rehabilitation is generally thought to be the most effective non-revascularization treatment option, and has been studied fairly extensively over the last 30 years. A formal exercise program will improve the primary endpoint of peak walking time by over 100% and demonstrates distinct improvements in the quality of life. However, this level of impact is achieved only in the context of a fully supervised and compliant program. Cilastazol is an approved drug for treating claudication, but it is not as effective as exercise. The net benefit over placebo is about 50%, and its use is also associated with improvements in the quality of life. The evidence for cilastazol was presented to the FDA for drug approval in 1998. Eight randomized trials all showed consistent response in terms of this treadmill endpoint, leading the FDA panel to conclude that efficacy was clear. Safety remained an issue and thus additional trials were requested of the sponsor. Cilastazol is a phosphodiesterase inhibitor, which as a class have been associated with increased risk of death, particularly in patients with heart failure.

There have also been a large number of negative symptom-based trials in PAD patients. Two prostaglandin trials with Beraprost, showed that oral prostaglandins do not improve exercise performance or quality of life. Studies of a 5-hydroxy tryptamine antagonist that had vasodilator and anti-platelet properties were solidly negative. Avasimibe is an acyl coenzyme A-cholesterol acyltransferase (ACAT) inhibitor that did not improve exercise performance in clinical trials.

Studies of statins have had mixed results. Hypothetically, statins have anti-inflammatory properties as well as metabolic effects on muscle metabolism that might be beneficial for PAD symptoms. Ongoing trials using a statin-niacin combination are being conducted to test this theory. Negative trials could occur simply because drugs did not work, but could possibly be explained by incorrect testing and methodology, with significant variability around the endpoint(s), and/or a significant placebo responder rate. Therefore, endpoint-driven trials have the advantage, since clinical endpoints are easy to discriminate.

For non-fatal events, typically an event committee performs adjudication, which differs from endpoint assessment in claudication trials, in which the primary endpoint is a performance-driven endpoint with the possibility for significant variability. If randomized patients are on placebo and six months later they walk significantly further, with considerable range in this measurement, this may relate to the type of treadmill test, investigative site performance and quality. In one example an initial peak walking time was 205 seconds and variability was 16%. With proper instruction and coaching, subsequent assessments in the same subject produced much higher peak walking times and lower variability. It is easy to understand how this might impact trial results, when the current medical therapy of choice produced an approximately 50% benefit in treated patients. Data from over a 100 sites under the management of the Colorado Prevention Center reveals common problems in PAD trials with treadmill equipment and testing procedures. The Colorado research team discovered that the variability and placebo response issues appear to be related to site quality, not patient demographics or patient factors. So if variability can be controlled, trial outcomes might similarly improve.

Antibiotic Treatment for PAD Symptoms

There are a number of putative mechanisms whereby antibiotic therapy might affect the clinical manifestations of atherosclerosis. Antibiotic therapy may affect the natural history and pathogenesis of atherosclerosis. It is felt that chronic vascular infection sets up localized inflammatory responses in the vasculature at discrete sites. Thus an antibiotic therapy directed at these vascular infections might exert a net anti-inflammatory effect on the vasculature. The anti-inflammatory component may be particularly important in claudication because claudication is not just muscle ischemia; there are also inflammatory affects on muscle metabolism that are detrimental. There is the potential to decrease cardiovascular risk and disease progression, as well as improve symptoms, which is unproven.

There is a suggestion that antichlamydial antibiotics may improve treadmill-walking performance. According to one European study, there was a marked decrease in the need for revascularization in treated patients, as well as reductions in carotid plaque and an improvement in painless walking distance. Thus, there are suggestions that, despite the negative signals in the event trials, there may be some positive data in PAD. The PROVIDENCE trial will study the effects of the antichlamydial rifamycin (Rifalazil) in patients with claudication. The proposed sample size is 274 patients, allowing greater than 90% power, which are to be studied over a 12-month duration. This trial is substantially smaller than previous event-based trials in cardiovascular disease that tested the hypothesis of anti-chlamydial antibiotic benefit in reducing cardiovascular events. The primary endpoint of PROVIDENCE 1 is to demonstrate improvement in peak walking time at six months in treated PAD patients testing positive for high levels of chlamydia antibodies, as compared to a placebo-treated group. Secondary aims are to demonstrate safety and any effects on claudication onset time, quality of life, and the comparative incidence of a combined endpoint of vascular death, non-fatal MI, non-fatal stroke, and revascularization in the two groups, although the study is not powered for these secondary endpoints.