Given that existing therapies for intermittent claudication are not appropriate for all patients, researchers are working to develop new therapies focused on improving patients’ ability to compensate for a vascular occlusion by expanding collateral artery pathways.
By Steven J. Miller, PhD; A. George Akingba, MD, PhD; and Joseph L. Unthank, PhD
The progression of atherosclerosis may lead to stroke and the development of coronary heart disease, peripheral arterial disease (PAD), or both. PAD has been identified as a global health issue, impacting 202 million people worldwide1,2 and approximately eight million in the US.3 Of the US PAD population, 10% to 12% eventually experience intermittent claudication as a result of arterial insufficiency or inadequate blood perfusion in the leg muscles.4,5 Approximately 20% to 30% of claudicants with subcritical limb ischemia progress to critical limb ischemia within 10 years of diagnosis, and the percentage is even higher if diabetes is present.6
As a result of the large population affected and the impact on lifestyle, new therapies to treat PAD are needed urgently.7,8 While surgery9 and exercise10 may be appropriate and highly efficacious for certain subsets of patients experiencing claudication, these approaches are not suitable in all cases. Experimental therapies using stem or progenitor cells are being investigated, but overall efficacy remains unclear in clinical trial results to date,11 and it will likely be several years before cell therapy becomes a viable treatment.
An effective pharmacological approach to the treatment of claudicants thus would be desirable, and a recent study12 suggests novel pharmacological approaches may provide significant benefit. However, the only pharmaceutical agents currently approved by the US Food and Drug Administration to treat intermittent claudication are the phosphodiesterase inhibitors pentoxifylline and cilostazol,8 and these drugs have been shown to provide only minimal improvement for claudicants in terms of maximal treadmill walking distance.13 In addition, they are expensive, have multiple side effects, and are contraindicated in patients with congestive heart failure.
A natural compensation for PAD-related vascular occlusions and resultant claudication is expansion of preexisting arterial pathways, known as collateral arteries, which bypass an occlusion and provide increased blood flow to peripheral tissues, especially during periods of increased metabolic demand. Although younger individuals have a high capacity to compensate for arterial occlusion through collateralization, compensation in those with atherosclerotic risk factors and comorbidities is greatly diminished.14 More research is needed to identify the mechanisms responsible for the impairment of the natural outward expansion of collateral arteries.
As reviewed by Ziegler et al,14 it has been demonstrated in multiple species that, following an acute femoral artery occlusion, the majority (about 70%) of the total vascular resistance resides in the collateral arteries rather than the distal microcirculation. Although the formation of new capillaries (angiogenesis) and arterioles in muscle tissue may contribute to correcting a perfusion deficit, overall increased flow to the extremities is largely dependent on the small arteries that form the collateral pathways. Thus, strategies to promote collateral formation should have a greater impact on intermittent claudication patients than therapies affecting only angiogenesis.
Cardiovascular risk factors associated with development of PAD and intermittent claudication include aging, hypertension, and diabetes or metabolic syndrome. These risk factors are associated with vascular dysfunction and impair the process of collateral development in both humans and animals.15-19 A common denominator for all these risk factors is the presence of elevated reactive oxygen species (ROS), which have been shown to be present in PAD patients20-22 and to inhibit collateral formation in animals.23-25 Thus, strategies for reducing production of or scavenging of excess ROS would be expected to promote collateral formation in patients with PAD. Indeed, results from trials using agents capable of altering vascular redox status (equivalent to ROS level) have shown significant impact on maximal walking distance in claudicants.12,26,27 Our research to date has focused on using preclinical models with cardiovascular risk factors to test the concept of altering vascular redox status to facilitate collateral growth.
Insight from preclinical models
There are many preclinical models for the study of collateral development in the context of arterial insufficiency.28 Our laboratory has utilized two different rodent models to study the role of ROS in the regulation of primary collateral formation.
One model uses serial ligations in the rat mesenteric vasculature to elevate blood flow in selected arteries and thus simulate formation of a collateral pathway.25,29 This model has the advantage of a readily accessible vasculature for repeated diameter measurements in situ in the same arteries, the ability to make hemodynamic measurements of flow and shear stress, and sufficient tissue to enable molecular studies of gene regulation and expression.
The second model is a mouse distal femoral artery ligation model that results in the gracilis arteries forming the primary collateral pathway.30 This mouse model is relevant to most claudication patients because it represents the most common site of focal occlusion, and also has the advantage of allowing the use of genetically modified animals to address mechanistic questions. The majority of studies using mouse femoral ligation have used a more severe model utilizing arterial ligation/excision techniques to focus on critical limb ischemia, and these models have been used primarily to assess hindlimb tissue perfusion and angiogenesis rather than remodeling of the resistance vessels that comprise the primary collateral pathways.
We have used the rat mesenteric and mouse femoral ligation models to investigate mechanisms regulating the promotion and impairment of primary collateral artery growth as well as agents to reverse collateral growth impairment in the presence of cardiovascular risk factors.
A recent study from our group investigated the role of the enzyme complex NADPH oxidase (nicotinamide adenine dinucleotide phosphate oxidase, or Nox) as an ROS source in physiological
collateral growth.31 Vascular Nox exists as three isoforms in the rodent, Nox1, Nox2, and Nox4, along with multiple associated subunits.32 Humans possess an additional isoform, Nox5.32
While Nox is thought to be the major source of vascular ROS, the roles of the various forms of Nox in different cell and tissue types and in various pathological conditions is not well delineated. ROS-mediated signaling is known to be essential for normal physiological vascular function,33-35 and the results from our work using both of the previously described models confirm that ROS are required for primary collateral artery development.31 These results, based on specific inhibitors of activity and expression, along with genetic ablation, also indicate that the NADPH oxidase Nox2 isoform is a primary source for the redox signaling associated with successful flow-mediated remodeling in healthy young animals.
Collateral flow modeling
We also have examined the role for ROS during periods of acute increases in collateral flow, which occur following an occlusion and precede the outward remodeling required for collateral formation. These experiments used acute vascular clamping and measurement of perivascular hydrogen peroxide (H2O2) concentrations with microelectrodes to study regulation of H2O2 that occurred with elevated flow in young, aged, and hypertensive rats. The results demonstrated that 1) perivascular H2O2 concentrations are higher in both aged and spontaneously hypertensive rats (SHR) than in young, normotensive rats; 2) H2O2 concentrations increase with elevated flow; and 3) Nox2 activation was responsible for its production.36,37
These results with acutely elevated flow likely parallel what occurs during early stages of collateral growth, and indeed, we have demonstrated that administration of antioxidants concurrent with creation of a mesenteric ligation model reverse impairment in both aging rats24 and SHR25 and also in diet-induced obese mice using the femoral ligation model.38 Results from these studies also have demonstrated that antioxidants administered up to a week after establishment of vessel occlusion in an aged animal could reverse impaired collateral growth by promoting flow-mediated remodeling.24 This methodology is analogous to the clinical situation in which therapies are initiated well after arterial insufficiency has been established. Evidence for a role of elevated Nox2 activation in collateral development is provided by a recent study in our lab indicating that activation of Nox2 by the p47phox subunit is an important factor involved in collateral growth impairment in the context of diet-induced obesity.38
Although there is much preclinical evidence for a role of Nox-mediated oxidant stress in impaired collateral development, there is a lack of knowledge in the area of the signaling pathways, molecules, and mechanisms involved. To begin to address this issue, we conducted a study using cDNA microarrays to examine global gene expression changes in mesenteric normal flow and high-flow collateral arteries of SHR compared with normotensive rats.39 An important finding was that major differences in collateral artery gene expression existed between groups; only 6% of genes had similar expression. These differences included mechano-sensitive, redox-regulated transcription regulators, which regulate expression of multiple proteins involved in vascular remodeling.
We determined that at least one of the transcription regulators was activated in endothelial cells of arteries in the normotensive rats, but not in the SHR. Interestingly, administration of the antioxidant apocynin stimulated this activation in the SHR, and we have previously shown that apocynin also reverses the collateral growth impairment in SHR.25 Other experiments have shown that the angiotensin type 1 receptor is involved in signaling necessary for collateral growth.39 These results suggest that collateral growth impairment results from an abnormality in a regulatory mechanism occurring between initial signal transduction and eventual gene transcription, and support redox-dependent modulation of transcription factors as a potential mechanism.
Our ongoing work in aged rats (unpublished data) indicates Nox2 inhibition or catalase administration reverses collateral growth impairment, implicating Nox2-derived H2O2 as an important ROS. Recent studies have shown that Nox2 activation and mitochondrial dysfunction both contribute to cellular oxidant stress, and the pathways involved in these events may interact.40,41 Future studies will focus on the involvement of mitochondrial oxidant stress and interactions with Nox2 as an important regulator of impaired collateral artery development. Other recent work in our laboratory has focused on developing a new technique using preclinical rodent models to allow assessment of the role of the collateral circulation in increased limb perfusion during periods of muscle activity. This type of model should allow us to better evaluate the effects of therapeutic agents designed to ameliorate subcritical limb ischemia.
Although much preclinical evidence exists for a role of oxidant stress in cardiovascular disease, trials in humans with various antioxidants have been largely unsuccessful. This can perhaps be explained by issues related to use of inappropriate or ineffective agents, suboptimal doses, or failure to screen patients for the presence of oxidant stress,42 among others.
A recent randomized placebo-controlled trial of 33 claudication patients (eight women, mean age 64.6 years) demonstrated that the angiotensin-converting enzyme (ACE) inhibitor ramipril was effective in increasing maximal walking distances.12 Compared with placebo, ramipril improved maximum treadmill walking distance by an adjusted mean of 131 m (62-199 m) (p = .001), treadmill intermittent claudication distance by 122 m (56-188 m) (p = .001), and patient-reported walking distance by 159 m (66-313 m) (p = .043). Measurements of pulse wave velocity indicated that a likely explanation for the improvement was a decrease in arterial stiffness. Research has shown that impaired arterial elasticity in humans is associated with oxidative stress.43
An additional meta-analysis of six randomized controlled trials with 821 claudicants has indicated that ACE inhibitors in general are effective in increasing maximal walking distance, and the effect is due at least in part to an improvement in endothelial function.27 Specifically, the meta-analysis found that treatment with ACE inhibitors improved maximum walking distance by a mean difference of 120.8 m (2.95-238.68 m) (p = .04) and pain-free walking distance by 74.87 m (25.24-124.5 m) (p = .003) compared with placebo.
Nox2, a primary source of vascular oxidant stress, is stimulated by angiotensin II;41 thus, ACE inhibitors may function at least in part by suppressing activity of Nox2, thereby improving endothelial function. PAD patients have elevated levels of circulating oxidant stress markers,20 and this includes a soluble form of Nox2.21 It is possible that agents like ramipril that prevent formation of ROS may be more effective than agents that scavenge ROS,42 which describes many antioxidants tried to date in clinical trials. Use of Nox inhibitors also may have the additional benefit of suppressing development of atherosclerosis,44 thereby potentially preventing onset of PAD-mediated claudication.
Certain problems will need to be addressed before redox modulation could be a viable therapy. Because Nox isoform expression and activity varies by cell and tissue type, it will be important to develop targeted therapies. In addition, there is the possibility of antioxidant agents interfering with physiological processes requiring redox signaling, such as flow-mediated dilation.34 Nevertheless, evidence from clinical trials, taken together with the preclinical evidence, suggests targeting Nox-derived ROS is a viable strategy for prevention and treatment of intermittent claudication, and perhaps even critical limb ischemia.
Steven J. Miller, PhD, is an associate professor in the Department of Surgery, Division of Vascular Surgery, and in the Department of Cellular and Integrative Physiology, at the Indiana University School of Medicine in Indianapolis. A. George Akingba, MD, PhD, is an assistant professor in the Department of Surgery, Division of Vascular Surgery, at the Indiana University School of Medicine and at the Eskenazi Hospital Surgery Clinic in Indianapolis. Joseph L. Unthank, PhD, is a professor in the Department of Surgery, Division of Vascular Surgery, and in the Department of Cellular and Integrative Physiology, at the Indiana University School of Medicine.
Disclosures: No conflicts of interest, financial or otherwise, are declared by the authors.
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- Shahin Y, Cockcroft JR, Chetter IC. Randomized clinical trial of angiotensin-converting enzyme inhibitor, ramipril, in patients with intermittent claudication. Br J Surg 2013;100(9):1154-1163.
- Stevens JW, Simpson E, Harnan S, et al. Systematic review of the efficacy of cilostazol, naftidrofuryl oxalate and pentoxifylline for the treatment of intermittent claudication. Br J Surg 2012;99(12):1630-1638.
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- Miller SJ, Coppinger BJ, Zhou X, Unthank JL. Antioxidants reverse age-related collateral growth impairment. J Vasc Res 2010;47(2):108-114.
- Miller SJ, Norton LE, Murphy MP, et al. The role of the renin-angiotensin system and oxidative stress in spontaneously hypertensive rat mesenteric collateral growth impairment. Am J Physiol Heart Circ Physiol 2007;292(5):H2523-H2531.
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