Peripheral arterial disease (PAD) is a common circulatory problem in which narrowed arteries reduce blood flow to your limbs. When you develop PAD, your extremities usually don’t receive enough blood flow to keep up with demand. This causes symptoms, most notably leg pain while walking, which can become so limiting that you cannot walk. This decreased blood flow can cause serious complications. Often, people with PAD have a higher risk of stroke and heart attack. It sometimes leads to loss of a limb. Unfortunately, due to the aging population and the high frequency of illnesses such as diabetes and heart disease, the number of patients with PAD will undoubtedly rise over the next decades. The morbidity and mortality associated with PAD are still on the rise, and it is a major public health priority to tackle this growing problem. Over the years, there have been considerable advances in the management of patients with PAD, especially in terms of pharmacological and endovascular treatment. However, there is still a high unmet need, and there are some patients with critical limb ischemia in which there are no revascularization options, and amputation is the only solution. In addition, some endovascular treatments have failed to show clear superiority over exercise and changes in lifestyle. Thus, the search for new treatments continues, and regenerative medicine has entered the arena.
Overview of Peripheral Arterial Disease (PAD)
Atherosclerosis is the predominant cause of PAD and occurs across a number of stages: beginning with the inflammatory response to a harmful stimulus, followed by the uptake of modified lipids by monocyte-derived macrophages to form foam cells, and then the extracellular deposition of lipids. Damage to endothelial cells, located at the lumenal border of all blood vessels, is widely considered to be a prerequisite lesion in atherosclerosis. Once initiated, an inflammatory and fibroproliferative cascade is triggered, resulting in the gradual development of a stenosis. The most detrimental outcome of atherosclerotic stenosis is the development of unstable atherosclerotic plaques which may rupture and induce thrombosis, commonly resulting in ischemic injury due to downstream embolization. Effects on the arterial system are diverse, largely dependent on the size and location of stenosis and the adequacy of collateral vessels. Symptoms may include claudication, ischemic pain at rest, ulcers or gangrene of the lower limb – the most severe consequence being amputation. Systemic effects of PAD are also a major concern; those afflicted demonstrate markedly increased mortality rates due to cardiovascular events such as myocardial infarction or stroke.
Importance of Regenerative Medicine in PAD Treatment
The prevalence of peripheral artery disease (PAD) rises with age and manifests as intermittent claudication or chronic limb threatening ischemia. It affects more than 200 million people globally, and its exact disease burden and prevalence is difficult to prove as many patients are asymptomatic. However, the incidence of PAD is increasing due to increasing age and incidence of diabetes, and it is also comorbid with other cardiovascular diseases such as coronary artery disease and cerebrovascular disease, and the projected global increase in diabetes over the next 20 years will also increase the incidence of PAD. Whilst treatments are available for PAD and the UK National Institute for Health and Clinical Excellence (NICE) and American College of Cardiology recommend revascularization by angioplasty or bypass grafting for certain patient groups, the global advancements in these treatment options have been slow. This is due to multiple factors including the lack of randomized trial data in certain patient groups, the lack of availability of the latest technology and techniques in less economically developed countries, and the fact that most PAD occurs in the elderly with multiple comorbidities who are at high surgical risk. Medical therapy such as antiplatelet agents, cholesterol reduction and blood pressure control is effective for secondary prevention of cardiovascular events in PAD patients, however such patients have high rates of adverse limb events and impaired quality of life. Therefore there is still a need for new effective treatment options for PAD, especially for patients who are poor candidates for revascularization and those with tissue loss in whom limb salvage is a priority.
Current Treatment Approaches for PAD
HMG-CoA reductase inhibitors, or statins, are effective in reducing the risk of fatal and nonfatal cardiovascular events in patients with PAD, although they have not been shown to improve symptoms of intermittent claudication or increase walking distance. Blood pressure control is also important in these patients, as hypertension is a major risk factor for progression of PAD and cardiovascular events. High cardiovascular risk suggests the use of antiplatelet agents such as aspirin or clopidogrel, which reduce the risk of myocardial infarction, stroke, and vascular death. Unfortunately, there are no medications that significantly improve symptoms of intermittent claudication or effectively treat advanced PAD.
Medications and Lifestyle Modifications Cilostazol and pentoxifylline are the two medications approved by the United States Food and Drug Administration for the treatment of intermittent claudication due to PAD. These drugs improve walking distance in a minority of patients and have little impact on other symptoms of PAD, but they remain a reasonable option for many patients. Less is known about their effects on progression of PAD. It should be noted, however, that cilostazol is contraindicated for patients with heart failure.
Over the past few decades, significant advances have been made in the treatment of cardiovascular disease, particularly in the fields of drug therapies and surgical interventions. In PAD, the primary goal of treatment is to relieve patient symptoms and prevent progression of the disease to critical limb ischemia. Secondary prevention of cardiovascular events and mortality is also an important goal, since patients with PAD are at increased risk for myocardial infarction and stroke.
Medications and Lifestyle Modifications
Modifications to lifestyle and risk factor reduction are as important with PAD as they are with any other cardiovascular disease and can be considered a treatment strategy. Patients with PAD who participate in a structured home-based exercise program have demonstrated up to a 6-fold increase in walking distance before the onset of leg pain. Regular supervised exercise consisting of walking and leg exercises can improve treadmill walking performance. Unfortunately, the benefits of exercise training are lost if patients do not maintain a long-term exercise program. Smoking cessation is paramount in halting the progression of PAD. Lifelong smokers with claudication have a 5-year mortality rate of 25% and a 10-year mortality rate of 54%. Smokers are also less likely to respond to treatment for intermittent claudication and will typically continue to decline in their ability to walk. A comprehensive program for smoking cessation can increase a smoker’s chance of quitting for good. Previous studies have also shown that the reduction of Homocysteine with vitamin therapy can reduce cardiovascular event rates in patients with PAD. Unfortunately, vitamin therapy is not very effective in the general population.
Today’s standard medications for PAD are aimed at reducing the risk of heart attack and stroke. Several medications can be effective in treating PAD. High blood pressure medicines can lower your risk of heart attack and stroke by protecting your heart and blood vessels. Cholesterol-lowering medicines may also reduce your risk of heart attack and stroke. If you have had a previous heart attack or stroke, aspirin and/or clopidogrel (Plavix) can lower your risk of having another heart attack or stroke. Medications for intermittent claudication can be effective in improving symptoms and increasing walking distance. Unfortunately, none of these medications improve the long-term prognosis of patients with PAD. Thus, in the future, we will depend on advances in regenerative medicine to help improve the long-term prognosis of patients with PAD.
Angioplasty and Stenting
Restenosis after angioplasty, a still common and important clinical problem, is now effectively treated with stent implantation. Stents are now used in more than 90% of all percutaneous transluminal coronary angioplasty (PTCA) procedures and in a large number of peripheral angioplasty procedures. The initial high radial force of self-expanding stents provides acute luminal gain and scaffolding for as long as the stent resists recoil and resists or contains dissection. The reduction in dissection with stenting leads to improved early results and a more predictable long-term outcome, especially in cases where antiplatelet therapy can be given. With adjunctive use of cutting balloon, directional atherectomy, or atherectomy devices, debulking of lesions before stenting has been possible in some cases and often improves stent and lesion outcome. High-pressure stent implantation and use of intravascular ultrasound or angiographic guidance have been several techniques to optimize stent deployment. Randomized trials have proven the clinical benefit of stenting over PTA alone in patients with iliac lesions, and stents have now become the treatment of choice for the iliac system. In the infrainguinal system, stents are successfully being used despite the limitation of excessive stent length, long-term restenosis, and difficulty of stenting smaller caliber arteries. Although trials have yet to compare the various devices with stenting, there is great interest in various new technologies to further improve the outcome of stenting in the treatment of femoropopliteal lesions.
Bypass Surgery
As mentioned, bypass surgery and angioplasty both have a common goal to improve blood flow to the affected area, thus relieving symptoms. At POBA or at bypass surgery, the bypass or treatment only works effectively if there is a suitable run off of the blood vessel. This, in effect, means will the treatment improve blood flow to the affected area, and if so, will it heal and reduce the symptoms. Without a suitable runoff, there is a higher leg amputation rate. This is why PAD can often lead to amputation, as the symptoms are not improved and infection ensues. Infections are common at the affected site because of reduced or poor blood flow, and since diabetes is another contributing factor for PAD, this increases infection rates even further.
Bypass surgery is recommended for patients with PAD who have severe leg pain that interferes with their daily life or is not relieved by other treatments. Bypass surgery creates a path for blood to flow around the blocked area. During the surgery, a vascular surgeon creates a new path for blood flow using a graft. The graft may be a vein from the patient’s leg or an artificial vessel. Compared to angioplasty and stenting, bypass surgery may be more effective in relieving symptoms of PAD in the long term. Bypass surgery carries some substantial risks, especially for older and sicker patients. These risks include an increased chance for cardiac or pulmonary complications and an increased chance for wound problems or infection.
Regenerative Medicine Approaches for PAD
Stem cell therapy is based on the premise that introducing stem cells into ischemic tissue will result in increased vascularity and ultimately reperfusion of the affected tissue. The stem cells used are typically bone marrow derived, and there are several methods of introduction including direct injection into the affected limb, administration into the circulatory system, and for limb ischemia the cells can be surgically implanted around the occluded artery. Once introduced into the tissue, the stem cells work to increase vascularity through either direct incorporation into vascular structures, or by secreting growth factors that induce angiogenesis. In addition to mature stem cell transplantation, another potential therapy is the use of growth factors to induce proliferation and differentiation of endogenous progenitor cells. This would have an advantage clinically by preventing the need to harvest stem cells from the patient, and growth factors could be delivered by way of injection or gene therapy. Overall, stem cell therapy has shown great promise in preclinical models, and several clinical trials are currently underway to determine the efficacy of this treatment in patients with PAD.
3.1 Stem Cell Therapy
Current conventional therapy for PAD is focused on either preventing or decreasing atherosclerosis and relieving symptoms, rather than restoring perfusion to ischemic tissue. Regenerative medicine offers a novel approach to treating PAD, by way of restoring blood flow to ischemic tissue through the use of progenitor cells, growth factors, and gene therapy. These novel therapies aim to lower amputation rates and decrease mortality due to cardiovascular events in patients with PAD.
Stem Cell Therapy
To date, clinical trials of adult BMACs cell therapy for PAD have provided evidence of varying degrees of efficacy, with several studies having demonstrated improvements in clinical symptoms and surrogate markers of ischemia. Currently, the most effective way of enriching for angiogenic cells is by density gradient centrifugation separation of mononuclear cells, followed by a short-term culture step to allow for cell adhesion and discard of non-angiogenic myeloid cells. However, this process is relatively cumbersome and may not be feasible for widespread cell therapy. ES cells and iPS cells hold great promise due to their ability to provide an unlimited source of angiogenic cells and secrete angiogenic growth factors; however, this approach is tempered by concerns of teratoma formation and the difficulty in differentiating these cells into a pure endothelial or smooth muscle cell phenotype.
Stem cell therapy constitutes the injection or incorporation of stem cells or progenitor cells into diseased tissues or organs, with the goal of replacing damaged tissue and curing disease. There are three possible approaches for stem cell-based therapies. The first of these involves the isolation and enrichment of circulating or bone marrow-derived angiogenic cells (BMACs) from patients, followed by the reinjection of these cells into ischemic tissue. The second approach is drug therapy with agents that mobilize BMACs from the bone marrow, increasing the numbers of these cells circulating in the peripheral blood to enhance their natural ability to home to sites of ischemia and form functional collateral vessels. Thirdly, there is the option of using embryonic stem (ES) cells or induced pluripotent stem cells instructed in vitro to differentiate into a specific mature cell type suitable for transplantation.
Gene Therapy
Gene therapy, or the introduction of new genes into the body in order to treat disease, is another promising option for PAD. Gene therapy has been studied in animal models with promising results. In a study, an animal model of hindlimb ischemia was treated with administration of hepatocyte growth factor (HGF) by gene transfer. HGF is a powerful mitogen for endothelial cells, and has already been shown to be angiogenic in vivo. The gene transfer of HGF resulted in a significant increase in blood flow in the ischemic limb, as compared to control groups. This was attributed to an increase in angiogenesis and arteriogenesis, which were also demonstrated histologically. Although direct growth factor therapy has had limited success in clinical trials, gene therapy represents a more prolonged and effective means of treatment. By targeting specific genes involved in angiogenesis, clinicians may be able to provide a safer and more efficient therapy for PAD. This may prove useful in patients with limb-threatening ischemia who are not candidates for revascularization.
Tissue Engineering
This tissue can then be surgically re-introduced into the patient and placed in situ or at an isolated site. Scaffold development is a dynamic field of study in PAD, especially as the ideal scaffold would be able to mimic the structure of the ECM it is replacing and have a lifespan that allows for slow degradation to be replaced by neo-tissue formation over time. This would ultimately lead to the complete regeneration of native tissue. Although achieving this is unlikely, the evolution of scaffold materials has led to those that can mimic certain aspects of native tissue and allow for control over degradation.
Tissue engineering, when possible, is undoubtedly a less invasive style of medical treatment for PAD. It may use cells from the patient’s own body to avoid immune response reactions and overcome the dilemmas associated with immunosuppressive therapy. The cells can be extracted mainly from the bone marrow and, in specific situations, from the skeletal muscle. These cells can be stimulated to enter a growth stage by other growth factors known to be produced in PAD. Then, these cells are introduced to a re-absorbable scaffold and cultured in vitro to encourage the development of an autologous bioengineered tissue.
Challenges and Future Directions
The future of regenerative therapy for PAD is potentially bright. In the next 5-10 years, the results of ongoing and future clinical trials will dictate the role of cell-based therapies in the treatment of PAD. The development of therapies involving cytokines, gene therapy, and tissue engineering will further build on the concept of direct angiogenesis and arteriogenesis stimulation seen with cell-based therapies and may offer simplified treatment options for patients. Should the safety and efficacy of regenerative therapies be realized, it is foreseeable that they could be used in combination with traditional revascularization techniques for patients with limb-threat, or as an attempt to salvage limbs that would otherwise be destined for amputation. Despite the long road ahead, the rapid pace of recent advancements in regenerative medicine suggests that new treatment options for PAD may be available within a generation.
The in-depth investigation above has revealed that the regenerative medicine options are auspicious for treatment of PAD, but still in the experimental phase. Clinical application of various current regenerative strategies is warranted before embarking upon further explorative basic science studies into novel regenerative techniques. Unfortunately, the progress of regenerative therapies into the clinical realm is limited by the lack of understanding of the mechanisms of action of cell therapies, availability of cells, and conflicting results from animal model experiments. Additionally, there has been no consensus for what should be the most appropriate control group for regenerative trials; in the current climate of evidence-based medicine, this is an essential consideration. Regulatory issues, funding, and industry commitment also pose significant barriers to regenerative therapies becoming part of standard clinical practice in the foreseeable future. All of these limitations will have a significant impact upon the pace of regenerative therapy development for PAD.
Limitations of Regenerative Medicine in PAD
Despite the promise of regenerative medicine in treating PAD, there are potential limitations that will need to be addressed. Much of the ongoing research is in preclinical trials. This is currently only a minor representation of what may be translated to clinical therapies. Most research is focusing on enhancing collateral vessel growth through angiogenesis or arteriogenesis. The translation to the clinic may involve administration of growth factors, gene therapy, or autologous cell transplantation. Problems may occur in safety, efficiency, and what specific patients and disease states these therapies may be appropriate for. It has yet to be determined what specific therapy may be indicated for a specific type of patient, and in the case of autologous cell therapy, it will need to be discerned whether taking cell donations from the patient’s bone marrow and expanding said cells ex vivo is cost efficient compared to isolating an angiogenic factor and producing it commercially. In order to apply any of these therapies, it will be necessary to have a more thorough understanding of PAD disease progression and the exact pathophysiology of ischemia in each case so that therapies can be tailored accordingly. This includes determining a specific time point at which it is less beneficial to attempt to revascularize an area of ischemic muscle and apply regenerative therapy instead.
Potential Strategies for Overcoming Challenges
The major challenge facing regenerative therapies in PAD is how to effectively use the patients’ own cells for stimulating regeneration, or combine cells with gene therapy or growth factors to achieve therapeutic angiogenesis. The failure of cell therapies to date in achieving clinically meaningful increases in limb perfusion has led to calls for new translational research and the testing of novel hypotheses. A major need is the identification of the natural signals for tissue regeneration, such as differences in the protein or gene expression profiles between non-healing and healing wounds. Understanding the body’s failure to heal wounds or ischemic tissues is a critical step to developing new therapies. An interesting example of this is the difference in the protease: protease inhibitor balance observed in chronic non-healing wounds. Other potential mechanisms to enhance the efficacy of regenerative therapies involve creating a pro-regeneration environment within the ischemic tissue. This can be done by physical methods such as shockwave therapy or external pressure to improve cell survival and the paracrine effects of injected cells. An entirely different approach is to perform targeted disruption of the HIF hydroxylase gene to enhance endogenous HIF-1 activity and thus increasing expression of HIF-1 responsive genes involved in angiogenesis and cell survival.
Future Perspectives in Regenerative Medicine for PAD
Major breakthroughs in each of these areas are likely to take several decades, and progress will be iterative with periodic revisions of strategies as new insights are gained.
Preclinical studies will also be required to develop and test gene transfer strategies to enhance the reparative potential of cells or to alter the functions of detrimental gene products in the ischemic milieu. These studies will necessitate the development of new gene transfer vectors and detailed investigation of their safety and efficacy profiles. In vitro and in vivo studies of combination therapies involving cell-based or gene transfer and traditional or novel pharmaceutical therapies, growth factors, or cytokines will also shed important light on rational therapeutic design.
Parallel studies must be performed to identify the optimal modes of delivery of cell therapies and develop and test new generations of biomaterial and bioengineered delivery systems. A detailed understanding of the pharmacokinetics and pharmacodynamics of cell therapy will be essential for the rational design of cell delivery strategies and dosing regimens. Studies to compare efficacy of different cell types and delivery strategies will need to be performed in animal models with close attention to quality control in cell production and standardization of cell dosing and delivery.
Elucidation of the mechanisms by which the PAD microenvironment impairs the function of reparative cells and/or stimulates maladaptive repair will identify targets for restoring normal repair and overcoming the adverse effects of ischemia and comorbidities. High-throughput screening technologies to identify compounds that inhibit detrimental effects on reparative cells or enhance their function will be an important adjunct to this work.
Studies need to be designed to identify the intrinsic properties of cells that mediate repair and to elucidate the molecular mechanisms by which these properties are expressed. This will allow for rational manipulation of cell phenotype and/or genotype to enhance their therapeutic potential or to identify small molecules or biologics that may be used to enhance endogenous repair or to act in lieu of cell therapies.
Numerous questions regarding all aspects of regenerative medicine for PAD will need to be addressed in future studies. This will require careful and consistent collaboration between basic scientists, translational researchers, and clinical investigators from multiple disciplines, including but not limited to cell biology, biochemistry, genetics, molecular biology, transplantation biology, biomaterial sciences, vascular biology and medicine, vascular surgery, interventional radiology, and cardiology.
