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. Author manuscript; available in PMC: 2019 Apr 1.
Published in final edited form as: Curr Opin Pharmacol. 2018 Feb 13;39:27–34. doi: 10.1016/j.coph.2018.01.005

Cell therapy for peripheral artery disease

Nikolaos G Frangogiannis 1,*
PMCID: PMC6019642  NIHMSID: NIHMS943273  PMID: 29452987

Abstract

Patients with severe peripheral artery disease (PAD) who are not candidates for revascularization have poor prognosis. Cell therapy using peripheral blood- or bone marrow-derived mononuclear cells, mesenchymal stem cells, or marker-specific subsets of bone marrow cells with angiogenic properties may hold promise for no-option PAD patients. Injected cells may exert beneficial actions by enhancing local angiogenesis (either through maturation of endothelial progenitors, or through secretion of angiogenic mediators), or by transducing cytoprotective signals that preserve tissue structure. Despite extensive research, robust clinical evidence supporting the use of cell therapy in patients with critical limb ischemia is lacking. Larger, well-designed placebo-controlled clinical trials did not support the positive results of smaller less rigorous studies. There is a need for high-quality clinical studies to test the effectiveness of cell therapy in PAD patients. Moreover, fundamental cell biological studies are needed to identify the optimal cell types, and to develop strategies that may enhance homing, survival and effectiveness of the injected cells.

Introduction

Lower extremity peripheral artery disease (PAD) is a major health burden, representing the third-leading cause of cardiovascular morbidity related to atherosclerotic disease after coronary disease and stroke. The prevalence of PAD rises sharply with age, affecting almost 20% of the US population at the age of 801,2. Epidemiologic studies have highlighted the global impact of the disease, suggesting dramatic recent increases in PAD prevalence in low and middle-income countries, and supporting the notion that we are faced with a global PAD pandemic, affecting more than 200 million men and women in both high-income countries and in the developing world3. Considering the mortality, morbidity and disability associated with PAD, there is an urgent need to develop new therapeutic strategies in order to prevent development and progression of the disease, and to treat life- or limb-threatening complications. Experimental studies and early stage clinical trials have suggested that cell therapy may be a promising new approach for patients with PAD4. The current review manuscript discusses the potential role of cell therapy approaches in the treatment of PAD.

The pathophysiologic basis of PAD

The clinical manifestations of PAD reflect the consequences of a mismatch between blood supply and demand5,6. The typical symptom of PAD is intermittent claudication, a characteristic squeezing leg pain associated with walking and relieved by rest. In normal subjects, exercise is associated with marked increases in peripheral artery blood flow and limb oxygen uptake, driven by increased metabolic demand. In contrast, in PAD patients, fixed stenotic lesions in peripheral arteries limit blood flow, reducing the supply of the affected territory and leading to ischemia. Although the main cause of supply and demand disequilibrium in PAD patients is structural, excessive vascular tone due to activation of neurohumoral pathways, or impaired vasodilatory responses due to endothelial dysfunction may increase vascular resistance, further limiting blood flow in the extremity7.

Repetitive limb ischemia followed by reperfusion causes mitochondrial dysfunction in skeletal myocytes and triggers generation of reactive oxygen species (ROS), leading to chronic structural changes in the skeletal muscle. ROS-driven apoptosis of skeletal myocytes leads to a reduction in skeletal muscle mass and is accompanied by fatty infiltration, impaired peripheral nerve function and fibrosis8,6,9,10. These pathologic alterations are associated with chronic skeletal muscle dysfunction and significant functional impairment. In a subset of patients, chronic ischemia follows an aggressive clinical course that culminates in the development of rest pain and significant tissue loss, a condition termed critical limb ischemia (CLI). Traditional treatment strategies in patients with CLI are focused on surgical bypass or endovascular interventions, aimed at restoring perfusion to prevent amputation of the affected limb11. However, a significant percentage of CLI patients do not have revascularization options; these patients have poor prognosis and often require amputation.

Cell therapy as a therapeutic approach in PAD

Considering the limited treatment options for patients with severe PAD, the rationale for cell therapy approaches is sound. In patients with severe atherosclerotic disease of the native arterial circulation, administration of cell populations capable of activating an angiogenic program may result in formation of neovessels, improving perfusion of the affected limb. Increased blood supply may prevent ischemic episodes and may even contribute to restoration of normal skeletal muscle structure. It should be emphasized that any beneficial effects of cell thepapy in PAD may not be necessarily due to incorporation of the cells into the vascular network, but may involve paracrine effects mediated through secretion of angiogenic mediators. Cell therapy may also activate yet unidentified cytoprotective and regenerative pathways that may improve limb function through effects independent of neovessel formation.

A growing body of experimental and clinical evidence suggests that cell-based therapy may hold promise in patients with severe PAD. Experimental investigations have used models of hindlimb ischemia to study the effectiveness of cell therapy approaches in promoting angiogenesis and in attenuating skeletal muscle injury. On the other hand most clinical studies investigating the effectiveness of cell therapy in patients with CLI are small phase I or II clinical trials. Considering the variable approaches used by different groups, the wide range of cell types used, and the absence of standardized protocols for characterization of the cells and for evaluation of clinical outcome, there is substantial uncertainty regarding the effectiveness of various cell types in PAD patients.

The therapeutic potential of endothelial progenitor cells (EPCs)

The identification of EPCs, as bone marrow-derived progenitors, that home to sites of injury and may contribute to angiogenesis12 provided a strong rationale for the use of cell therapy in PAD patients. It should be noted that, despite progress in understanding the mechanistic basis of the angiogenic response, the contribution of blood-derived progenitors in neovessel formation following injury remains controversial. In a mouse model of hindlimb ischemia, both marrow-derived and non-marrow derived endothelial progenitor populations have been implicated in formation of neovessels13. Despite the recent use of lineage tracing approaches in mouse models, the origin of neovascular endothelial cells in sites of injury remains controversial. Studies in the ischemic myocardium suggested a significant contribution of mesenchymal cells that undergo conversion into endothelial cells through a p53-dependent mechanism14. In contrast, other investigations suggested that practically all neovessels in the injured myocardium are derived from pre-existing endothelial cells, and not through lineage transdifferentiation15. It is plausible that the relative contributions of various cellular sources in the angiogenic response may be dependent on the pathophysiologic context and on the site of injury. Unfortunately, lineage tracing studies investigating the cellular origin of angiogenic vascular cells in ischemic skeletal muscle have not been performed.

Regardless of the origin of endogenous angiogenic endothelial cells in the ischemic limb, local injection of circulating endothelial progenitors would be expected to enrich the ischemic site with a pool of angiogenic cells, promoting neovessel formation and improving function. To achieve this goal, several different approaches have been used, injecting unselected or marker-specific mononuclear cells from the bone marrow, or the peripheral blood. These populations may contain a subset of bona fide endothelial progenitors that incorporate to the vascular network forming new vessels, and other cell types that may contribute to the angiogenic process by secreting cytokines, angiogenic growth factors, matrix metalloproteinases, matricellular proteins, or miRNA-containing exosomes (Figure 1)16,17,18.

Figure 1.

Figure 1

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Cell biological mechanisms responsible for the protective effects of cell therapy in peripheral artery disease. Most clinical studies have tested the effects of intramuscular or intra-arterial injections of bone marrow (BM) or peripheral blood-derived mononuclear cells (BM-MNC and PB-MNC respectively), marker-specific mononuclear cells, or bone marrow-derived mesenchymal stem cells (BM-MSC). These cells contain subsets of endothelial progenitor cells (EPCs) that may promote angiogenesis by incorporating into the vascular system. Injected cells may also act through paracrine actions by secreting angiogenic cytokines, growth factors, matrix metalloproteinases and mi-RNA-containing exosomes. Moreover, cell therapy may protect ischemic skeletal myocytes (SM), modulate inflammatory activity and inhibit fibrogenic pathways, preserving structure and function. Additional symbols: Ma, macrophage; F, fibroblast; EC, endothelial cell; P, pericyte.

Bone marrow-derived mononuclear cells (BM-MNC) and peripheral blood-derived mononuclear cells (PB-MNC) in the treatment of PAD

Unselected mononuclear cells harvested, derived either from the bone marrow or the peripheral blood, represent a mixture of monocytes, non-hematopoietic stromal cells (including mesenchymal stem cells), and EPCs and have been used in both experimental models of limb ischemia and in patients with PAD. In the TACT (Therapeutic Angiogenesis using Cell Transplantation) study, injection with autologous BM-MNCs in the gastrocnemius of the ischemic limb reduced rest pain and increased transcutaneous oxygen pressure in patients with CLI; improvement was sustained for at least 24 weeks19. Over the last few years, several additional clinical trials suggested that intramuscular or intra-arterial injections of BM-MNCs or PB-MNCs in patients with CLI are safe, and may reduce rest pain and improve ulcer healing (Table 1)20,21. In many studies, improved clinical outcome was associated with objective evidence of enhanced perfusion. However, in most studies, effects on amputation rates did not reach statistical significance. Some of the larger, more rigorous, and well-designed studies failed to support the beneficial effects22, suggested by smaller nonplacebo controlled investigations20. The conflicting findings may reflect the clinical improvement observed in placebo-treated patients22, and emphasize the importance of rigorous design, large population size and accurate blinding in order to test the effectiveness of therapy. The optimal cell type, concentration of cells and route of delivery remain unclear. Moreover the mechanistic basis for any protective effects remains obscure. Prolonged survival of the injected cells in the hostile environment of the ischemic limb has not been documented. Thus, it is unclear whether any benefit related to cell therapy is due to direct involvement of the cells in angiogenesis, or reflects paracrine effects that may include indirect stimulation of angiogenic pathways, modulation of inflammatory cascades, or cytoprotective actions on the ischemic limb.

Table 1.

Findings of randomized controlled trials examining the effects of cell therapy in patients with Critical Limb Ischemia (CLI)

Type of cell
therapy		 Patient population Follow
-up		 Main findings Ref.
Autologous BM-MNCs (im). Patients (84% males, 69% diabetic) with chronic limb ischemia, rest pain, non-healing ulcers (22%), gangrene (40%) without revascularization options. Group A: 25 patients with unilateral ischemia receiving BM-MNCs (vs. saline infusion in the less ischemic limb). Group B: 22 patients with bilateral ischemia, receiving BM-MNCs (in one limb) vs. PB-MNCs (in the other). 24w-3y Cell therapy increased ankle-brachial index (ABI), reduced rest pain, improved walking time, and increased transcutaneous oxygen pressure (TcO2). Increases in ABI were higher in patients receiving BM-MNCs (vs. PB-MNCs). Follow-up suggested sustained improvement in leg pain and ulcer size for at least 2y. 19
Autologous PB-MNCs (im).
Autologous G-CSF-mobilized PB-MNC (im) 28 diabetic patients (cell therapy 14, control 14) Gender: 18M, 10W. Mean age: 71y 3mo Cell therapy reduced leg pain, reduced amputation rate (AR) (treatment: 0%, control: 20%), improved ulcer healing, increased perfusion, improved angiographic scores, and increased ABI. 20
Autologous G-CSF-mobilized PB-MNC (im) 40 diabetic patients (cell therapy 20, control 20). Gender: 29M, 11W. Mean age: 71.4y 12w Cell therapy reduced pain, improved Fontaine score, improved ulcer healing, and increased ABI and TcO2. No significant effects on AR (treatment: 15%, control: 25%). 38
Autologous G-CSF mobilized PB-MNCs (im) 21 diabetic patients (control 14, cell therapy 7) Mean age: 64y 3mo Cell therapy improved walking ability, reduced AR (treatment: 0%, control: 50%), improved blood flow and increased ABI. There was a trend towards a reduction in ulcer size. 39
Autologous CD34+ cells (im, low and high dose protocols) 28 patients (control 12, low dose 7, high dose 9; 54% diabetic) Gender: 19M, 9F Mean age: 66y. 12mo Cell therapy was safe and well-tolerated. There was a trend towards a reduction in AR (control: 75%, low dose: 43%, high dose: 22%) 23
Autologous expanded blood-derived “angiogenic cell precursors” (VesCell) 20 patients (control 10, cell therapy 10, 60% diabetic) Gender: 13M, 7F Mean age: 61.8y. 3mo–2y Cell therapy reduced AR (major amputations in the treatment group: 0% at 3mo and 30% at 2y and in the control group 60% at 3mo and 2y), increased ABI at 3mo and 2y, and increased TcO2. 40
Autologous CD133+ cells sorted from G-CSF-mobilized PB-MNCs 10 patients (2:1 randomization) Gender: 8M, 2W. Mean age: control 85y, treatment 65y. 12mo Subject enrollment was suspended due to a high rate of mobilization failure. Cell therapy was associated with non-significant trends towards lower AR (control: 66%, treatment: 14%), reduced walking impairment and improved quality of life 41
Autologous BM-MNCs (im) 25 patients (treatment group n=13, control n=12). Age: treatment 62y, control 68y. 1mo Cell therapy improved rest pain, and increased ABI and TcO2. There were no reports of amputations in control or treatment groups. 42
Autologous bone marrow aspirate concentrate (im) 96 patients Cell therapy: n=42 (36M, 6F), Control: n=54 (42M, 12F) Age: treatment 66y, control 64y. Diabetes: treatment 88.1%, control 98.2%. 120d Cell therapy reduced rest pain, decreased AR (treatment 21%, control: 44%), and increased ABI and TcO2. The bone marrow concentrate of patients who failed therapy exhibited lymphopenia. 43
Autologous bone marrow aspirate concentrate (im) 48 patients. Treatment n=34 (23M, 11F), control n=14 (9M, 5F). Age: treatment 72.5y, control 65.7y. Diabetes: treatment 52.9%, control 42.8%. 6mo Cell therapy was well tolerated. There were trends towards reduced AR (treatment: 17.6%, control: 28.6%), reduced pain, increased ABI and better quality of life in the cell therapy group. 44,45
Autologous BM-MNCs (im) 41 diabetic patients, mean age: 64y BM-MNC group (n=21 limbs, males: 42%), BM-MSC group (n=20 limbs, males: 39%), Normal saline (n=41 limbs). 24w Administration of BM-MNCs and BM-MSCs was well-tolerated. Cell therapy reduced AR (0% in BMMSC and BM-MNC group, vs. 16.2% in control group) and improved rest pain. Ulcer healing was accelerated in the BM-MSC group in comparison with BM-MNC patients. 21
Autologous BM-MSCs (im)
Autologous BM-MNC (ia) 39 patients (treatment n=19, control n=20) Age: treatment 64.4y, control 64.5y. Diabetes: treatment 53%, control 48%. Male: treatment 84%, control 62%. 3mo Cell therapy did not significantly affect AR (treatment 21%, control 5%) and did not significantly increase ABI. However, cell therapy improved ulcer healing and reduced rest pain. 46
Autologous BM-MNCs (im)+ VEGF gene therapy 32 non-diabetic patients. Treatment: n=16, 11M 5W, mean age 66.8y. Control: n=16, 10M, 6W, mean age 68.3y. 3mo No statistically significant difference in AR between groups (treatment: 25%, control: 50%). Only patients enrolled in the treatment group exhibited increased ABI (75% of treated patients) and ulcer healing (69% of treated patients). 47
Autologous BM-MNCs (im) 58 patients BM-MNC: (n=29, 22M, 7F), Placebo: (n=29, 23M, 6F). Diabetes: treatment 44.8%, control 41.4%. Mean age: treatment 61y, control 63y. 6mo Cell therapy improved rest pain and ulcer healing and increased ABI. There was no significant difference in the rate of major amputations (treatment 10%, control 17%). 48
Autologous expanded BM-MNCs, containing CD90+ cells and a subset of macrophages (Ixmyelocel-T) 72 patients Treatment: n=48, 34M 14F. Control: n=24 14M 10F. Diabetes: treatment 44%, control 63%. Age: treatment 69.2y, control 67.3y. 12mo Cell therapy was well-tolerated, but did not affect AR (control: 25%, treatment 21%). 24
Repetitive (3 times in a 3-week interval) autologous BM-MNC (ia) 160 patients. Treatment: n=81 (57M, 24F), control: n=79, (51M 28F). Age: treatment 69y, control 65y. Diabetes: treatment 36%, control 39%. 6mo Cell therapy had no effects on AR (control 13%, treatment 19%), rest pain, ABI and TcO2. 22
Autologous BM-MNC (im) 38 patients (treatment n=18, placebo n=20). Diabetes: treatment 55%, control 35%. 6–12mo Cell therapy has no effects on AR (placebo: 26%, treatment: 18%). Cell therapy and control groups had comparable improvements in rest pain and in TcO2. 49
Allogeneic expanded BM-MSC derived from healthy donors (im) 20 patients. MSC group (n=10), placebo (n=10) Patients had either atherosclerotic disease or thrombangitis obliterans 6mo Cell therapy was safe and increased ABI. There were no significant effects on AR. 26
Allogeneic expanded BM-MSC derived from healthy donors (high and low dose groups - im) 90 patients with CLI due to thrombangitis obliterans. Control (n=18), low dose (n=36), high dose (n=36). Age range: 38–42y. 6mo AR was comparable between groups. High dose BM-MSCs reduced rest pain and improved ulcer healing. Both low and high-dose groups exhibited improved quality of life scores. 50
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CLI, critical limb ischemia; Ref., Reference; G-CSF, granulocyte-colony stimulating factor; AR, amputation rate; ABI, ankle-brachial index; TcO2, transcutaneous oxygen pressure; PB-MNC, peripheral blood-derived mononuclear cells; BM-MNC, bone marrow-derived mononuclear cells; BM-MSC, bone marrow-derived mesenchymal stem cells; im, intramuscular; ia, intra-arterial; M, men; W, women; mo, months; w, weeks; d, days; y, years

Administration of marker-specific cells

Considering the functional and phenotypic heterogeneity of MNCs, identification of specific MNC subsets with angiogenic or cytoprotective properties represents a rational approach for further development of cell therapy strategies for PAD patients. Evidence suggests that CD34 may mark a subset of cells with angiogenic potential; a subset of CD34+ cells may be capable of differentiation to mature endothelial cells. In a randomized controlled pilot study, intramuscular injection of Granulocyte-Colony Stimulating Factor (G-CSF)-mobilized CD34+ cells in patients with CLI was safe and was associated with trends towards reduced amputation rates23. Strategies using multicellular subsets of bone marrow-derived cells have also been tested. Ixmyelocel-T is an expanded population of bone marrow cells comprised predominantly of CD90+ mesenchymal cells and a subset of bone marrow macrophages. In a randomized phase 2 trial in patients with CLI, treatment with Ixmyelocel-T was safe, but did not significantly affect major amputation rates24. Expression of high levels of the cytosolic enzyme aldehyde dehydrogenase (ALDH) has also been used to select a subset of angiogenic progenitors derived from bone marrow cells. In a randomized double-blind phase 2 clinical trial, intramuscular injection of cells with high ALDH activity did not improve peak walking time and perfusion in patients with claudication25.

Mesenchymal stem cells (MSC)

Because of their potential for transdifferentiation and their potent effects in modulation of cell survival, inflammation and angiogenesis, MSCs are promising candidates for PAD cell therapy. A randomized controlled trial showed that infusion of autologous bone marrow-derived MSCs (BM-MSC) improved symptoms, accelerated ulcer healing and accentuated collateral blood vessel growth in diabetic patients with CLI21. Moreover, administration of allogeneic bone marrow-derived MSCs from healthy donors in patients with end-stage PAD was safe and well-tolerated, but did not have significant effects on amputation rates26.

Appraisal of the clinical evidence on the effects of cell therapy in PAD

A recent meta-analysis of randomized, nonrandomized and noncontrolled studies for treatment of PAD suggested that although cell therapy did not affect all-cause mortality, it may have significantly improved the chances of amputation-free survival and ameliorated endpoints related to limb perfusion, pain and functional capacity in comparison with control treatment27. However, efficacy of cell therapy on all endpoints was no longer significant in placebo-controlled studies and disappeared in randomized controlled trials with a low risk of bias27. Thus, there is currently no robust evidence to support the effectiveness of cell therapy in patients with PAD.

PAD patients who are not candidates for revascularization strategies due to high risk, unfavorable vascular involvement, or failed endovascular approaches (no-option patients) represent a major therapeutic challenge and have a poor prognosis. It has been argued that, because there is no alternative to amputation in patients with end-stage CLI, cell therapy should be administered if available, even in the absence of robust evidence to support effects on amputation-free survival27,4. However, implementation of expensive treatment strategies with minimal or no benefit to the affected patient population is of limited value, and may be harmful by reallocating healthcare resources, depriving other patient populations from highly effective therapies.

What are the reasons for the limited success of cell therapy in PAD patients? First, design of cell therapy approaches has been based predominantly on empiricism, and much less on sound cell biological insights. The lack of experimental animal models that recapitulate the pathophysiology of human PAD greatly complicates design of new strategies and limits our ability to test their effectiveness. Experimental evidence, when available, is often based on studies performed in young healthy animals that may not be relevant to the human PAD populations, typically comprised of older subjects with a high prevalence of diabetes, smoking and chronic atherosclerotic disease. Second, BM-MNCs and PB-MNCs typically used for treatment are highly heterogeneous, and may contain several different subsets with a wide range of effects. Specific subpopulations with protective angiogenic properties need to be defined. Third, the use of autologous cells in patients with comorbid conditions (such as diabetes) that may perturb their reparative and angiogenic properties may greatly limit effectiveness. Fourth, the fate of the cells following injection is unclear. Survival of the cells in the hostile environment of the ischemic limb may be limited; persistence of the cells in ischemic regions has not been consistently documented. Fifth, human PAD is characterized by anatomical heterogeneity, resulting in marked regional differences in the severity of ischemia and tissue damage. Refined administration strategies taking into account the regional distribution of ischemic lesions in PAD patients may be required to improve effectiveness.

There is little doubt that cell therapy in PAD treatment has a bright future. To achieve the full potential of this highly promising strategy, there is a need for a concerted effort to advance our knowledge on the fundamental cellular mechanisms of angiogenesis, while investing in robust clinical studies to test the most promising strategies in PAD patients.

Conclusions and future directions

The need for randomized double-blind placebo controlled studies to document any effects of cell therapy approaches in PAD patients cannot be overemphasized. The improvement observed in placebo-treated patients in PAD clinical trials22,28 emphasizes the need for rigorously designed and well-controlled studies in order to derive robust conclusions. Moreover, interpretation of the findings of clinical trials is dependent on introduction of endpoints for assessment of the cell biological consequences of the strategy. Assessment of cell homing and survival, and quantitative analysis of the effects of therapy on the vasculature in the ischemic area can provide critical information to understand the basis for success or failure, and to identify patient subpopulations with favorable responses.

Most importantly, we need to introduce new cell biological concepts in the design of cell therapy approaches. Dissection of the cell biological mechanisms of angiogenesis is critical to design an effective cell therapy approach for PAD patients. Understanding the phenotypic profile, properties, and mobilization mechanisms of endothelial progenitor populations and of mononuclear cell subsets with angiogenic properties is needed to define optimal cell types for therapy. Moreover, treatment with mediators that improve mobilization, homing and survival of endogenous progenitor cells may be useful to maximize benefit of cell therapy.

Chemokines are a family of chemotactic cytokines with an important role in leukocyte trafficking following ischemia29. Several members of the chemokine family play essential roles in mobilization and migration of endogenous EPCs and may regulate their recruitment in ischemic sites30, 31. Accentuation of chemokine signaling may be a promising strategy to enhance infiltration of the ischemic tissue with angiogenic cells. The CXC chemokine Stromal cell-derived factor (SDF)-1/CXCL12 is a key regulator of bone marrow cell mobilization and is critically involved in recruitment of progenitor cells in ischemic tissues32,33. It has been suggested that in certain pathologic conditions, such as diabetes, hyperglycemia-mediated downmodulation of chemokine receptor expression in EPCs and other progenitor cells may reduce their homing in sites of injury, resulting in defective angiogenesis and impaired reparative responses. A recent study demonstrated that manipulation of EPCs to increase expression of the chemokine receptor CXCR7 (one of the receptors that mediate CXCL12 actions) improved outcome in a model of limb ischemia in diabetic mice, enhancing the angiogenic function of the cells34. Other members of the chemokine family may act indirectly, increasing the angiogenic capacity of MNCs. The CX3C chemokine Fractalkine/CX3CL1 has been suggested to increase angiogenic potential of bone marrow-derived macrophages by accentuating expression of platelet factor-4/CXCL435. Pre-treatment of MNCs with mediators inducing an angiogenic program may stimulate their therapeutic potential in PAD. Moreover, genetic manipulations activating a pro-survival program in EPCs36 or incorporation of matrix substrates that prolong survival and promote differentiation37 may accentuate the beneficial actions of cell therapy.

Acknowledgments

Dr Frangogiannis’ laboratory is supported by NIH grants R01 HL76246 and R01 HL85440, and by Department of Defense grants PR151134 and PR151029.

Footnotes

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