Improving Progenitor Cell Selection to Promote Therapeutic Angiogenesis in Patients With Critical Limb Ischemia

Peripheral artery disease (PAD) is a major health problem, representing the 3rd leading cause of cardiovascular morbidity after coronary artery disease and stroke. The prevalence of PAD rises sharply with aging, so it can affect up to 20% of the population at the age of 80 years. Epidemiologic studies have documented a recent increase in the prevalence of PAD, highlighting the notion that we are now faced with a PAD pandemic, affecting more than 200 million men and women worldwide.^1–3

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In its more severe and aggressive form, PAD is associated with chronic ischemia that leads to the development of rest pain, non-healing ulcers and tissue necrosis with gangrene, a condition known as critical limb ischemia (CLI). Treatment strategies in patients with CLI are essentially focused on surgical bypass or endovascular interventions aimed at restoring perfusion to prevent amputation of the affected limb.⁴ However, a significant proportion of CLI patients do not have revascularization options because of high operative risk and/or unfavorable vascular anatomy. These patients have a poor prognosis and it is estimated that 25% of patients diagnosed with CLI will die within 1 year and an additional 30% will require limb amputation.⁵ The isolation of bone marrow-derived endothelial progenitor cells (EPCs) that can home in to sites of ischemia and contribute to angiogenesis⁶ provided a strong rationale for the use of cell therapy in PAD patients. The landmark Therapeutic Angiogenesis by Cell Transplantation (TACT) trial was the first study to demonstrate that intramuscular transplantation of autologous bone marrow-derived mononuclear cells (BM-MNCs) could improve the ankle-brachial index (ABI) and tissue oxygen saturation (TcPO₂) in patients with CLI.⁷ In the past 2 decades, there have been more than 50 reported therapeutic cell trials in patients with PAD.^8–10 Most of these have been small phase 1 or 2 studies. Nonetheless, a recent meta-analysis of randomized, nonrandomized and noncontrolled trials for treatment of PAD suggested that although cell therapy did not affect all-cause death, it may have significantly improved amputation-free survival rates and ameliorated endpoints related to limb perfusion, rest pain and functional capacity.¹¹

The majority of cell therapy trials in PAD patients have used unselected autologous MNCs harvested from the bone marrow, or peripheral blood MNCs isolated by leukapheresis after mobilization with G-CSF or GM-CSF.^8–10 In this issue of the Journal, Liotta et al¹² report the results of the SCELTA trial, which compared the security and efficacy of direct muscular injections of nonmobilized enriched circulating EPCs isolated from the blood (ECEPCs) vs. BM-MNCs in patients with CLI. In a previous study, the same group demonstrated the existence of a subpopulation of circulating CD14⁺ cells exhibiting low expression of CD34. These CD14⁺CD34^low cells could differentiate into endothelial cells in vitro and constituted a major source of EPCs in the peripheral blood.¹³ In the SCELTA study, the authors used leukapheresis and antibody-coated magnetic beads to isolate CD14⁺ cells (including CD14⁺CD34^low cells) and CD34⁺ cells. Although 60 patients were initially planned, the enrollment was stopped after 40 patients, as 1 primary endpoint (improvement of muscle perfusion) was already achieved. This was a single-center study and the recruitment period was quite long (>5 years), highlighting the difficulty of enrolling patients in this type of complex study with strict inclusion and exclusion criteria. In the end, 23 patients were treated with ECEPCs and 17 with BM-MNCs. It was found that both therapies were safe and provided similar levels of improvement in terms of rest pain, consumption of analgesics, pain-free walking distance, wound healing and quality of life, as well as toe-brachial index (TBI), ABI, TcPO₂ and muscle perfusion assessed by contrast-enhanced ultrasound.¹² Compared with historical clinical controls, both groups showed reduced number of deaths and major amputations, although these data should be seen as exploratory, considering the small number of patients and the lack of a control group not receiving cell therapy.

The trial raises several important questions. The major issue is the mechanism by which ECEPCs can improve tissue perfusion in patients with CLI. It is now recognized that only a very small pool of circulating or tissue resident cells are ‘true’ EPCs that can differentiate into mature endothelial cells, integrate into vascular structures and participate in the formation of neovessels.¹⁴ On the other hand, most circulating pro-angiogenic cells (including the originally described ‘early-outgrowth EPCs’) do not differentiate into endothelial cells but stimulate angiogenesis and neovascularization through paracrine secretion of angiogenic factors.¹⁴ Are CD14⁺CD34^low cells ‘true’ EPCs? Although these cells have been described as bona fide EPCs in vitro,¹³ whether they act as true EPCs in vivo is currently unknown. Moreover, their ability to stimulate neovascularization and improve tissue perfusion has not been established. The ECEPCs described in the SCELTA study were a mixture of several cell types, including CD14⁺CD34⁻ cells (81% on average), CD14⁺CD34^low cells (18%) and CD34⁺CD14⁻ cells (0.26%). Therefore, although ‘enriched’ in CD14⁺CD34^low cells, ECEPCs are not a pure population of progenitor cells. The authors found that the number of CD14⁺CD34^low cells positively correlated with calf perfusion values, but no correlation was found with the other cell types. However, it is still possible that the other cell types (e.g., CD14⁺CD34⁻ cells) act synergistically with CD14⁺CD34^low cells to promote angiogenesis via different mechanisms (Figure). In a similar fashion, it was previously shown that coculture of CD34⁺ cells with CD34⁻ cells led to robust enhancement of neovascularization in vitro.¹⁵

Figure.

Potential mechanisms involved in the angiogenic effects of enriched circulating endothelial progenitor cells (ECEPCs). EC, endothelial cells; MMPs, matrix metalloproteinases; MVs, microvesicles.

The investigators of the SCELTA trial injected all the cells that could be retrieved, so each patient received a different number of cells. It will be important in future studies to determine the dose of cells that is needed to improve perfusion in patients with CLI. In addition, the route of administration (i.e., intramuscular vs. intra-arterial) and the frequency of administration are other important parameters to investigate. It is unlikely that a single injection of cells (with a short survival time in ischemic tissues) is optimal to address chronic diseases such as PAD and CLI. Whereas initial cell therapy trials used crude cell populations harvested from the bone marrow, it seems appropriate to identify, isolate and use as therapeutic agents the progenitor cells that exhibit the highest angiogenic activities in vitro and in vivo. In this regard, the SCELTA trial is a step in the right direction. In the future, randomized double-blind placebo controlled studies are needed with a systematic approach to characterize the cell therapy product, including its isolation, dosing, frequency, and delivery method. The endemic crisis of PAD warrants new therapies. The so-called ‘no-option’ patients with CLI require novel approaches such as cell therapy to improve perfusion, reduce pain and avoid tissue damage and limb amputation.

References

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