Angiogenic potential of BM MSCs derived from patients with critical leg ischemia

Critical leg ischemia (CLI) is the most severe form of peripheral arterial disease and is characterized by the inability of arterial blood flow to meet the metabolic demands of resting muscle or tissue, resulting in rest pain and/or tissue necrosis, and frequently necessitating amputation. As a result of the discovery of the angiogenic potential of BM mononuclear cells (BM-MNCs), cell therapy has been proposed to treat peripheral arterial disease. The therapeutic angiogenesis by cell transplantation (TACT) study was the first trial to demonstrate a significant improvement after i.m. injection of total autologous BM-MNCs.1 In the French OPTIPEC trial (Clinical trial registration: NCT00377897), we have shown the development of a proliferative angiogenic process in the distal part of the legs in amputation specimens from patients with CLI who had received local therapeutic injections of BM-MNCs.2 Strong data from preclinical models strongly suggest that BM-MNCs do not differentiate into endothelial cells, and that their paracrine effect is most likely responsible for the angiogenic process.3 However, BM-MNCs isolated from patients with cardiovascular disease have a significantly reduced neovascularization capacity in vivo, despite similar content of hematopoietic stem cells.4 Notably, BM-MNCs contain a complex assortment of angiogenic cells, including hematopoietic progenitors as well as MSCs. An important issue is to define, among BM-MNCs, what is the sub-population of pro-angiogenic cells that could be further isolated and expanded to develop an autologous and efficient cell therapy product. Among these cell types, MSCs, which are easily obtained in culture from BM, are potential candidates. MSCs are multipotent stem cells with the ability to differentiate into mesoderm-derived cells that exhibit immunoregulatory properties, improve hematopoietic engraftment, prevent graft failure, and reduce the incidence or severity of acute GVHD. Given their capacity to secrete angiogenic growth factors, MSCs have been proposed to improve angiogenesis, but their endothelial differentiation potential is a matter of debate. Furthermore, no data exist on the angiogenic potential of MSCs isolated from patients with peripheral arterial disease. We studied 11 CLI patients enrolled in the OPTIPEC clinical trial, which is a multicenter phase I non-randomized study (median age: 65 years). As described elsewhere,2 the cell therapy protocol was similar to those initially published by Tateishi-Yuyama et al.1 For this biological study, we used 3 mL of the concentrated BM-MNCs that are used as the cell therapy product. We successfully isolated MSCs from BM-MNCs in all the 11 patients enrolled. These cells displayed a typical MSC phenotype (positive for CD90, CD73 and CD44, negative for CD14, 45 and 31, Figure 1a). We tested their ability to increase blood flow recovery in a nude mouse model of hindlimb ischemia.5 MSCs from CLI patients showed an effect comparable to that of MSCs isolated from four control patients of the same age, diagnosed for a peripheral thrombocytopenia with no peripheral arterial disease or cardiovascular disease (Figure 1b). These data demonstrate that MSCs, isolated from the BM-MNCs in patients with CLI, have a strong ability to promote neovascularization in nude mice after induction of hindlimb ischemia (P<0.05 for control MSCs and MSCs from CLI patients vs PBS patients; P=0.36 between control MSCs and MSCs from CLI patients; Mann and Whitney test). To decipher the mechanism of the angiogenic effect of MSCs, we tested the ability of MSCs to promote neovascularization in our model versus endothelial progenitor cells (endothelial colony forming cells: ECFCs) isolated from cord blood. Indeed, ECFCs have been proposed as the active cell type in vasculogenic processes.6 To identify the species origin of cells in the neo-vessels 14 days after surgery, ischemic and non-ischemic muscles from mice that received MSCs, ECFCs or PBS were harvested, and human as well as murine cells were enumerated. We found that MSC administration promoted the infiltration of murine CD31-positive cells, in contrast to ECFCs that increased human β2-microglobulin and CD31-positive cells (Figure 1c). These results are in agreement with the concept of a paracrine angiogenic effect of MSCs on local murine endothelial cells, whereas ECFCs are able to build vessels by themselves. A future challenge will be to develop an autologous cell therapy product dedicated to treat CLI. To date, no homogeneous cell preparation has been proposed and the angiogenic potential of MSCs isolated from patients with peripheral arterial disease has never been tested. We have hereby shown the unaltered angiogenic capacity of MSCs from CLI patients compared with control adults. This result contrasts with the decreased angiogenic potential of MNCs observed in patients with cardiovascular disease.4 The angiogenic potential of MSCs seems to be preserved, despite cumulative cardiovascular risk factors in CLI that make this cell population really attractive for the development of an autologous cell therapy product. Interestingly, rat MSCs have been shown to have better angiogenic potential than MNCs.7 This result combined with the efficacy of MSCs from CLI patients is in favor of their use as a cell therapy product instead of MNCs. The present study was designed as a pilot and feasibility trial. The functional characteristics of MSCs need to be confirmed in large and randomized trials, which are currently ongoing. In case of failure to isolate/cultivate MSCs from a given CLI patient, the use of MSC banks would be a therapeutic option, given the lack of HLA class II Ag on MSCs. To our knowledge, our study is the first one to test MSCs from CLI patients in a preclinical model. Autologous human MSCs, previously tested in a heart model of revascularization and clinically approved for other indications, have been administered in four patients with Buerger disease, a non atherosclerotic ischemic disease. Efficacy has been tested in hindlimb ischemia model and cells seemed to be efficient in patient healing and in the hind limb preclinical model of ischemia.8 One patient with systemic sclerosis has received autologous MSCs.9 Causality cannot be established by a single case, but i.v. infusion of expanded autologous MSCs may foster the recovery of the vascular network, restore blood flow and reduce skin necrosis. Recently, MSCs isolated from iPS cell lines have been used and showed to attenuate limb ischemia in mice.10 In conclusion, the MSCs isolated from the BM of CLI patients are able to induce blood flow recovery in vivo to the same degree as controls, in contrast to the reduced ability that has been described for BM-MNCs and endothelial progenitor cells from patients with cardiovascular disorders. The prior existence of clinical trials and safety procedures applicable for this cell type would facilitate the rapid testing of MSCs as an autologous cell therapy product. The authors declare no conflict of interest. C d’Audigier was supported by the Fondation pour la recherche medicale (FRM). We thank Kayle Shapiro for helpful comments and proofreading of our manuscript. This work was supported by research grants from AP-HP PHRC Optipec (03-034), Fondation pour la recherche medicale (FRM) and Fondation de France. Author contributions: DMS designed the study, performed the research and wrote the letter. Cd’A, CLG, LM and BD performed the research. JSS designed research protocols. LDC prepared BM-MNCs. PG and JE designed the OPTIPEC study, harvested BM from CLI patients and wrote the letter.