CD34+/M-cadherin+ Bone Marrow Progenitor Cells Promote Arteriogenesis in Ischemic Hindlimbs of ApoE-/- Mice

Background: Cell-based therapy shows promise in treating peripheral arterial disease (PAD); however, the optimal cell type and long-term efficacy are unknown. In this study, we identified a novel subpopulation of adult progenitor cells positive for CD34 and M-cadherin (CD34(+)/M-cad(+) BMCs) in mouse and human bone marrow. We also examined the long-lasting therapeutic efficacy of mouse CD34(+)/M-cad(+) BMCs in restoring blood flow and promoting vascularization in an atherosclerotic mouse model of PAD. Methods and Findings: Colony-forming cell assays and flow cytometry analysis showed that CD34(+)/M-cad(+) BMCs have hematopoietic progenitor properties. When delivered intra-arterially into the ischemic hindlimbs of ApoE(-/-) mice, CD34(+)/M-cad(+) BMCs alleviated ischemia and significantly improved blood flow compared with CD34(+)/M-cad(-) BMCs, CD34(-)/M-cad(+) BMCs, or unselected BMCs. Significantly more arterioles were seen in CD34(+)/M-cad(+) cell-treated limbs than in any other treatment group 60 days after cell therapy. Furthermore, histologic assessment and morphometric analyses of hindlimbs treated with GFP(+) CD34(+)/M-cad(+) cells showed that injected cells incorporated into solid tissue structures at 21 days. Confocal microscopic examination of GFP(+) CD34(+)/M-cad(+) cell-treated ischemic legs followed by immunostaining indicated the vascular differentiation of CD34(+)/M-cad(+) progenitor cells. A cytokine antibody array revealed that CD34(+)/M-cad(+) cell-conditioned medium contained higher levels of cytokines in a unique pattern, including bFGF, CRG-2, EGF, Flt-3 ligand, IGF-1, SDF-1, and VEGFR-3, than did CD34(+)/M-cad(-) cell-conditioned medium. The proangiogenic cytokines secreted by CD34(+)/M-cad(+) cells induced oxygen-and nutrient-depleted endothelial cell sprouting significantly better than CD34(+)/M-cad(-) cells during hypoxia. Conclusion: CD34(+)/M-cad(+) BMCs represent a new progenitor cell type that effectively alleviates hindlimb ischemia in ApoE(-/-) mice by consistently improving blood flow and promoting arteriogenesis. Additionally, CD34(+)/M-cad(+) BMCs contribute to microvascular remodeling by differentiating into vascular cells and releasing proangiogenic cytokines and growth factors.