Cd47 Activation By Thrombospondin Peptides Enhances Bone Marrow Mononuclear Cell Adhesion, Recruitment During Thrombosis, Endothelial Differentiation And Stimulates Pro-Angiogenic Cell Therapy

Bone marrow-mononuclear cells (BMC) contain endothelial progenitor cells (EPC) useful in cell therapy to enhance ischemic neovascularization. There is growing interest in the role of thrombosis in EPC engraftment and function. We hypothesized that activation of CD47 on the surface of BMC by platelet thrombospondin-1 (TSP1) and derived peptides modulates their proliferation, survival, EPC differentiation and pro-angiogenic potential. Culture of BMC with peptide 4N1–1 that recapitulates the CD47-binding site of TSP1 (1 to 100 μM) stimulated changes in cell shape by FACS within 90 seconds and cytoskeletal rearrangement within minutes (Phalloidin-stained actin stress fibers). 4N1–1 stimulated a striking increase in adhesion to fibrin (8 fold increase above control; p < 0.05), increased EPC differentiation (50% above BSA; p < 0.05), but did not modulate proliferation or apoptosis. In a model of FeCl3-induced intravascular thrombosis combined with systemic injection of fluorescent BMC and intra-vital microscopy, we showed specific BMC recruitment to sites of intra-vascular thrombosis, with increased BMC-to-vessel wall interactions (4 fold increase; p < 0.05) and decreased rolling speeds. BMC recruitment was strongly stimulated by 4N1–1 treatment for 1h prior to injection, with further decreased local rolling speeds (−90% vs BSA-treatment; p < 0.05) and the emergence of permanent cell engraftment (12 fold increase; p < 0.05). In a mouse model of hindlimb ischemia after femoral artery ligation, systemic injection of BMC promoted neovascularization significantly compared to saline (+51.5%, p < 0.05). BMC pretreatment with 4N1–1 for 1h promoted neovascularization beyond BSA-treated cells (+94.4%, p < 0.05), by cutaneous flow, capillary density and microangiographic score assessment 14d after ligation. In summary, we showed that CD47 activation stimulates strong BMC adhesion, specific recruitment to sites of thrombosis in vivo and ischemic neovascularization. CD47 activation and TSP1-derived peptides may thus play a critical role in EPC engraftment to sites of vascular injury and in the onset of vasculogenesis. We suggest that 4N1–1 could be useful in the clinic to stimulate progenitor cell preparations, and to enhance pro-angiogenic cell therapy.