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Background: Recruitment and entrapment of bone marrow-derived endothelial progenitor cells (EPCs) is important in vascular endothelial growth factor (VEGF)-induced angiogenesis. EPC mobilization and differentiation are modulated by stromal-derived factor-1[alpha] (SDF-1[alpha]/CXCL12), another important chemokine. In this study, we investigated the hypothesis that SDF-1[alpha] and VEGF might act synergistically on EPC-mediated vasculogenesis.

Methods: EPCs were isolated and cultured from human peripheral blood, then transduced with retroviral vectors pBabe containing human VEGF165 complimentary DNA (Td/V-EPCs) and pBabe wild-type (Td/p-EPCs). EPC migration activity was investigated with a modified Boyden chamber assay. EPC apoptosis induced by serum starvation was studied by annexin V assays. The combined effect of local administration of SDF-1[alpha] and Td/V-EPC transplantation on neovascularization was investigated in a murine model of hind limb ischemia.

Results: Over-expression of hVEGF165 increased SDF-1[alpha]-mediated EPC migration. SDF-1[alpha]-mediated migration was significantly increased when EPCs were modified with VEGF (Td/V-EPCs) vs when VEGF was not present (Td/p-EPCs) or when VEGF alone was present (Td/V-EPCs; 196.8 /- 15.2, 81.2 /- 9.8, and 67.4 /- 7.4/mm2, respectively P < .001). SDF-1[alpha] combined with VEGF reduced serum starvation-induced apoptosis of EPCs more than SDF-1[alpha] or VEGF alone (P < .001). To determine the effect of this combination in vivo, SDF-1[alpha] was locally injected alone into the ischemic hind limb muscle of nude mice or combined with systemically injected Td/V-EPCs. The SDF-1[alpha] plus VEGF group showed significantly increased local accumulation of EPCs, blood-flow recovery, and capillary density compared with the other groups. The ratio of ischemic/normal blood flow in Td/V-EPCs plus SDF-1[alpha] group was significantly higher (P < .01), as was capillary density (capillaries/mm2), an index of neovascularization (Td/V-EPCs plus SDF-1[alpha] group, 863 /- 31; no treatment, 395 /-13; SDF-1[alpha], 520 /- 29; Td/p-EPCs, 448 /- 28; Td/p-EPCs plus SDF-1[alpha], 620 /- 29; Td/V-EPCs, 570 /- 30; P < .01). To investigate a possible mechanistic basis, we showed that VEGF up-regulated the receptor for SDF-1[alpha], CXCR4, on EPCs in vitro.

Conclusion: The combination of SDF-1[alpha] and VEGF greatly increases EPC-mediated angiogenesis. The use VEGF and SDF-1[alpha] together, rather than alone, will be a novel and efficient angiogenesis strategy to provide therapeutic neovascularization.

Clinical Relevance: The stimulation of neovascularization using growth factors is a promising experimental treatment for arterial occlusive disease. Combined therapy could be better than the use of single growth factors for enhancing therapeutic angiogenesis, especially in severely ischemic tissue. This study suggests that stromal-derived factor-1[alpha] (SDF-1[alpha]) and vascular endothelial growth factor (VEGF) form a synergistic angiogenic pathway that is critical for endothelial progenitor cell-induced neovascularization. Targeting both SDF-1[alpha] and VEGF signals may be a novel, efficient strategy for treating ischemic diseases.

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