Putative endothelial cell (EC) progenitors or angioblasts were isolated from human peripheral blood by magnetic bead selection on the basis of cell surface antigen expression. In vitro, these cells differentiated into ECs. In animal models of ischemia, heterologous, homologous, and autologous EC progenitors incorporated into sites of active angiogenesis. These findings suggest that EC progenitors may be useful for augmenting collateral vessel growth to ischemic tissues (therapeutic angiogenesis) and for delivering anti- or pro-angiogenic agents, respectively, to sites of pathologic or utilitarian angiogenesis.

Background —Vascular endothelial growth factor (VEGF), an endothelial cell mitogen that promotes angiogenesis, was initially identified as a vascular permeability factor (VPF). Abundant evidence suggests that angiogenesis is preceded and/or accompanied by enhanced microvascular permeability. The mechanism by which VEGF/VPF increases vascular permeability (VP), however, has remained enigmatic. Accordingly, we used an in vivo assay of VP (Miles assay) to study the putative mediators of VEGF/VPF-induced permeability. Methods and Results —VEGF/VPF and positive controls (platelet-activating factor [PAF], histamine, and bradykinin) all increased vascular permeability. Prior administration of the tyrosine kinase inhibitors genistein or herbimycin A prevented VEGF/VPF-induced permeability. Placenta growth factor, which binds to Flt -1/VEGF-R1 but not Flk -1/KDR/VEGF-R2 receptor tyrosine kinase, failed to increase permeability. Other growth factors such as basic fibroblast growth factor (FGF), acidic FGF, platelet-derived growth factor-BB, transforming growth factor-β, scatter factor, and granulocyte macrophage-colony stimulating factor (8 to 128 ng) failed to increase permeability. VEGF/VPF-induced permeability was significantly attenuated by the nitric oxide (NO) synthase inhibitors N ω -nitro- l -arginine (10 mg/kg) or N ω -nitro- l -arginine methyl ester (20 mg/kg) and the cyclooxygenase inhibitor indomethacin (5 mg/kg). The inactive enantiomer N ω -nitro- d -arginine methyl ester (20 mg/kg) did not inhibit VEGF/VPF-induced permeability. In vitro studies confirmed that VEGF/VPF stimulates synthesis of NO and prostaglandin metabolites in microvascular endothelial cells. Finally, NO donors and the prostacyclin analogue taprostene administered together but not alone reproduced the increase in permeability observed with VEGF/VPF. Conclusions —These results implicate NO and prostacyclin produced by the interaction of VEGF/VPF with its Flk -1/KDR/VEGF-R2 receptor as mediators of VEGF/VPF-induced vascular permeability. Moreover, this property appears unique to VEGF/VPF among angiogenic cytokines.

These findings thus underscore the extent to which hyperlipidemia adversely affects native collateral development but does not preclude augmented collateral vessel growth in response to exogenous cytokines. Moreover, results obtained in the apoE-/- and athymic nude mice imply a critical role for infiltrating T cells as a source of VEGF in neovascularization of ischemic tissues.