Abstract 819: Critical Role of Vascular Endothelial Growth Factor Receptor-1 in Endothelial Integrity
Vascular endothelial growth factor (VEGF) binds both VEGF receptor-1 (Flt-1) and VEGF receptor-2 (Flk-1). Activation of Flk-1 was thought to play a major role in the regulation of endothelial function by VEGF. Recently, Flt-1 specific ligands have been reported to exert beneficial effects for the treatment of cardiovascular disease. However, the role of Flt-1 in angiogenesis has not been fully understood. To elucidate the role of Flt-1, we constructed retroviral vectors expressing a small interfering RNA targeted for either the Flt-1 gene or the Flk-1 gene and examined the effects of silencing these genes on endothelial function. Introduction of the constructs into human endothelial cells (EC) effectively and stably down-regulated expression of these genes. Depletion of Flt-1 markedly reduced cell growth as compared with mock-infection, whereas silencing the Flk-1 gene decreased cell growth to a lesser extent. The ability to survive after serum starvation in response to VEGF was significantly impaired in Flk-1-depleted but not in Flt-1-depleted EC. The study of long-term culture showed that depletion of Flt-1 but not Flk-1 induced expression of negative cell cycle regulators such as p53 and p21 and thereby shortened the lifespan of EC. As a result of such impairment, depletion of these genes reduced the angiogenic ability of EC in response to VEGF as compared with mock-infected EC. Flt-1-depleted EC displayed constitutive activation of Akt, while its activity was significantly reduced in Flk-1-depleted EC. Inhibition of Akt activation prevented premature senescence as well as induction of p53 and p21 expression and thus improved impairment of angiogenesis in Flt-1-depleted EC. To examine the effects of Flt-1-depletion on neovascularization in vivo, we produced models of hindlimb ischemia and matrigel implantation in Flt-1-deficient heterozygote (Flt-1 KO) mice. Consistent with the in vitro data, increased activity of Akt was found in EC of Flt-1 KO mice. Moreover, neovascularization was significantly impaired in Flt-1 KO mice compared with wild-type mice. This impairment was markedly improved in Akt1/Flt-1 double KO mice. These results suggest that Flt-1 has a critical role in endothelial integrity by modulating the VEGF-Akt signaling pathway.