Abstract 224: Regulation of VEGF-Mediated Angiogenesis by the Akt/PKB Substrate Girdin
The serine/threonine kinase Akt is an essential regulator of angiogenesis stimulated by vascular endothelial growth factor (VEGF). Akt activation promotes cell survival, migration, tube formation and release of NO in endothelial cells. However, it is unknown how Akt regulates the migration of endothelial cells, a crucial process for vessel sprouting, branching, and the formation of networks. Recently, we found a novel Akt substrate, Girdin, that featured actin cross-link properties. In the present study, we investigated the role of Girdin in the signaling pathway induced by VEGF in human umbilical vein endothelial cells (HUVECs). Western blot analyses using an antiphospho Girdin antibody revealed that Girdin is phosphorylated at Ser-1416 by Akt activation after VEGF stimulation. In addition, phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 and depletion of Akt using short interference RNA (siRNA) abolished Girdin phosphorylation. Immunocytochemistry and electron microscope analysis showed that Girdin co-localized with actin filaments in lamellipodia and stress fibers. Immunocytochemistry also showed phosphorylated Girdin accumulated in the leading edge of migrating cells. After treatment with Girdin siRNA, lamellipodia formation was disrupted. The depletion of Girdin by siRNA also attenuated the cell migration and tube formation on matrigel activated by VEGF. We constructed siRNA resistant (siRNAr) versions of Girdin harboring silent mutations. Restoration of Girdin activity with adenovirus siRNAr-Girdin wild type in siRNA Girdin transfected HUVECs reversed the expression of Girdin and tube formation on matrigel. In contrast, HUVECs cotransfected with siRNA Girdin and adenovirus mutants in which the Ser-1416 is mutated to Ala that is resistant to phosphorylation, failed to complete tube formation. In vivo matrigel plug assay, the number of outgrowth microvessels were reduced by short hairpin RNA-expressing adenoviruses targeted against Girdin. In conclusions, Girdin is essential for the integrity of the cytoskeleton and cell motility in endothelial cells. Those results strongly suggest that the PI3K-Akt-Girdin pathway in endothelial cells is essential for VEGF induced angiogenesis.