Abstract 5592: Role of Afadin in Vascular Endothelial Growth Factor- and Sphingosine 1-Phosphate-Induced Angiogenesis

[Background] Better understanding of mechanism of angiogenesis and angiogenic signaling of pro-angiogenic factors such as vascular endothelial growth factor (VEGF) and sphingosine 1-phosphate (S1P) will aid in developing therapeutic angiogenesis for ischemic heart disease and peripheral arterial disease. Recently, Rap1 small G protein was identified as a key angiogenic signaling molecule. However, mechanisms involved in the Rap1 regulation of angiogenesis and downstream effectors of Rap1 are not known. Afadin is an adherens junction protein that connects immunoglobulin-like adhesion molecule nectins to the actin cytoskeleton and binds activated Rap1. Recently, we identified that, besides the role in cell-cell adhesion formation in epithelial cells, afadin plays a role in the regulation of directional cell movement of fibroblasts. We therefore attempted to investigate the role of endothelial afadin in angiogenesis and clarify the underlying molecular mechanism. [Method and Results] Knockdown of Rap1 or afadin by siRNA inhibited the VEGF- and S1P-induced tubulogenesis, migration and proliferation, and conversely, increased the serum deprivation-induced apoptosis of human umbilical vein endothelial cells (HUVECs). Immunoprecipitation and immunofluorescence studies revealed that Rap1 regulated the interaction between afadin and phosphatidyl-inositol 3-kinase (PI3K) and recruitment of the afadin-PI3K complex to the leading edge, suggesting that Rap1 and afadin regulates the PI3K signaling. Indeed, knockdown of Rap1 or afadin by siRNA inhibited the VEGF- and S1P-induced phosphorylation of Akt. Thus, afadin regulated the PI3K-Akt signaling. To examine the role of afadin in angiogenesis in vivo, we developed endothelial cell-specific afadin-knockout mice using the Cre/loxP system. Majority (96%) of afadin-knockout mice were embryonically lethal, suggesting an essential role of endothelial afadin in embryonic vascular development and angiogenesis. We performed Matrigel plug assay in born adult afadin-knockout mice and observed that VEGF- and S1P-induced angiogenesis was impaired in afadin-knockout mice. [Conclusion] These results demonstrate that afadin regulates VEGF- and S1P-induced angiogenesis downstream of Rap1.