Abstract 3946: Molecular Identification and Characterization of a Novel Gene that Regulates Endothelial Cell Apoptosis and Angiogenesis

Abstract

Endothelial apoptosis is a pivotal process for angiogenesis during embryogenesis as well as postnatal life. Here, we identified and characterized a novel gene (ARIA) that regulates endothelial apoptosis and angiogenesis. ARIA was expressed in a variety of human cultured endothelial cells as well as in blood vessels of e9.5d mouse embryos, suggesting its role in angiogenesis. Amino acid sequence analysis revealed that ARIA is a novel membrane protein with no homology to conserved domain structures reported before. Knockdown of ARIA in HUVEC using siRNA resulted in significant reduction of endothelial apoptosis. In contrast, ARIA-knockdown did not affect either endothelial cell migration or proliferation. Among factors associated with apoptosis, inhibitor of apoptosis (cIAP)-1 and cIAP-2 protein expression was significantly increased by ARIA-knockdown whereas their mRNA level was not changed. cIAP-1 and cIAP-2 play as ‘gardians’ of the cell death machinery by directly bind to and neutralize caspases as well as by modulate death signaling of TNF receptors. Simultaneous knockdown of cIAP-1 and cIAP-2 abolished the anti-apoptotic effect of ARIA-knockdown. On the other hand, yeast two-hybrid screening using the putative intracellular domain of ARIA as the bait identified the 20S proteasome α subunit-7 as a binding partner of ARIA. Proteasomal inhibition significantly increased cIAP-1 and cIAP-2 expression in HUVEC, indicating their proteasomal degradation in endothelial cells. ARIA-knockdown cancelled this effect of proteasomal inhibition. ARIA-knockdown also mimicked proteasomal inhibition with respect to the expression and subcellular localization of cIAP-1 and cIAP-2 in HUVEC. These results strongly suggest that ARIA plays crucial roles in the proteasomal degradation of cIAP-1 and cIAP-2 in endothelial cells via its interaction with the 20S proteasome α subunit-7. Furthermore, in vivo angiogenesis assessed by Matrigel-plug assay was significantly enhanced by ARIA-knockdown. Taken together, ARIA is a novel factor regulating endothelial apoptosis and angiogenesis through modulating proteasomal degradation of cIAP-1 and cIAP-2. Inhibition of ARIA could be a novel target for developing the better therapeutic angiogenesis.