Endothelial Cell Survival

The a v b 3 integrin plays a fundamental role during the angiogenesis process by inhibiting endothelial cell apoptosis. However, the mechanism of inhibition is unknown. In this report, we show that integrinmediated cell survival involves regulation of nuclear factor-kappa B (NFk B) activity. Different extracellular matrix molecules were able to protect rat aortaderived endothelial cells from apoptosis induced by serum withdrawal. Osteopontin and b 3 integrin ligation rapidly increased NFk B activity as measured by gel shift and reporter activity. The p65 and p50 subunits were present in the shifted complex. In contrast, collagen type I (a b 1 -integrin ligand) did not induce NFk B activity. The a v b 3 integrin was most important for osteopontin-mediated NFk B induction and survival, since adding a neutralizing antib 3 integrin antibody blocked NFk B activity and induced endothelial cell death when cells were plated on osteopontin. NFk B was required for osteopontinand vitronectin-induced survival since inhibition of NFk B activity with nonphosphorylatable I k B completely blocked the protective effect of osteopontin and vitronectin. In contrast, NFk B was not required for fibronectin, laminin, and collagen type I–induced survival. Activation of NFk B by osteopontin depended on the small GTP-binding protein Ras and the tyrosine kinase Src, since NFk B reporter activity was inhibited by Ras and Src dominant-negative mutants. In contrast, inhibition of MEK and PI3-kinase did not affect osteopontin-induced NFk B activation. These studies identify NFk B as an important signaling molecule in a v b 3 integrin-mediated endothelial cell survival. I n recent years it has become evident that integrinmediated adhesion to extracellular matrix (ECM) 1 proteins is required for growth and survival of many cell types. Adhesion to ECM is required for progression of cells through the cell cycle by regulating cyclinD1, cyclinECdk2, and Rb protein activities (Fang et al., 1996). Disruption of adhesion arrests cells in the G1 phase and causes apoptosis (Boudreau et al., 1996; Frisch and Francis, 1994; Howlett and Bissell, 1993; Ingber et al., 1995; Meredith et al., 1993; Re et al., 1994). The requirement of cell–ECM adhesive interactions for cell cycle progression and cell survival is likely to be important in tissue development and involution as a mechanism to regulate cell positioning and cell number (Lin and Bissell, 1993). In addition, anchorage dependence of survival may serve to limit tumor progression by preventing invasion or metastasis of tumor cells (Varner and Cheresh, 1996). Integrin-regulated survival properties have also been shown to be relevant in wound repair since integrin antagonists induced apoptosis of migrating endothelial cells, thereby blocking angiogenesis (Brooks et al., 1994 a ; Brooks et al., 1994 b ; Friedlander et al., 1996). The mechanism by which integrin-mediated ECM adhesion is able to prevent apoptosis is unclear and under intense investigation. Focal adhesion kinase (FAK) is phosphorylated in response to cell adhesion, and constitutively activated membrane-targeted FAK was able to rescue cells from suspension-induced cell death (Frisch et al., 1996 b ). The JNK pathway is active in detached epithelial cell (Frisch et al., 1996 a ), and the Ras, PI3-kinase, Akt pathways are activated and functionally implicated in cell attachment–induced survival (Khwaja et al., 1997). Two groups suggest that integrin engagement positively regulates expression of the antiapoptotic gene bcl-2 in COS and endothelial cells (Stromblad et al., 1996; Zhang et al., 1995). In addition, Stromblad et al. showed that a v b 3 engagement and clustering in endothelial cells, but not b 1 or a v b 5 ligation, conferred an antiapoptotic phentoype to endothelial cells. Importantly, the same group showed that inhibition of angiogenesis by antia v b 3 antibody correlates Address all correspondence to Marta Scatena, Department of Pathology, University of Washington, Box 357335, Seattle, WA. Tel.: 206 685 4288; Fax: 206 685 3662; E-mail: [email protected] 1. Abbreviations used in the paper : cIAP, baculovirus inhibitors of apoptosis mammalian homologs; ECM, extracellular matrix; EMSA, electrophoretic mobility shift assay; FAK, focal adhesion kinase; NFk B, nuclear factor-kappa B; PARP, poly(ADP-ribose) polymerase; PI3, phosphotidylinositol 3; RAEC, rat aortic endothelial cells. on M ay 2, 2017 D ow nladed fom Published May 18, 1998