Identification of a role of the vitronectin receptor and protein kinase C in the induction of endothelial cell vascular formation

When cultured on a basement membrane substratum, endothelial cells undergo a rapid series of morphological and functional changes which result in the formation of histotypic tube‐like structures, a process which mimics in vivo angiogenesis. Since this process is probably dependent on several cell adhesion and cell signaling phenomena, we examined the roles of integrins and protein kinase C in endothelial cell cord formation. Polyclonal antisera directed against the entire vitronectin (αvβ3) and fibronectin (α5β1) receptors inhibited cord formation. Subunit‐specific monoclonal antibodies to αv, β3, and β1 integrin subunits inhibited cord formation, while monoclonal antibodies to α3 did not, which implicated the vitronectin receptor, and not the fibronectin receptor, in vascular formation. Protein kinase C inhibitors inhibited cord formation, while phorbol 12‐myristate 13‐acetate (PMA) caused endothelial cells to form longer cords. Since the vitronectin receptor has been shown to be phosphorylated in an in vitro system by protein kinase C, the possible functional link between the vitronectin receptor and protein kinase C during cellular morphogenesis was examined. The vitronectin receptor was more highly phosphorylated in cord‐forming endothelial cells on basement membrane than in monolayer cells on vitronectin. Furthermore, this phosphorylation was inhibited by protein kinase C inhibitors, and PMA was required to induce vitronectin receptor phosphorylation in endothelial cells cultured on vitronectin. Colocalization studies were also performed using antisera to the vitronectin receptor and antibodies to protein kinase C. Although no strict colocalization was found, protein kinase C was localized in the cytoskeleton of endothelial cells initially plated on basement membrane or on vitronectin, and it translocated to the plasma membrane of C‐shaped cord‐forming cells on basement membrane. Thus, both the vitronectin receptor and protein kinase C play a role in in vitro cord formation. © 1993 Wiley‐Liss, Inc.