Kininostatin, an Angiogenic Inhibitor, Inhibits Proliferation and Induces Apoptosis of Human Endothelial Cells

Abstract

We recently reported that domain 5 (D5) of high-molecular-weight kininogen inhibited critical steps required for angiogenesis. Thus, it was named kininostatin. To understand its mechanism of action, we further investigated the effects of D5 on basic fibroblast growth factor (bFGF)-induced endothelial cell proliferation and cell viability. We report here that D5-inhibited cell proliferation of human endothelial cells stimulated by bFGF was associated with a significant reduction of cyclin D1 expression, which is a critical component required for the transition from G 1 to S phase of the cell cycle. However, inhibition of cell proliferation by D5 was not due to an inhibition of extracellular signal–regulated protein kinase activity. Endothelial cells underwent apoptosis when cultured in a serum-free medium, which was prevented by bFGF. D5 reversed the protective effect of bFGF by 80%. Cells treated with D5 in the presence of bFGF showed typical morphological features of apoptosis, which was further confirmed by 2 additional assays: Hoechst 33258 cell staining and DNA fragmentation analysis. We conclude that the inhibition of endothelial cell proliferation and induction of apoptosis together represent a major contribution to the antiangiogenic activity of D5.