Kininostatin, an angiogenic inhibitor, inhibits proliferation and induces apoptosis of human endothelial cells

We recently reported that domain 5 (D5) of high-molecular-weight kininogen inhibited critical steps required for angiogenesis. Thus, it was named kini nostatin. To understand its mechanism of action, we further investigated th e effects of D5 on basic fibroblast growth factor (bFGF)-induced endothelia l cell proliferation and cell viability. We report here that D5-inhibited c ell proliferation of human endothelial cells stimulated by bFGF was associa ted with a significant reduction of cyclin D1 expression, which is a critic al component required for the transition from G(1) to S phase of the cell c ycle. However, inhibition of cell proliferation by D5 was not due to an inh ibition of extracellular signal-regulated protein kinase activity. Endothel ial cells underwent apoptosis when cultured in a serum-free medium, which w as prevented by bFGF. D5 reversed the protective effect of bFGF by 80%. Cel ls treated with D5 in the presence of bFGF showed typical morphological fea tures of apoptosis, which was further confirmed by 2 additional assays: Hoe chst 33258 cell staining and DNA fragmentation analysis. We conclude that t he inhibition of endothelial cell proliferation and induction of apoptosis together represent a major contribution to the antiangiogenic activity of D 5.