Definition of the Apoptotic Pathway Induced in Proliferating Endothelial Cells by Cleaved High Molecular Weight Kininogen.

Previous studies from our laboratory and others have demonstrated that cleaved high molecular weight kininogen (HKa) induces selective apoptosis of proliferating endothelial cells and inhibits angiogenesis. Recently we reported that the induction of endothelial cell apoptosis by HKa was matrix dependent and requires the generation of reactive oxygen species. To further define the mechanisms of HKa-induced endothelial cell apoptosis, we have investigated the pathway through which apoptosis occurs. HKa-induced endothelial cell apoptosis was caspase-dependent, as demonstrated by the cleavage of pro-caspase 3 and the inhibition of apoptosis by the broad spectrum caspase inhibitor z-VAD-fmk. Activation of the intrinsic pathway of apoptosis was also confirmed by demonstrating cleavage of pro-caspase 9 as well as the release of cytochrome C from mitochondria. These results are consistent with our previous work, as apoptosis induced through oxidative stress is a well known activator of the intrinsic apoptotic pathway. Given the resistance to HKa-induced endothelial cell apoptosis conferred by culture of endothelial cells on collagen I or IV, we questioned whether MMPs may be involved in this process. Interestingly, we observed that the broad spectrum MMP inhibitor GM6001 completely protected endothelial cells from HKa-induced apoptosis. Moreover, we observed that HKa-induced apoptosis was associated with PECAM-1 cleavage within 1 hour of exposure, and that inhibition of apoptosis by either GM6001 or z-VAD-fmk also inhibited the cleavage of PECAM-1. These findings are likely to be relevant to the apoptotic pathway, as intact PECAM-1 has been previously reported to inhibit cytochrome c release after exposure of cells to cytotoxic stimuli, while a cleaved cytoplasmic domain of PECAM-1 enhances the susceptibility of cells to apoptosis. In summary, our studies demonstrate that the antiangiogenic activity of HKa may be critically dependent on MMP activation, which in turn leads to ROS generation and caspase activation. The subsequent cleavage of PECAM-1, presumably by caspases, may be another important event in HKa-induced apoptosis.