High molecular weight kininogen (HK) is a single-chain glycoprotein that plays a central role in contact activation. We previously reported that the cleaved form of high molecular weight kininogen (HKa), which lacks bradykinin, induces apoptosis of proliferating endothelial cells and inhibits angiogenesis in vivo. This activity involves binding of HKa to tropomyosin exposed on the endothelial cell surface. We have also demonstrated that endothelial cells cultured on types I or IV collagen are endowed with relative resistance to the apoptotic effects of HKa. We hypothesized that one mechanism that might account for this specific effect could be the ability of collagen to inhibit hydroxyl radical induced apoptosis through scavenging of hydroxyl radicals and inhibition of cellular lipid peroxidation. To address this issue, we assessed the ability of HKa to inhibit endothelial cell proliferation in the absence or presence of glutathione (GSH), an intracellular thiol antioxidant which scavenges reactive oxygen species (ROS) and prevents ROS-induced cell damage. The results of these experiments demonstrated that GSH blocked the ability of HKa to inhibit endothelial proliferation and induce endothelial cell apoptosis on all ECM proteins tested, including gelatin, laminin, vitronectin, fibronectin, and collagens I and IV. Similar results were obtained when N-acetylcysteine (NAC), a cell membrane permeable GSH precursor, was employed. However, neither GSH nor NAC protected endothelial cells from apoptosis induced by 2-methoxyestradiol, which also induces selective apoptosis of proliferating endothelial cells, suggesting that the ability of GSH to block HKa-induced endothelial cell apoptosis is specific. To further examine the role of ROS in HKa induced endothelial cell apoptosis, we measured the cellular GSH level and content of lipid peroxidation products MDA and 4-HNE following exposure to HKa. These studies demonstrated that exposure of endothelial cells cultured on gelatin to HKa led to a rapid fall in intracellular GSH, accompanied by a three fold increase in MDA and 4-HNE. In contrast, little change in the levels of oxidation products were observed when cells were cultured on collagen type I or IV. These results demonstrate that the ability of HKa to induce apoptosis of proliferating endothelial cells is associated with the production of reactive oxygen species (ROS) and a change in intracellular redox status. To further test this conclusion, we examined another redox sensitive signaling regulator, thioredoxin (TRX), which normally occurs primarily in the cytoplasm but translocates to the nucleus in response to oxidative stress. In response to HKa, thioredoxin quickly translocated from the cytoplasm to the nucleus of endothelial cells cultured on gelatin, though nuclear translocation of thioredoxin did not occur when cells were cultured on type I collagen. These results suggest that nuclear accumulation of TRX may be intimately involved in HKa induced endothelial cell apoptosis by sensing oxidative stress. In conclusion, our data suggests that HKa induces endothelial cell apoptosis through an oxidation dependent pathway, which is co-regulated through additional signals emanating from the extracellular matrix. Ongoing studies are focused on defining the mechanisms by which ROS are generated, and the role of HKa-tropomyosin interactions in stimulating this oxidant pathway.
LncRNA LUADT1 sponges miR-195 to prevent cardiac endothelial cell apoptosis in sepsis
