Ethanol-lnduced Apoptosis Mediated by Mitochondrial Permeability Transition in Primary Cultured Sinusoidal Endothelial Cells

Abstract

Accumulating evidence suggests that ethanol delays liver regeneration in response to liver injury. The responsible mechanisms have been less well understood in sinusoidal endothelial cells (SECs) than in hepatocytes. To test this theory in vitro we prepared primary cultured SECs in rats and investigated the effect of ethanol on apoptosis and mitochondrial permeability transition (MPT), which is involved in the process of apoptosis. SECs were isolated based on a modification of the method of Wisse et al. SECs were cultured in EBM-2 containing vascular endothelial growth factor (20 ng/ml) at 37°C for 5 days. SECs displayed a confluent cell sheet at day 5 in culture. The bromodeoxyuridine labeling index was decreased whereas the apoptotic cells increased during the time in culture. Addition of 100 mM ethanol to the medium for 6h induced two- and four-fold increases in apoptosis at days 2 and 5, respectively. This apoptotic effect was augmented in a dose-dependent manner by ethanol (50–150 mM). When SECs were exposed to ethanol for 3h, MPT was observed, but there was no significant increase in apoptotic cells. Furthermore, cyclosporin A, an MPT inhibitor, suppressed apoptosis in SECs exposed to ethanol for 6h. These results indicate that ethanol induces apoptosis in SECs, a process is mediated by MPT. It may therefore be hypothesized that ethanol-induced apoptosis in SECs causes impaired angiogenesis in liver, contributing to the delay in liver regeneration.