Mitochondrial Mechanism of Microvascular Endothelial Cell Apoptosis Induced by Homocysteine

Abstract

An elevated level of homocysteine (Hcy) limit the growth and induces apoptosis. The mechanism of Hcy-induced programmed cell death in endothelial cells is largely unknown. We hypothesize that Hcy induces intracellular reactive oxygen species (ROS) production that leads to the loss of mitochondrial potential (MP) accompanied by the release of mitochondrial cyto-c. Cyto-c release contributes to caspase-9, -6 and -3 activation, which results in the degradation of nuclear proteins including poly (ADP-ribose) polymerase(PARP), which subsequently leads to the cleavage of DNA and cell death. In this study rat heart microvascular endothelial cells (MVEC) were treated with different doses of Hcy at different time intervals. Apoptosis was measured by DNA laddering and TUNEL assay. ROS production and MP were determined using fluorescent probes (2,7-dichlorofluorescein and JC-1 respectively by confocal microscopy. Differential gene expression for apoptosis was analyzed by cDNA array. The results showed that Hcy-mediated ROS production preceded the loss of MP, the release of cyto-c and the activation of caspase-9 and -3. Moreover, the Hcy treatment resulted in a decrease in Bcl2/Bax ratio. Caspase-9 and -3 were activated, causing cleavage of PARP and DNA fragmentation. The cytotoxic effect of Hcy was blocked by using small interfering RNA (siRNA) mediated suppression of caspase-9 in MVEC. Suppressing the activation of caspase-9 inhibited caspase-3 and enhanced the cell viability and MP. Our data suggested that Hcy-mediated ROS production promotes cell death in part by disturbing MP which results in subsequent release of cytochrome-c and activation of caspase-9 and 3, leading to cell death. Supported by NHLBI.