Mitochondrial mechanism of microvascular endothelial cells apoptosis in hyperhomocysteinemia

Abstract

An elevated level of homocysteine (Hcy) limits the growth and induces apoptosis. However, 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 transmembrane mitochondrial potential (Δψ m ) accompanied by the release of cytochrome-c from mitochondria. Cytochrome-c release contributes to caspase activation, such as caspase-9, caspase-6, and caspase-3, which results in the degradation of numerous nuclear proteins including poly (ADP-ribose) polymerase (PARP), which subsequently leads to the internucleosomal cleavage of DNA, resulting 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 transferase-mediated dUTP nick-end labeling (TUNEL) assay. ROS production and MP were determined using florescent probes (2,7-dichlorofluorescein (DCFH-DA) and 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzamidazolocarbocyanin iodide (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 cytochrome-c, and the activation of caspase-9 and -3. Moreover the Hcy treatment resulted in a decrease in Bcl 2 /Bax ratio, evaluated by mRNA levels. Caspase-9 and -3 were activated, causing cleavage of PARP, a hallmark of apoptosis and internucleosomal 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 the activation of caspase -3 and enhanced the cell viability and MP. Our data suggested that Hcy-mediated ROS production promotes endothelial 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. KeywordsPARP; cytochrome-c; reactive oxygen species; siRNA; cardiac microvascular endothelial cells; oxidative stress; mitochondrial membrane potential; siRNA; cDNA array; TUNEL; Bax; Bcl 2 ; caspase Hyperhomocysteinemia is an independent risk factor for cardiovascular disease, stroke, and peripheral vascular disease [Jacobsen, 1998;Hankey and Eikelboom, 1999;Fallon et al., 2001;Suhara et al., 2004] [Tsai et al., 1994].Hcy is a thiol-containing amino acid formed during the intracellular demethylation of methionine and causes multifold effects through the reactivity of its sufhydryl group. Increase in levels of Hcy impairs cellular function and leads to loss of endothelial antithrombotic function, lipid peroxidation, impairment of platelet aggregation, enhanced oxidative stress, and apoptosis (programmed cell death) [Blom et al., 1995;Tawakol et al., 1997;Carsten and Kenneth, 2004]. These unfavorable vascular effects of Hcy are a result of the generation of reactive oxygen species (ROS) [...

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