Mitochondrial mechanism of microvascular endothelial cells apoptosis in hyperhomocysteinemia

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]. There are three ranges of hyperhomocysteinemia: moderate (16–30 μM), intermediate (31–100 μM), and severe (>100 μM) [Cheng and Kaplowitz, 2004]. Extracellular thiols are oxidized, and only a fraction of total plasma homocysteine (Hcy) is in the reduced form in vivo and in vitro. Previous studies from our laboratory and others have suggested the ability of Hcy to damage the endothelium and cause cardiovascular disease and stroke. However, this effect of Hcy is multifactorial with differential and specific effects on both the endothelial and vascular smooth muscle cells. For example, Hcy at doses of 0.1–1.0 mM markedly inhibits endothelial cell growth over time in vitro; in contrast, vascular smooth muscle cells respond to similar concentrations of Hcy with an increase in cyclin D1 and cyclin A mRNA expression and a resulting marked increase in cell proliferation [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 anti-thrombotic 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) [Kotamraju et al., 2000; Tyagi et al., 2005], which play a critical role in endothelial damage and dysfunction [Olszewski and McCully, 1993; Abdelfatah et al., 2002]. ROS acts as an upstream factor for mitochondrial membrane depolarization [C. Sidoti-de Fraisse et al., 1998]. Increased production of ROS is implicated in loss of mitochondrial membrane potential (MP) (ΔΨm) [Leist et al., 1997; Crompton, 1999] and induction of members of Bcl2 protein family. Mitochondrial releases cytochrome-c and caspases that leads to eventual endothelial cell apoptosis and cardiac dysfunction [Kumar and Jugdutt, 2003]. Apoptosis or programmed cell death, an evolutionarily conserved and genetically controlled active cell suicide process, maintains cellular and tissue homeostasis of multicellular organisms. Apoptosis can be initiated through two pathways. First, the extrinsic pathway, is activated by extracellular signals that interact with cell surface receptors (surface receptor-mediated). Second, the intrinsic pathway, is mitochondrial-dependent, involving Bcl2 protein family, and the caspases (i.e., caspase-9, caspase-6, caspase-3) [Danial and Korsmeyer, 2004; Lowe et al., 2004a,b]. The Bcl2 family of proteins consists of anti-apoptotic members, Bcl2 and Bcl-Xl and pro-apoptotic members, Bax, Bak, and BH3. Caspases, a group of cysteine proteases, have been identified in both Caenorhabditis elegans (ced-3) and mammalian cells; and are known to be essential for apoptosis [Cohen, 1997; Thornberry, 1998; Budihardjo et al., 1999]. All caspases are synthesized as enzymatically inert zymogens and are activated by autocatalytic cleavage or by activation of other caspases. Among the various caspases, caspase-9 and caspase-3 appear to be important in apoptosis [Porter and Janicke, 1999; Kuida, 2000]. Activation of Bax and Bak results in the release of cytochrome-c from mitochondria [Cheng et al., 2001; Wei et al., 2001], which binds to Apaf-1 and promotes activation of procaspase-9, forming the apoptosome and resulting in caspase-9 activation [Li et al., 1997; Green and Reed, 1998]. The activated caspase-9 cleaves downstream caspases such as caspase-3 and caspase-6. Effector caspases like caspase-3, 6, and 7 are responsible for initiating the events that lead to the hallmarks of apoptosis, including chromatin condensation, DNA fragmentation, cleavage of poly (ADP-ribose) polymerase (PARP), and formation of apoptotic bodies [Porter and Janicke, 1999]. Previously, we demonstrated that Hcy-mediated endothelial dysfunction was ameliorated by caspase inhibitor [Mujumdar et al., 2001]. In this study, we show that Hcy induces ROS production, which in part instigates the intrinsic apoptotic pathway during hyperhomocysteinemia leading to programmed cell death.