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Mitochondrial mechanism of microvascular endothelial cells apoptosis in hyperhomocysteinemia
Author(s) -
Tyagi Neetu,
Ovechkin Alexander V.,
Lominadze David,
Moshal Karni S.,
Tyagi Suresh C.
Publication year - 2006
Publication title -
journal of cellular biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.028
H-Index - 165
eISSN - 1097-4644
pISSN - 0730-2312
DOI - 10.1002/jcb.20837
Subject(s) - apoptosis , cytochrome c , dna laddering , microbiology and biotechnology , poly adp ribose polymerase , programmed cell death , biology , dna fragmentation , tunel assay , chemistry , biochemistry , dna , polymerase
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′‐tetraethyl‐benzamidazolocarbocyanin 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. J. Cell. Biochem. 98: 1150–1162, 2006. © 2006 Wiley‐Liss, Inc.