Journal
ATHEROSCLEROSIS
Volume 221, Issue 1, Pages 48-54Publisher
ELSEVIER IRELAND LTD
DOI: 10.1016/j.atherosclerosis.2011.11.032
Keywords
Homocysteine; S-Adenosylhomocysteine; NADPH oxidase; Endothelial cells
Funding
- ICaR-VU [2003V070, 2008V076]
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Objective: We have previously shown that homocysteine (Hcy) induces phosphatidylserine (PS) exposure, apoptosis and necrosis in human endothelial cells. Since it has been suggested that S-Adenosylhomocysteine (SAH) is the main causative factor in Hcy-induced pathogenesis of cardiovascular disease, we evaluate here whether the cytotoxic Hcy effect in endothelial cells is also SAH dependent. Methods and results: Human umbilical vein endothelial cells (HUVECs) were exposed to the following conditions: (1) non-treated control (resulting in 2.8 nM intracellular SAH and 3.1 mu M extracellular l-Hcy); and incubation with (2) 50 mu M adenosine-2,3-dialdehyde (ADA; resulting in 17.7 nM intracellular SAH and 3.1 mu M extracellular l-Hcy), (3) 2.5 mM Hcy (resulting in 20.9 nM intracellular SAH and 1.8 mM extracellular l-Hcy), and (4) 1, 10 and 100 mu M SAH. We then determined the effect of treatment on annexin V-positivity, caspase-3 activity, cytochrome c release (sub) cellular expression of NOX2, NOX4, p47(phox) and nitrotyrosine, and H2O2. Both Hcy and ADA significantly increased PS exposure (n = 5), caspase-3 activity (n = 6) and cytochrome c release (n = 3). Incubation with extracellular SAH alone did not affect cell viability. Both Hcy and ADA also induced similar increases in nuclear NOX2 and (peri) nuclear NOX4, coinciding with (peri) nuclear p47(phox) expression and local reactive oxygen species (ROS) (n = 3). Inhibition of NOX-mediated ROS by the flavoenzyme inhibitor diphenylene iodonium (DPI) significantly decreased apoptosis induction (n = 3) and ROS production (n = 3). Conclusion: SAH induces PS exposure and apoptosis in endothelial cells independently of Hcy. Our study therefore shows that Hcy-mediated endothelial dysfunction, as determined in the cell model used, is mainly due to SAH accumulation. (C) 2011 Elsevier Ireland Ltd. All rights reserved.
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