S-Adenosylhomocysteine induces apoptosis and phosphatidylserine exposure in endothelial cells independent of homocysteine

Abstract

ObjectiveWe 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 resultsHuman umbilical vein endothelial cells (HUVECs) were exposed to the following conditions: (1) non-treated control (resulting in 2.8nM intracellular SAH and 3.1μM extracellular l-Hcy); and incubation with (2) 50μM adenosine-2,3-dialdehyde (ADA; resulting in 17.7nM intracellular SAH and 3.1μM extracellular l-Hcy), (3) 2.5mM Hcy (resulting in 20.9nM intracellular SAH and 1.8mM extracellular l-Hcy), and (4) 1, 10 and 100μM SAH. We then determined the effect of treatment on annexin V-positivity, caspase-3 activity, cytochrome c release (sub)cellular expression of NOX2, NOX4, p47phox 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 p47phox 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). ConclusionSAH 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.