Cellular Hypomethylation Impairs Major Antioxidant Systems and Induces Endothelial Activation

Abstract

Hyperhomocysteinemia (HHcy) is a risk factor for vascular disease, but the underlying mechanisms remain undefined. The homocysteine precursor, S-adenosylhomocysteine (AdoHcy), which accumulates in the setting of HHcy, is a strong inhibitor of cellular methyltransferases. Given the importance of methylation reactions, we hypothesized that cellular hypomethylation might contribute to HHcy-induced endothelial dysfunction and vascular disease. We investigated the role of AdoHcy-induced hypomethylation on oxidative stress and cell activation in human endothelial cells. We induced intracellular AdoHcy accumulation by inhibiting the AdoHcy hydrolase (SAHH) reaction using both pharmacological and siRNA approaches. Extracellular hydrogen peroxide (H2O2) was measured using Amplex Red, and intracellular H2O2 flux was monitored with the biosensor HyPer2. Expression of antioxidants and adhesion molecules were monitored by quantitative RT-PCR and immunoblotting. SAHH inhibition caused a 2.6± 0.3 fold (p<0.005) increase in cellular H2O2 release with a 3.9± 0.9 fold (p<0.05) increase in intracellular H2O2. Concomitant with this increase in ROS production, glutathione peroxidase-1 (GPx-1) expression and activity were decreased 51± 9% and 39± 15%, respectively (p<0.05), whereas expression of thioredoxin-interacting protein, an inhibitor of thioredoxin activity, was increased by 3.3± 0.1 fold (p<0.001). Additionally, SAHH inhibition caused a significant increase in ICAM (2.4± 0.13 fold, p<0.05) and VCAM (2.1± 0.7 fold, p<0.05) protein expression. These pro-atherogenic effects of SAHH inhibition were significantly lessened by adenoviral-mediated GPx-1 overexpression (p<0.05) or by treatment with the antioxidant N-acetyl-cysteine (8 mM) (p<0.05), suggesting that SAH-modulated endothelial activation is ROS-dependent. Previous studies have associated hyperhomocysteinemia with vascular oxidative stress. Our findings suggest that the methylation inhibitor and homocysteine precursor S-adenosylhomocysteine importantly contributes to oxidative imbalance and thereby triggers endothelial cell activation to promote a pro-atherogenic state.

Bibliografische Daten

OriginalspracheEnglisch
ISSN0009-7322
DOIs
StatusVeröffentlicht - 28.03.2018
Extern publiziertJa