Differential expression and role of hyperglycemia induced oxidative stress in epigenetic regulation of [beta]1, [beta]2 and [beta]3-adrenergic receptors in retinal endothelial cells

Aberrant epigenetic profiles are concomitant with a spectrum of developmental defects and diseases. Role of methylation is an increasingly accepted factor in the pathophysiology of diabetes and its associated complications. This study aims to examine the correlation between oxidative stress and methylation of [beta]1, [beta]2 and [beta]3-adrenergic receptors and to analyze the differential variability in the expression of these genes under hyperglycemic conditions. Human retinal endothelial cells were cultured in CSC complete medium in normal (5 mM) or high (25 mM) glucose to mimic a diabetic condition. Reverse transcription PCR and Western Blotting were performed to examine the expression of [beta]1, [beta]2 and [beta]3-adrenergic receptors. For detections, immunocytochemistry was used. Bisulfite sequencing method was used for promoter methylation analysis. Apoptosis was determined by the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. Dichlorodihydrofluorescein diacetate (DCFH-DA) assay was used to measure reactive oxygen species (ROS) production in the cells. [beta]1 and [beta]3-adrenergic receptors were expressed in retinal endothelial cells while [beta]2-adrenergic receptor was not detectable both at protein and mRNA levels. Hyperglycemia had no significant effect on [beta]1 and [beta]2-adrenergic receptors methylation and expression however [beta]3-adrenergic receptors showed a significantly higher expression (p < 0.05) and methylation (p < 0.01) in high and low glucose concentration respectively. Apoptosis and oxidative stress were inversely correlated with [beta]3-adrenergic receptors methylation with no significant effect on [beta]1 and [beta]2-adrenergic receptors. [beta]2-adrenergic receptor was hypermethylated with halted expression. Our study demonstrates that [beta]1 and [beta]3-adrenergic receptors expressed in human retinal endothelial cells. Oxidative stress and apoptosis are inversely proportional to the extent of promoter methylation, suggesting that methylation loss might be due to oxidative stress-induced DNA damage.