Differential expression and role of hyperglycemia induced oxidative stress in epigenetic regulation of β1, β2 and β3-adrenergic receptors in retinal endothelial cells

Background: 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. Methods: 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. Results: beta 1 and beta-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. Conclusion: 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.