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

Sher Zaman Safi 0 Rajes Qvist 0 Gracie Ong Siok Yan 1 Ikram Shah Bin Ismail 0 0 Department of Medicine, Faculty of Medicine, University of Malaya , 50603 Kuala Lumpur , Malaysia 1 Department of Anesthesiology, Faculty of Medicine, University of Malaya , 50603 Kuala Lumpur , Malaysia 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 1, 2 and 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 1, 2 and 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: 1 and 3-adrenergic receptors were expressed in retinal endothelial cells while 2-adrenergic receptor was not detectable both at protein and mRNA levels. Hyperglycemia had no significant effect on 1 and 2-adrenergic receptors methylation and expression however 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 3-adrenergic receptors methylation with no significant effect on 1 and 2-adrenergic receptors. 2-adrenergic receptor was hypermethylated with halted expression. Conclusion: Our study demonstrates that 1 and 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. - Background Diabetes is a growing epidemic, caused by excess glucose levels and bodys inability to produce or regulate insulin [1]. It is predicted that the number of people with diabetes will increase from 171 million in 2000 to 366 million by 2030 [2,3]. Diabetes is linked to several vascular pathologies, including severe blindness, atherosclerosis, stroke and renal failure. The growing number of people with diabetes suggests that diabetic retinopathy (DR) and diabetic macular edema (DME) will continue to be sight threatening diseases. Distinct morphological abnormalities in the retinal microvasculature either remain stable or progress to diabetic macular edema or proliferative diabetic retinopathy, the leading causes of severe visual impairment in working-age adults in industrialized countries [4,5]. In vitro and in vivo studies have revealed that hyperglycemic environment induces a number of cellular changes which affect the function and viability of cells [6,7]. Changes in the diabetic retina are due to a variety of factors including high glucose, oxidative stress and high levels of inflammatory markers [8,9]. Reactive oxygen species (ROS), especially mitochondrial ROS, play a significant role in modulating the cellular redox status [10]. Excessive production of ROS and the impairment of the oxidant/antioxidant balance may in part underlie the pathogenesis of diabetes and its associated complications [11]. Actions of such key pathological mediators of diabetes can lead to dysregulated epigenetic mechanisms that affect chromatin structure and gene expression profiles [12]. DNA methylation is one of the mechanisms for the epigenetic control of gene expression and aberrant DNA methylation patterns of CpG islands can influence normal transcriptional regulation in various diseases [13]. Beta adrenergic receptors are G protein coupled receptors (GPCRs), initially characterized by Ahlquist in 1948. [14]. Activation of these receptors takes place at the transmembrane region, which allows ligand binding and elicit a range of cellular actions such as phosphorylation and activation of various signaling pathways [15]. Upon binding of specific ligands, 1 and 2-adrenergic receptors activate the stimulatory G protein resulting in the dissociation of G protein subunits from . The subunits are linked to the stimulation of intracellular adenylyl cyclase, followed by the conversion of adenosine triphosphate into cyclic adenosine monophosphate (cAMP) and consequently leads to activation of protein kinase A and phosphorylation of several other substrates [16,17]. However contrary to 1 and 2-adrenergic receptors, 3-adrenergic receptor couple through the inhibitory G protein (Gi), causing a reduced generation of cAMP [18]. Work in animal models suggests that adrenergic receptor agents may promote corneal wound healing and it is known that cornea has an abundance of -adrenergic receptors in both the epithelium and endothelium of the cornea [19]. Previous studies demonstrate that -adrenergic receptors agonists have positive effects on the retina by reducing the inflammatory markers [20-22]. Studies on rats have demonstrated that highly selective agonists for -ARs partially reversed obesity [23] and insulin resistance [24]. Similarly -adrenergic receptor agonists like isoproterenol inhibits the formation of degenerative capillaries, prevents apoptosis of cells and reduces tumor necrosis factor (TNF) while anatagonists increase retinal dysfunction [25]. The role of Beta-adrenergic receptor agonists and antagonists in the treatments of glaucoma, diabetic retinopathy, and their potential mechanisms of actions, are still under investigation. Type 2 diabetes is a multifactorial disease caused by a number of genetic, epigenetic and environmental factors [26]. In mammalian cells, DNA methylation takes place at cytosine in CpG dinucleotides and has been associated with transcriptional silencing [27]. Recent studies have shown that epigenetic modifications such as DNA methylation and histone modifications may affect the pathogenesis of type 2 diabetes [28,29]. Differential gene expressions show dynamic alterations in gene transcription and mRNA stability that can be influenced by the epigenetic modification of the genome in response to chronic hyperglycemic stress [30,31]. A number of studies have evaluated the epigenetic mechanisms in various developmental defects and diseases. Oxidative stress and alterations in DNA methylation have also been observed in diabetes, but no clear correlation between these events has been demonstrated until now. Very little research has focused on the epigenetics of adrenergic rece (...truncated)