title: Differential expression and role of hyperglycemia induced oxidative stress in epigenetic regulation of β1, β2 and β3-adrenergic receptors in retinal endothelial cells.
abstract: 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 β1, β2 and β3-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 β1, β2 and β3-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
  β1 and β3-adrenergic receptors were expressed in retinal endothelial cells while β2-adrenergic receptor was not detectable both at protein and mRNA levels. Hyperglycemia had no significant effect on β1 and β2-adrenergic receptors methylation and expression however β3-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 β3-adrenergic receptors methylation with no significant effect on β1 and β2-adrenergic receptors. β2-adrenergic receptor was hypermethylated with halted expression.
  
  
  CONCLUSIONS
  Our study demonstrates that β1 and β3-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.
authors.affiliation: Department of Medicine, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia. safi.nust@yahoo.com.
authors.forename: Sher Zaman
authors.initials: SZ
authors.lastname: Safi
authors.forename: Rajes
authors.initials: R
authors.lastname: Qvist
authors.forename: Gracie Ong Siok
authors.initials: GO
authors.lastname: Yan
authors.forename: Ikram Shah Bin
authors.initials: IS
authors.lastname: Ismail
mesh_terms: D017209:Apoptosis / Q000235:genetics; D001483:Base Sequence; D018899:CpG Islands / Q000235:genetics; D019175:DNA Methylation / Q000235:genetics; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D044127:Epigenesis, Genetic / Q000187:drug effects; D020869:Gene Expression Profiling; D005947:Glucose / Q000494:pharmacology; D006801:Humans; D006943:Hyperglycemia / Q000235:genetics* / Q000473:pathology; D008969:Molecular Sequence Data; D018384:Oxidative Stress / Q000187:drug effects / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011941:Receptors, Adrenergic / Q000235:genetics* / Q000378:metabolism; D018342:Receptors, Adrenergic, beta-1 / Q000235:genetics / Q000378:metabolism; D012160:Retina / Q000473:pathology*
chemical_list: D011941:Receptors, Adrenergic; D018342:Receptors, Adrenergic, beta-1; D005947:Glucose