Hyperglycemia-induced oxidative stress and epigenetic regulation of ET-1 gene in endothelial cells

Abstract

Introduction: Hyperglycemia-induced endothelial dysfunction and the subsequent increase of oxidative stress could lead to aberrant regulation of various genes which are responsible for a range of functions. This study aims to find out how hyperglycemia affect oxidative stress and then the expression and methylation of endothelin 1 (ET-1) gene in in human umbilical vein endothelial cells (HUVEC).Methods: Cells were cultured in growth medium and exposed to low and high glucose concentrations to mimic normal and diabetic condition respectively. Computational analysis were performed using UCSC genome browser and eukaryotic promoter database (EPD). The expression of ET-1 gene was investigated by real time PCR. Cytotoxicity and oxidative stress were determined by MTT and DCFH-DA assays respectively. Promoter methylation was assessed by the bisulfite sequencing method.Results: DCFH-DA assay showed that hyperglycemia can significantly increase the regulation of reactive oxygen species synthesis. The relative expression of ET-1 gene was increased due to exposure to high glucose concentration. MTT assay revealed reduced viability of cells due to the glucose induced damage. Methylation analysis revealed hypomethylation of the promoter of ET-1 however the difference was not significant. Out of 175 CpGs at 25 CpG sites, only 36 CpGs were methylated (20.5% methylation) in cell treated with normal glucose. Upon exposure to high glucose only 30 CpGs were methylated in 175 CpGs at 25 CpG sites (17.1% methylation).Discussion: Our study concludes a significantly high expression of ET-1 gene in response to high glucose exposure in HUVECs. It also reports that hyperglycemic condition leads to elevated oxidative stress. No significant change was found in methylation when cells were treated with high and low glucose concentrations.

DNA Methylation is associated with oxidative stress and genomic deregulation in diabetes mellitus

“…Biswas et al reported the presence of hypomethylation in the proximal promoter and 5' UTR/ rst exon regions of EDN1 in high glucose-induced endothelial cells (HUVECs and HRECs) [60]. Another group reported about the hypomethylation of the promoter of ET-1 in the high glucose-induced HUVECs, though the difference with low glucose-treated HUVECs was not signi cant [61].…”

Causal relationship between type 2 diabetes and common respiratory system diseases: a two-sample Mendelian randomization analysis

“…Reactive oxygen species can damage cells and tissues, and stimulate inflammatory responses. Prolonged oxidative stress can disrupt the immune system, making it difficult to cope with inflammation ( 37 ). Finally, a high blood sugar level can impair the function of endothelial cells (inner lining of blood vessels), leading to vascular inflammation ( 38 ).…”

Factors Important in the Use of Fluorescent or Luminescent Probes and Other Chemical Reagents to Measure Oxidative and Radical Stress

“…Even accounting for the papers which simply mention or review the use of DCFH2 or related probes, it seems likely that well over 6000 studies have utilized such probes. Despite well-known shortcomings, which have been repeatedly stressed by numerous authors, e.g., [5,6,8,9,13,19,23,24,27,30,54,55,[79][80][81][82][83][84][85][86][87][88][89], these probes continue to be widely recommended and used in many investigations, e.g., [90][91][92][93][94][95], including a recent study that attracted quite widespread press coverage [96]. There are three basic problems with this and similar 'leuco' dyes such as the chloromethyl analogue and other variants promoted by manufacturers; while DCFH2 is the best characterized, it seems likely that all exhibit many of the problems discussed below.…”