Advanced Glycation End Product (AGE)- Mediated Reactive Oxygen Species Production in Cultured Endothelial Cells is Dependent on NADPH Oxidase Activation

Abstract

Introduction: The interaction of Advanced Glycation Endproduct (AGE) with Receptor for Advanced Glycation End product (RAGE) on endothelial cells enhances the intracellular Reactive Oxygen Species (ROS) production. Activation of the NADPH Oxidase (NOX) is one of the important mechanisms for ROS generation in the endothelial cells. Aim: To determine the role of AGE in ROS production via NOX activation in endothelial cells. To assess the ameliorating action of drugs, ramipril, and losartan, as well as the antioxidants, resveratrol and N-Acetyl-Cysteine (NAC) on AGE-mediated effects in endothelial cells. Materials and Methods: The present experimental in-vitro study was conducted at Department of Biochemistry, University College of Medical Sciences and Guru Teg Bahadur Hospital, Delhi, India. The cultured Human Umbilical Vein Endothelial Cells (HUVECs) were treated with advanced glycation end productbovine serum albumin (AGE-BSA) 200 μg/mL and unmodified BSA in the same concentration for 24 hours. The HUVECs were also co-treated with losartan (5 μM), ramipril (5 μM), resveratrol (5 μM) and NAC (5 μM) with AGE-BSA for 24 hour. ROS generation was assessed by using 2’, 7’-dichlorodihydrofluorescein diacetate (H2DCFDA) method. For the activation of NOX, NOX p47phox subunit mRNA expression was analysed by Real Time Polymerase Chain Reaction (qPCR). Significant difference between the two groups was determined by the Student’s t-test. A value of p<0.05 was considered significant. Results: A significant increase (p<0.01) in ROS production was observed at a concentration of 200 μg/mL of AGE-BSA as compared with control (cells treated with unmodified BSA). NOX expression in cells was found to be significantly increased (p<0.01) 2-fold at mRNA level after 24 hours treatment with AGE-BSA. Losartan, ramipril, resveratrol and NAC significantly scavenged ROS production, and decreased the AGE-mediated increased NOX mRNA expression was observed. Conclusion: The present in-vitro study signifies the role of AGE in enhanced ROS generation by activation of NOX in endothelial cells. Losartan, ramipril, resveratrol, and NAC may attenuate the AGE-mediated endothelial dysfunction by counteracting the NOX-mediated increased ROS production.

Keywords

• antioxidant
• endothelial dysfunction
• oxidative stress
• polymerase chain reaction