Reactive Oxygen Species Differentially Modulate the Metabolic and Transcriptomic Response of Endothelial Cells.

Niklas Müller,Kurt Noack,Katrin Schröder,Pedro Felipe Malacarne,Timothy Warwick,Arthur J L Cooper,Norbert Weissmann,Ralf P Brandes,Flávia Rezende

doi:10.3390/antiox11020434

Abstract

Reactive oxygen species (ROS) are important mediators of both physiological and pathophysiological signal transduction in the cardiovascular system. The effects of ROS on cellular processes depend on the concentration, localization, and duration of exposure. Cellular stress response mechanisms have evolved to mitigate the negative effects of acute oxidative stress. In this study, we investigate the short-term and long-term metabolic and transcriptomic response of human umbilical vein endothelial cells (HUVEC) to different types and concentrations of ROS. To generate intracellular H2O2, we utilized a lentiviral chemogenetic approach for overexpression of human D-amino acid oxidase (DAO). DAO converts D-amino acids into their corresponding imino acids and H2O2. HUVEC stably overexpressing DAO (DAO-HUVEC) were exposed to D-alanine (3 mM), exogenous H2O2 (10 µM or 300 µM), or menadione (5 µM) for various timepoints and subjected to global untargeted metabolomics (LC-MS/MS) and RNAseq by MACE (Massive analysis of cDNA ends). A total of 300 µM H2O2 led to pronounced changes on both the metabolic and transcriptomic level. In particular, metabolites linked to redox homeostasis, energy-generating pathways, and nucleotide metabolism were significantly altered. Furthermore, 300 µM H2O2 affected genes related to the p53 pathway and cell cycle. In comparison, the effects of menadione and DAO-derived H2O2 mainly occurred at gene expression level. Collectively, all types of ROS led to subtle changes in the expression of ribosomal genes. Our results show that different types and concentration of ROS lead to a different metabolic and transcriptomic response in endothelial cells.

Highlights

The vascular system is entirely lined by a single layer of endothelial cells (EC), which facilitate the exchange of nutrients and oxygen between the blood and surrounding tissues.Angiogenic endothelial cells migrate into hypoxic tissue, and endothelial nitric oxide (NO)production inhibits complex IV of the respiratory chain
D-amino acid oxidase (DAO) expression after transduction was readily detected in human umbilical vein endothelial cells (HUVEC) by immunofluorescence
To test the activity of DAO, H2 O2 was measured by luminol/ HRP chemiluminescence

Summary

Introduction

The vascular system is entirely lined by a single layer of endothelial cells (EC), which facilitate the exchange of nutrients and oxygen between the blood and surrounding tissues.Angiogenic endothelial cells migrate into hypoxic tissue, and endothelial nitric oxide (NO)production inhibits complex IV of the respiratory chain. The vascular system is entirely lined by a single layer of endothelial cells (EC), which facilitate the exchange of nutrients and oxygen between the blood and surrounding tissues. Angiogenic endothelial cells migrate into hypoxic tissue, and endothelial nitric oxide (NO). Production inhibits complex IV of the respiratory chain. Endothelial ATP production highly relies on glycolysis instead of oxidative phosphorylation. EC have a relative small mitochondrial volume [1,2]. The endothelial production of ROS is triggered by oxidants produced from activated immune cells, cytokines, or physical stimuli, such as oscillatory flow [3,4]. Since ROS can lead to oxidative stress, various anti-oxidant defense mechanisms have evolved, such as detoxifying enzymes, redox-sensitive gene expression, and a dynamic metabolic response

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