Simultaneous Manipulation and Imaging of Chemogenetically Induced Hydrogen Peroxide in Hardly Transfectable Endothelial Cells

Abstract

Hydrogen peroxide (H2O2) is a critical signaling molecule in vascular cells, which controls signaling events, yet it can cause pathological oxidative stress in excess. The lack of suitable tools undermined experimental approaches to study the role of oxidative eu- and distress in cellular ultra-locales. This study exploits a yeast-derived D-amino acid oxidase (mDAAO) as a chemogenetic tool to induce, visualize and test the cytotoxicity of H2O2 in hardly transfectable endothelial cells. Due to the poor transfectability of endothelial cells, lentiviral vectors have been used to generate cell lines stably expressing mDAAOs. mDAAOs are substrate-based chemogenetic enzymes that convert D-amino acids to their corresponding alpha-keto acids and generate H2O2 as a byproduct, which can be visualized with a novel ultrasensitive, and ratiometric H2O2 biosensor termed HyPer7. This study tested the suitability of two different D-amino acids, including D-alanine and D-methionine, to induce oxidative stress in endothelial cells. Live-cell imaging experiments unveiled that 10 mM D-methionine generated significantly higher and faster H2O2 signals than D-alanine. However, both D-amino acids induced comparable levels of cell death documented by a colorimetric cell metabolic activity assay (MTT). This study provides a guide for manipulating and monitoring the cytotoxic effect of H2O2 in endothelial cells.

Visualizing H2O2and NO in endothelial cells: strategies and pitfalls

“…Among various biosensors, we selected the green fluorescent protein (GFP)-based H 2 O 2 sensitive biosensor HyPer7 [24] and the orange fluorescent protein (OFP)-based NO probe O-geNOp [25]–[27] to simultaneously measure these two reactive molecules in vascular endothelial cells using multispectral imaging approaches. However, endothelial cells are known to be difficult to transfect [28]. To overcome these challenges, we have recently developed new strategies to improve transfection efficiency in endothelial cells by using viral gene delivery methods [23], [28].…”

“…However, endothelial cells are known to be difficult to transfect [28]. To overcome these challenges, we have recently developed new strategies to improve transfection efficiency in endothelial cells by using viral gene delivery methods [23], [28].…”