Visualizing hydrogen peroxide and nitric oxide dynamics in endothelial cells using multispectral imaging under controlled oxygen conditions

Abstract

The complex interplay between hydrogen peroxide (H2O2) and nitric oxide (NO) in endothelial cells presents challenges due to technical limitations in simultaneous measurement, hindering the elucidation of their direct relationship. Previous studies have yielded conflicting findings regarding the impact of H2O2 on NO production. To address this problem, we employed genetically encoded biosensors, HyPer7 for H2O2 and geNOps for NO, allowing simultaneous imaging in single endothelial cells. Optimization strategies were implemented to enhance biosensor performance, including camera binning, temperature regulation, and environmental adjustments to mimic physiological normoxia. Our results demonstrate that under ambient oxygen conditions, H2O2 exhibited no significant influence on NO production. Subsequent exploration under physiological normoxia (5 kPa O2) revealed distinct oxidative stress levels characterized by reduced basal HyPer7 signals, enhanced H2O2 scavenging kinetics, and altered responses to pharmacological treatment. Investigation of the relationship between H2O2 and NO under varying oxygen conditions revealed a lack of NO response to H2O2 under hyperoxia (18 kPa O2) but a modest NO response under physiological normoxia (5 kPa O2). Importantly, the NO response was attenuated by L-NAME, suggesting activation of eNOS by endogenous H2O2 generation upon auranofin treatment. Our study highlights the intricate interplay between H2O2 and NO within the endothelial EA.hy926 cell line, emphasizing the necessity for additional research within physiological contexts due to differential response observed under physiological normoxia (5 kPa O2). This further investigation is essential for a comprehensive understanding of the H2O2 and NO signaling considering the physiological effects of ambient O2 levels involved.