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

The complex interplay between hydrogen peroxide (HO) 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 HO on NO production. To address this problem, we employed genetically encoded biosensors, HyPer7 for HO 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, HO exhibited no significant influence on NO production. Subsequent exploration under physiological normoxia (5 kPa O) revealed distinct oxidative stress levels characterized by reduced basal HyPer7 signals, enhanced HO scavenging kinetics, and altered responses to pharmacological treatment. Investigation of the relationship between HO and NO under varying oxygen conditions revealed a lack of NO response to HO under hyperoxia (18 kPa O) but a modest NO response under physiological normoxia (5 kPa O). Importantly, the NO response was attenuated by l-NAME, suggesting activation of eNOS by endogenous HO generation upon auranofin treatment. Our study highlights the intricate interplay between HO 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 O). This further investigation is essential for a comprehensive understanding of the HO and NO signaling considering the physiological effects of ambient O levels involved.