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

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.