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

The complex interplay between hydrogen peroxide (H ₂ O ₂ ) 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 H ₂ O ₂ on NO production. To address this problem, we employed genetically encoded biosensors, HyPer7 for H ₂ O ₂ 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, H ₂ O ₂ exhibited no significant influence on NO production. Subsequent exploration under physiological normoxia (5 kPa O ₂ ) revealed distinct oxidative stress levels characterized by reduced basal HyPer7 signals, enhanced H ₂ O ₂ scavenging kinetics, and altered responses to pharmacological treatment. Investigation of the relationship between H ₂ O ₂ and NO under varying oxygen conditions revealed a lack of NO response to H ₂ O ₂ under hyperoxia (18 kPa O ₂ ) but a modest NO response under physiological normoxia (5 kPa O ₂ ). Importantly, the NO response was attenuated by l-NAME, suggesting activation of eNOS by endogenous H ₂ O ₂ generation upon auranofin treatment. Our study highlights the intricate interplay between H ₂ O ₂ 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 ₂ ). This further investigation is essential for a comprehensive understanding of the H ₂ O ₂ and NO signaling considering the physiological effects of ambient O ₂ levels involved.
Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Emrah Eroglu reports financial support was provided by European Molecular Biology Organization. Hamza Yusuf Altun reports financial support and travel were provided by European Molecular Biology Organization. Hamza Yusuf Altun reports financial support and travel were provided by European Cooperation in Science and Technology. Emrah Eroglu reports financial support was provided by Scientific and Technological Research Council of Turkey. Hamza Yusuf Altun reports financial support was provided by Scientific and Technological Research Council of Turkey. Melike Secilmis reports financial support was provided by Scientific and Technological Research Council of Turkey. Tuba Akgul Caglar reports financial support was provided by Scientific and Technological Research Council of Turkey. The authors declare that they have no known competing financial intersts or personal relationships that could have appeared to influence the work reported in this paper. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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