Search

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., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Iron is an essential metal for cellular metabolism and signaling, but it has adverse effects in excess. The physiological consequences of iron deficiency are well established, yet the relationship between iron supplementation and pericellular oxygen levels in cultured cells and their downstream effects on metalloproteins has been less explored. This study exploits the metalloprotein geNOps in cultured HEK293T epithelial and EA.hy926 endothelial cells to test the iron-dependency in cells adapted to standard room air (18 kPa O 2 ) or physiological normoxia (5 kPa O 2 ). We show that cells in culture require iron supplementation to activate the metalloprotein geNOps and demonstrate for the first time that cells adapted to physiological normoxia require significantly lower iron compared to cells adapted to hyperoxia. This study establishes an essential role for recapitulating oxygen levels in vivo and uncovers a previously unrecognized requirement for ferrous iron supplementation under standard cell culture conditions to achieve geNOps functionality., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

A common approach to investigate oxidant-regulated intracellular pathways is to add exogenous H 2 O 2 to living cells or tissues. However, the addition of H 2 O 2 to the culture medium of cells or tissues approach does not accurately replicate intracellular redox-mediated cell responses. d-amino acid oxidase (DAAO)-based chemogenetic tools represent informative methodological advances that permit the generation of H 2 O 2 on demand with a high spatiotemporal resolution by providing or withdrawing the DAAO substrate d-amino acids. Much has been learned about the intracellular transport of H 2 O 2 through studies using DAAO, yet these valuable tools remain incompletely characterized in many cultured cells. In this study, we describe and characterize in detail the features of a new modified variant of DAAO (termed mDAAO) with improved catalytic activities. We tested mDAAO functionality in several cultured cell lines employing live-cell imaging techniques. Our imaging experiments show that mDAAO is suitable for the generation of H 2 O 2 under hypoxic conditions imaged with the novel ultrasensitive H 2 O 2 sensor (HyPer7). Moreover, this approach was suitable for generating H 2 O 2 in a reversible and concentration-dependent manner in subcellular locales. Furthermore, we show that the choice of d-amino acids differentially affects mDAAO-dependent intracellular H 2 O 2 generation. When paired with the hydrogen sulfide (H 2 S) sensor hsGFP, administration of the sulfur-containing amino acid d-cysteine to cells expressing mDAAO generates robust H 2 S signals. We also show that chemogenetic H 2 O 2 generation in different cell types yields distinct HyPer7 profiles. These studies fully characterize the new mDAAO as a novel chemogenetic tool and provide multiparametric approaches for cell manipulation that may open new lines of investigations for redox biochemists to dissect the role of ROS signaling pathways with high spatial and temporal precision., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Multispectral live-cell imaging is an informative approach that permits detecting biological processes simultaneously in the spatial and temporal domain by exploiting spectrally distinct biosensors. However, the combination of fluorescent biosensors with distinct spectral properties such as different sensitivities, and dynamic ranges can undermine accurate co-imaging of the same analyte in different subcellular locales. We advanced a single-color multiparametric imaging method, which allows simultaneous detection of hydrogen peroxide (H 2 O 2 ) in multiple cell locales (nucleus, cytosol, mitochondria) using the H 2 O 2 biosensor HyPer7. Co-culturing of endothelial cells stably expressing differentially targeted HyPer7 biosensors paved the way for co-imaging compartmentalized H 2 O 2 signals simultaneously in neighboring cells in a single experimental setup. We termed this approach COMPARE IT, which is an acronym for co-culture-based multiparametric imaging technique. Employing this approach, we detected lower H 2 O 2 levels in mitochondria of endothelial cells compared to the cell nucleus and cytosol under basal conditions. Upon administering exogenous H 2 O 2 , the cytosolic and nuclear-targeted probes displayed similarly slow and moderate HyPer7 responses, whereas the mitochondria-targeted HyPer7 signal plateaued faster and reached higher amplitudes. Our results indicate striking differences in mitochondrial H 2 O 2 accumulation of endothelial cells. Here, we present the method's potential as a practicable and informative multiparametric live-cell imaging technique.