Background: Current paradigms suggest that nitric oxide (NO) produced by endothelial cells (ECs) via endothelial nitric oxide synthase (eNOS) in the vessel wall is the primary regulator of blood flow and blood pressure. However, red blood cells (RBCs) also carry a catalytically active eNOS, but its role is controversial and remains undefined. This study aimed to elucidate the functional significance of red cell eNOS compared to EC eNOS for vascular hemodynamics and NO metabolism. Methods: We generated tissue-specific "loss-" and "gain-of-function" models for eNOS by using cell-specific Cre-induced gene inactivation or reactivation. We created two founder lines carrying a floxed eNOS (eNOS flox/flox ) for Cre-inducible knock out (KO), as well as gene construct with an inactivated floxed/inverted exon (eNOS inv/inv ) for a Cre-inducible knock in (KI), which respectively allow targeted deletion or reactivation of eNOS in erythroid cells (RBC eNOS KO or RBC eNOS KI mice) or endothelial cells (EC eNOS KO or EC eNOS KI mice). Vascular function, hemodynamics, and NO metabolism were compared ex vivo and in vivo . Results: The EC eNOS KOs exhibited significantly impaired aortic dilatory responses to acetylcholine, loss of flow-mediated dilation (FMD), and increased systolic and diastolic blood pressure. RBC eNOS KO mice showed no alterations in acetylcholine-mediated dilation or FMD but were hypertensive. Treatment with the NOS inhibitor L-NAME further increased blood pressure in RBC eNOS KOs, demonstrating that eNOS in both ECs and RBCs contributes to blood pressure regulation. While both EC eNOS KOs and RBC eNOS KOs had lower plasma nitrite and nitrate concentrations, the levels of bound NO in RBCs were lower in RBC eNOS KOs as compared to EC eNOS KOs. Crucially, reactivation of eNOS in ECs or RBCs rescues the hypertensive phenotype of the eNOS inv/inv mice, while the levels of bound NO were restored only in RBC eNOS KI mice. Conclusions: These data reveal that eNOS in ECs and RBCs contribute independently to blood pressure homeostasis.

Several diseases are associated with perturbations in redox signaling and aberrant hydrogen sulfide metabolism, and numerous analytical methods exist for the measurement of the sulfur-containing species affected. However, uncertainty remains about their concentrations and speciation in cells/biofluids, perhaps in part due to differences in sample processing and detection principles. Using ultrahigh-performance liquid chromatography in combination with electrospray-ionization tandem mass spectrometry we here outline a specific and sensitive platform for the simultaneous measurement of 12 analytes, including total and free thiols, their disulfides and sulfide in complex biological matrices such as blood, saliva and urine. Total assay run time is < 10 min, enabling high-throughput analysis. Enhanced sensitivity and avoidance of artifactual thiol oxidation is achieved by taking advantage of the rapid reaction of sulfhydryl groups with N-ethylmaleimide. We optimized the analytical procedure for detection and separation conditions, linearity and precision including three stable isotope labelled standards. Its versatility for future more comprehensive coverage of the thiol redox metabolome was demonstrated by implementing additional analytes such as methanethiol, N-acetylcysteine, and coenzyme A. Apparent plasma sulfide concentrations were found to vary substantially with sample pretreatment and nature of the alkylating agent. In addition to protein binding in the form of mixed disulfides (S-thiolation) a significant fraction of aminothiols and sulfide appears to be also non-covalently associated with proteins. Methodological accuracy was tested by comparing the plasma redox status of 10 healthy human volunteers to a well-established protocol optimized for reduced/oxidized glutathione. In a proof-of-principle study a deeper analysis of the thiol redox metabolome including free reduced/oxidized as well as bound thiols and sulfide was performed. Additional determination of acid-labile sulfide/thiols was demonstrated in human blood cells, urine and saliva. Using this simplified mass spectrometry-based workflow the thiol redox metabolome can be determined in samples from clinical and translational studies, providing a novel prognostic/diagnostic platform for patient stratification, drug monitoring, and identification of new therapeutic approaches in redox diseases.

The main function of red blood cells (RBCs) is the transport of respiratory gases along the vascular tree. To fulfill their task, RBCs are able to elastically deform in response to mechanical forces and, pass through the narrow vessels of the microcirculation. Decreased RBC deformability was observed in pathological conditions linked to increased oxidative stress or decreased nitric oxide (NO) bioavailability, like hypertension. Treatments with oxidants and with NO were shown to affect RBC deformability ex vivo, but the mechanisms underpinning these effects are unknown. In this study we investigate whether changes in intracellular redox status/oxidative stress or nitrosation reactions induced by reactive oxygen species (ROS) or NO may affect RBC deformability. In a case-control study comparing RBCs from healthy and hypertensive participants, we found that RBC deformability was decreased, and levels of ROS were increased in RBCs from hypertensive patients as compared to RBCs from aged-matched healthy controls, while NO levels in RBCs were not significantly different. To study the effects of oxidants on RBC redox state and deformability, RBCs from healthy volunteers were treated with increasing concentrations of tert-butylhydroperoxide (t-BuOOH). We found that high concentrations of t-BuOOH (≥ 1 mM) significantly decreased the GSH/GSSG ratio in RBCs, decreased RBC deformability and increased blood bulk viscosity. Moreover, RBCs from Nrf2 knockout (KO) mice, a strain genetically deficient in a number of antioxidant/reducing enzymes, were more susceptible to t-BuOOH-induced impairment in RBC deformability as compared to wild type (WT) mice. To study the role of NO in RBC deformability we treated RBC suspensions from human volunteers with NO donors and nitrosothiols and analyzed deformability of RBCs from mice lacking the endothelial NO synthase (eNOS). We found that NO donors induced S-nitrosation of the cytoskeletal protein spectrin, but did not affect human RBC deformability or blood bulk viscosity; moreover, under unstressed conditions RBCs from eNOS KO mice showed fully preserved RBC deformability as compared to WT mice. Pre-treatment of human RBCs with nitrosothiols rescued t-BuOOH-mediated loss of RBC deformability. Taken together, these findings suggest that NO does not affect RBC deformability per se, but preserves RBC deformability in conditions of oxidative stress.

The transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a key master switch that controls the expression of antioxidant and cytoprotective enzymes, including enzymes catalyzing glutathione de novo synthesis. In this study, we aimed to analyze whether Nrf2 deficiency influences antioxidative capacity, redox state, NO metabolites, and outcome of myocardial ischemia reperfusion (I/R) injury. In Nrf2 knockout (Nrf2 KO) mice, we found elevated eNOS expression and preserved NO metabolite concentrations in the aorta and heart as compared to wild types (WT). Unexpectedly, Nrf2 KO mice have a smaller infarct size following myocardial ischemia/reperfusion injury than WT mice and show fully preserved left ventricular systolic function. Inhibition of NO synthesis at onset of ischemia and during early reperfusion increased myocardial damage and systolic dysfunction in Nrf2 KO mice, but not in WT mice. Consistent with this, infarct size and diastolic function were unaffected in eNOS knockout (eNOS KO) mice after ischemia/reperfusion. Taken together, these data suggest that eNOS upregulation under conditions of decreased antioxidant capacity might play an important role in cardioprotection against I/R. Due to the redundancy in cytoprotective mechanisms, this fundamental antioxidant property of eNOS is not evident upon acute NOS inhibition in WT mice or in eNOS KO mice until Nrf2-related signaling is abrogated.