Hypoxia increases persulfide and polysulfide formation by AMP kinase dependent cystathionine gamma lyase phosphorylation.

Hydropersulfide and hydropolysulfide metabolites are increasingly important reactive sulfur species (RSS) regulating numerous cellular redox dependent functions. Intracellular production of these species is known to occur through RSS interactions or through translational mechanisms involving cysteinyl t-RNA synthetases. However, regulation of these species under cell stress conditions, such as hypoxia, that are known to modulate RSS remain poorly understood. Here we define an important mechanism of increased persulfide and polysulfide production involving cystathionine gamma lyase (CSE) phosphorylation at serine 346 and threonine 355 in a substrate specific manner, under acute hypoxic conditions. Hypoxic phosphorylation of CSE occurs in an AMP kinase dependent manner increasing enzyme activity involving unique inter- and intramolecular interactions within the tetramer. Importantly, both cellular hypoxia and tissue ischemia result in AMP Kinase dependent CSE phosphorylation that regulates blood flow in ischemic tissues. Our findings reveal hypoxia molecular signaling pathways regulating CSE dependent persulfide and polysulfide production impacting tissue and cellular response to stress.

Elevated plasma sulfides are associated with cognitive dysfunction and brain atrophy in human Alzheimer's disease and related dementias.

Emerging evidence indicates that vascular stress is an important contributor to the pathophysiology of Alzheimer's disease and related dementias (ADRD). Hydrogen sulfide (H2S) and its metabolites (acid-labile (e.g., iron-sulfur clusters) and bound (e.g., per-, poly-) sulfides) have been shown to modulate both vascular and neuronal homeostasis. We recently reported that elevated plasma sulfides were associated with cognitive dysfunction and measures of microvascular disease in ADRD. Here we extend our previous work to show associations between elevated sulfides and magnetic resonance-based metrics of brain atrophy and white matter integrity. Elevated bound sulfides were associated with decreased grey matter volume, while increased acid labile sulfides were associated with decreased white matter integrity and greater ventricular volume. These findings are consistent with alterations in sulfide metabolism in ADRD which may represent maladaptive responses to oxidative stress.

Methamphetamine causes cardiovascular dysfunction via cystathionine gamma lyase and hydrogen sulfide depletion.

Methamphetamine (METH) is an addictive illicit drug used worldwide that causes significant damage to blood vessels resulting in cardiovascular dysfunction. Recent studies highlight increased prevalence of cardiovascular disease (CVD) and associated complications including hypertension, vasospasm, left ventricular hypertrophy, and coronary artery disease in younger populations due to METH use. Here we report that METH administration in a mouse model of 'binge and crash' decreases cardiovascular function via cystathionine gamma lyase (CSE), hydrogen sulfide (H2S), nitric oxide (NO) (CSE/H2S/NO) dependent pathway. METH significantly reduced H2S and NO bioavailability in plasma and skeletal muscle tissues co-incident with a significant reduction in flow-mediated vasodilation (FMD) and blood flow velocity revealing endothelial dysfunction. METH administration also reduced cardiac ejection fraction (EF) and fractional shortening (FS) associated with increased tissue and perivascular fibrosis. Importantly, METH treatment selectively decreased CSE expression and sulfide bioavailability along with reduced eNOS phosphorylation and NO levels. Exogenous sulfide therapy or endothelial CSE transgenic overexpression corrected cardiovascular and associated pathological responses due to METH implicating a central molecular regulatory pathway for tissue pathology. These findings reveal that therapeutic intervention targeting CSE/H2S bioavailability may be useful in attenuating METH mediated cardiovascular disease.

The ataxia-telangiectasia mutated gene product regulates the cellular acid-labile sulfide fraction.

The ataxia-telangiectasia mutated (ATM) protein regulates cell cycle checkpoints, the cellular redox state, and double-stranded DNA break repair. ATM loss causes the disorder ataxia-telangiectasia (A-T), distinguished by ataxia, telangiectasias, dysregulated cellular redox and iron responses, and an increased cancer risk. We examined the sulfur pool in A-T cells, with and without an ATM expression vector. While free and bound sulfide levels were not changed with ATM expression, the acid-labile sulfide faction was significantly increased. ATM expression also increased cysteine desulfurase (NFS1), NFU1 iron-sulfur cluster scaffold homolog protein, and several mitochondrial complex I proteins' expression. Additionally, ATM expression suppressed cystathionine ß-synthase and cystathionine γ-synthase protein expression, cystathionine γ-synthase enzymatic activity, and increased the reduced to oxidized glutathione ratio. This last observation is interesting, as dysregulated glutathione is implicated in A-T pathology. As ATM expression increases the expression of proteins central in initiating 2Fe-2S and 4Fe-4S cluster formation (NFS1 and NFU1, respectively), and the acid-labile sulfide faction is composed of sulfur incorporated into Fe-S clusters, our data indicates that ATM regulates aspects of Fe-S cluster biosynthesis, the transsulfuration pathway, and glutathione redox cycling. Thus, our data may explain some of the redox- and iron-related pathologies seen in A-T.

Decreased availability of nitric oxide and hydrogen sulfide is a hallmark of COVID-19.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is involved in a global outbreak affecting millions of people who manifest a variety of symptoms. Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 is increasingly associated with cardiovascular complications requiring hospitalizations; however, the mechanisms underlying these complications remain unknown. Nitric oxide (NO) and hydrogen sulfide (H2S) are gasotransmitters that regulate key cardiovascular functions. METHODS: Blood samples were obtained from 68 COVID-19 patients and 33 controls and NO and H2S metabolites were assessed. H2S and NO levels were compared between cases and controls in the entire study population and subgroups based on race. The availability of gasotransmitters was examined based on severity and outcome of COVID-19 infection. The performance of H2S and NO levels in predicting COVID-19 infection was also analyzed. Multivariable regression analysis was performed to identify the effects of traditional determinants of gasotransmitters on NO and H2S levels in the patients with COVID-19 infection. RESULTS: Significantly reduced NO and H2S levels were observed in both Caucasian and African American COVID-19 patients compared to healthy controls. COVID-19 patients who died had significantly higher NO and H2S levels compared to COVID-19 patients who survived. Receiver-operating characteristic analysis of NO and H2S metabolites in the study population showed free sulfide levels to be highly predictive of COVID-19 infection based on reduced availability. Traditional determinants of gasotransmitters, namely age, race, sex, diabetes, and hypertension had no effect on NO and H2S levels in COVID-19 patients. CONCLUSION: These observations provide the first insight into the role of NO and H2S in COVID-19 infection, where their low availability may be a result of reduced synthesis secondary to endotheliitis, or increased consumption from scavenging of reactive oxygen species.

Suppression of mitochondrial respiration by hydrogen sulfide in hibernating 13-lined ground squirrels.

Hibernating mammals may suppress their basal metabolic rate during torpor by up to 95% to reduce energy expenditure during winter, but the underlying mechanisms remain poorly understood. Here we show that hydrogen sulfide (H2S), a ubiquitous signaling molecule, is a powerful inhibitor of respiration of liver mitochondria isolated from torpid 13-lined ground squirrels, but has a weak effect on mitochondria isolated during summer and hibernation arousals, where metabolic rate is normal. Consistent with these in vitro effects, we find strong seasonal variations of in vivo levels of H2S in plasma and increases of H2S levels in the liver of squirrels during torpor compared to levels during arousal and summer. The in vivo changes of liver H2S levels correspond with low activity of the mitochondrial H2S oxidizing enzyme sulfide:quinone oxidoreductase (SQR) during torpor. Taken together, these results suggest that during torpor, H2S accumulates in the liver due to a low SQR activity and contributes to inhibition of mitochondrial respiration, while during arousals and summer these effects are reversed, H2S is degraded by active SQR and mitochondrial respiration rates increase. This study provides novel insights into mechanisms underlying mammalian hibernation, pointing to SQR as a key enzyme involved in the control of mitochondrial function.

Plasma hydrogen sulfide: A biomarker of Alzheimer's disease and related dementias.

While heart disease remains a common cause of mortality, cerebrovascular disease also increases with age, and has been implicated in Alzheimer's disease and related dementias (ADRD). We have described hydrogen sulfide (H2 S), a signaling molecule important in vascular homeostasis, as a biomarker of cardiovascular disease. We hypothesize that plasma H2 S and its metabolites also relate to vascular and cognitive dysfunction in ADRD. We used analytical biochemical methods to measure plasma H2 S metabolites and MRI to evaluate indicators of microvascular disease in ADRD. Levels of total H2 S and specific metabolites were increased in ADRD versus controls. Cognition and microvascular disease indices were correlated with H2 S levels. Total plasma sulfide was the strongest indicator of ADRD, and partially drove the relationship between cognitive dysfunction and white matter lesion volume, an indicator of microvascular disease. Our findings show that H2 S is dysregulated in dementia, providing a potential biomarker for diagnosis and intervention.

Decreased bioavailability of hydrogen sulfide links vascular endothelium and atrial remodeling in atrial fibrillation.

Oxidative stress drives the pathogenesis of atrial fibrillation (AF), the most common arrhythmia. In the cardiovascular system, cystathionine γ-lyase (CSE) serves as the primary enzyme producing hydrogen sulfide (H2S), a mammalian gasotransmitter that reduces oxidative stress. Using a case control study design in patients with and without AF and a mouse model of CSE knockout (CSE-KO), we evaluated the role of H2S in the etiology of AF. Patients with AF (n = 51) had significantly reduced plasma acid labile sulfide levels compared to patients without AF (n = 65). In addition, patients with persistent AF (n = 25) showed lower plasma free sulfide levels compared to patients with paroxysmal AF (n = 26). Consistent with an important role for H2S in AF, CSE-KO mice had decreased atrial sulfide levels, increased atrial superoxide levels, and enhanced propensity for induced persistent AF compared to wild type (WT) mice. Rescuing H2S signaling in CSE-KO mice by Diallyl trisulfide (DATS) supplementation or reconstitution with endothelial cell specific CSE over-expression significantly reduced atrial superoxide, increased sulfide levels, and lowered AF inducibility. Lastly, low H2S levels in CSE KO mice was associated with atrial electrical remodeling including longer effective refractory periods, slower conduction velocity, increased myocyte calcium sparks, and increased myocyte action potential duration that were reversed by DATS supplementation or endothelial CSE overexpression. Our findings demonstrate an important role of CSE and H2S bioavailability in regulating electrical remodeling and susceptibility to AF.

Nicotinamide nucleotide transhydrogenase (NNT) regulates mitochondrial ROS and endothelial dysfunction in response to angiotensin II.

Endothelial dysfunction is a critical, initiating step in the development of hypertension (HTN) and mitochondrial reactive oxygen species (ROS) are important contributors to endothelial dysfunction. Genome-wide association studies (GWAS) have identified single nucleotide polymorphisms (SNPs) in the nicotinamide nucleotide transhydrogenase (Nnt) gene that are associated with endothelial dysfunction and increased risk for HTN. NNT is emerging as an important enzyme that regulates mitochondrial NADPH levels and mitochondrial redox balance by supporting the thiol dependent peroxidase systems in the mitochondria. We have previously shown that the absence of NNT in C57Bl/6J animals promotes a more severe hypertensive phenotype through reductions in •NO and endothelial dependent vessel dilation. However, the impact of NNT on human endothelial cell function remains unclear. We utilized NNT directed shRNA in human aortic endothelial cells to test the hypothesis that NNT critically regulates mitochondrial redox balance and endothelial function in response to angiotensin II (Ang II). We demonstrate that NNT expression and activity are elevated in response to the mitochondrial dysfunction and oxidative stress associated with Ang II treatment. Knockdown of NNT led to a significant elevation of mitochondrial ROS production and impaired glutathione peroxidase and glutathione reductase activities associated with a reduction in the NADPH/NADP+ ratio. Loss of NNT also promoted mitochondrial dysfunction, disruption of the mitochondrial membrane potential, and impaired ATP production in response to Ang II. Finally, we observed that, while the loss of NNT augmented eNOS phosphorylation at Ser1177, neither eNOS activity nor nitric oxide production were similarly increased. The results from these studies clearly demonstrate that NNT is critical for the maintenance of mitochondrial redox balance and mitochondrial function. Loss of NNT and disruption of redox balance leads to oxidative stress that compromises eNOS activity that could have a profound effect on the endothelium dependent regulation of vascular tone.

Hydrogen sulfide stimulates xanthine oxidoreductase conversion to nitrite reductase and formation of NO.

Cardiovascular disease is the leading cause of death and disability worldwide with increased oxidative stress and reduced NO bioavailability serving as key risk factors. For decades, elevation in protein abundance and enzymatic activity of xanthine oxidoreductase (XOR) under hypoxic/inflammatory conditions has been associated with organ damage and vascular dysfunction. Recent reports have challenged this dogma by identifying a beneficial function for XOR, under similar hypoxic/acidic conditions, whereby XOR catalyzes the reduction of nitrite (NO2-) to nitric oxide (NO) through poorly defined mechanisms. We previously reported that hydrogen sulfide (H2S/sulfide) confers significant vascular benefit under these same conditions via NO2- mediated mechanisms independent of nitric oxide synthase (NOS). Here we report for the first time the convergence of H2S, XOR, and nitrite to form a concerted triad for NO generation. Specifically, hypoxic endothelial cells show a dose-dependent, sulfide and polysulfide (diallyl trisulfide (DATS)-induced, NOS-independent NO2- reduction to NO that is dependent upon the enzymatic activity of XOR. Interestingly, nitrite reduction to NO was found to be slower and more sustained with DATS compared to H2S. Capacity for sulfide/polysulfide to produce an XOR-dependent impact on NO generation translates to salutary actions in vivo as DATS administration in cystathionine-γ-lyase (CSE) knockout mice significantly improved hindlimb ischemia blood flow post ligation, while the XOR-specific inhibitor, febuxostat (Febx), abrogated this benefit. Moreover, flow-mediated vasodilation (FMD) in CSE knockout mice following administration of DATS resulted in greater than 4-fold enhancement in femoral artery dilation while co-treatment with Febx completely completely abrogated this effect. Together, these results identify XOR as a focal point of convergence between sulfide- and nitrite-mediated signaling, as well as affirm the critical need to reexamine current dogma regarding inhibition of XOR in the context of vascular dysfunction.

Tissue-dependent variation of hydrogen sulfide homeostasis in anoxic freshwater turtles.

Hydrogen sulfide (H2S) controls numerous physiological responses. To understand its proposed role in metabolic suppression, we measured free H2S and bound sulfane sulfur (BSS) in tissues of the freshwater turtle Trachemys scriptaelegans, a species undergoing strong metabolic suppression when cold and anoxic. In warm normoxic turtles, free H2S was higher in red blood cells (RBCs) and kidney (∼9-10 µmol l-1) than in brain, liver and lung (∼1-2 µmol l-1). These values overall aligned with the tissue H2S-generating enzymatic activity. BSS levels were similar in all tissues (∼0.5 µmol l-1) but ∼100-fold higher in RBCs, which have a high thiol content, suggesting that RBCs function as a circulating H2S reservoir. Cold acclimation caused significant changes in free and bound H2S in liver, brain and RBCs, but anoxia had no further effect, except in the brain. These results show tissue-dependent sulfide signaling with a potential role in brain metabolic suppression during anoxia in turtles.

The Ataxia telangiectasia-mutated and Rad3-related protein kinase regulates cellular hydrogen sulfide concentrations.

The ataxia telangiectasia-mutated and Rad3-related (ATR) serine/threonine kinase plays a central role in the repair of replication-associated DNA damage, the maintenance of S and G2/M-phase genomic stability, and the promotion of faithful mitotic chromosomal segregation. A number of stimuli activate ATR, including persistent single-stranded DNA at stalled replication folks, R loop formation, hypoxia, ultraviolet light, and oxidative stress, leading to ATR-mediated protein phosphorylation. Recently, hydrogen sulfide (H2S), an endogenous gasotransmitter, has been found to regulate multiple cellular processes through complex redox reactions under similar cell stress environments. Three enzymes synthesize H2S: cystathionine-ß-synthase, cystathionine γ-lyase, and 3-mercaptopyruvate sulfurtransferase. Since H2S can under some conditions cause DNA damage, we hypothesized that ATR activity may regulate cellular H2S concentrations and H2S-syntheszing enzymes. Here we show that human colorectal cancer cells carrying biallelic knock-in hypomorphic ATR mutations have lower cellular H2S concentrations than do syngeneic ATR wild-type cells, and all three H2S-synthesizing enzymes show lower protein expression in the ATR hypomorphic mutant cells. Additionally, ATR serine 428 phosphorylation is altered by H2S donor and H2S synthesis enzyme inhibition, while the oxidative-stress induced phosphorylation of the ATR-regulated protein CHK1 on serine 345 is increased by H2S synthesis enzyme inhibition. Lastly, inhibition of H2S production potentiated oxidative stress-induced double-stranded DNA breaks in the ATR hypomorphic mutant compared to ATR wild-type cells. Our findings demonstrate that the ATR kinase regulates and is regulated by H2S.

Cystathionine γ-Lyase Modulates Flow-Dependent Vascular Remodeling.

Objective- Flow patterns differentially regulate endothelial cell phenotype, with laminar flow promoting vasodilation and disturbed flow promoting endothelial proinflammatory activation. CSE (cystathionine γ-lyase), a major source of hydrogen sulfide (H2S) in endothelial cells, critically regulates cardiovascular function, by both promoting vasodilation and reducing endothelial activation. Therefore, we sought to investigate the role of CSE in the endothelial response to flow. Approach and Results- Wild-type C57Bl/6J and CSE knockout ( CSE-/-) mice underwent partial carotid ligation to induce disturbed flow in the left carotid. In addition, endothelial cells isolated from wild-type and CSE -/- mice were exposed to either laminar or oscillatory flow, an in vitro model of disturbed flow. Interestingly, laminar flow significantly reduced CSE expression in vitro, and only disturbed flow regions show discernable CSE protein expression in vivo, correlating with enhanced H2S production in wild-type C57BL/6J but not CSE-/- mice. Lack of CSE limited disturbed flow-induced proinflammatory gene expression (ICAM-1[intercellular adhesion molecule 1], VCAM-1 [vascular cell adhesion molecular 1]) and monocyte infiltration and CSE-/- endothelial cells showed reduced NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) activation and proinflammatory gene expression in response to oscillatory flow in vitro. In addition, CSE-/- mice showed reduced inward remodeling after partial carotid ligation. CSE-/- mice showed elevated vascular nitrite levels (measure of nitric oxide [NO]) in the unligated carotids, suggesting an elevation in baseline NO production, and the NO scavenger 2-(4-carboxyphenyl)-4,5-dihydro-4,4,5,5-tetramethyl-1H-imidazolyl-1-oxy-3-oxide normalized the reduced inward remodeling, but not inflammation, of ligated carotids in CSE-/- mice. Conclusions- CSE expression in disturbed flow regions critically regulates both endothelial activation and flow-dependent vascular remodeling, in part through altered NO availability.

Total sulfane sulfur bioavailability reflects ethnic and gender disparities in cardiovascular disease.

Hydrogen sulfide (H2S) has emerged as an important physiological and pathophysiological signaling molecule in the cardiovascular system influencing vascular tone, cytoprotective responses, redox reactions, vascular adaptation, and mitochondrial respiration. However, bioavailable levels of H2S in its various biochemical metabolite forms during clinical cardiovascular disease remain poorly understood. We performed a case-controlled study to quantify and compare the bioavailability of various biochemical forms of H2S in patients with and without cardiovascular disease (CVD). In our study, we used the reverse-phase high performance liquid chromatography monobromobimane assay to analytically measure bioavailable pools of H2S. Single nucleotide polymorphisms (SNPs) were also identified using DNA Pyrosequencing. We found that plasma acid labile sulfide levels were significantly reduced in Caucasian females with CVD compared with those without the disease. Conversely, plasma bound sulfane sulfur levels were significantly reduced in Caucasian males with CVD compared with those without the disease. Surprisingly, gender differences of H2S bioavailability were not observed in African Americans, although H2S bioavailability was significantly lower overall in this ethnic group compared to Caucasians. We also performed SNP analysis of H2S synthesizing enzymes and found a significant increase in cystathionine gamma-lyase (CTH) 1364 G-T allele frequency in patients with CVD compared to controls. Lastly, plasma H2S bioavailability was found to be predictive for cardiovascular disease in Caucasian subjects as determined by receiver operator characteristic analysis. These findings reveal that plasma H2S bioavailability could be considered a biomarker for CVD in an ethnic and gender manner. Cystathionine gamma-lyase 1346 G-T SNP might also contribute to the risk of cardiovascular disease development.

Role of thiosulfate in hydrogen sulfide-dependent redox signaling in endothelial cells.

Recent reports have revealed that hydrogen sulfide (H2S) exerts critical actions to promote cardiovascular homeostasis and health. Thiosulfate is one of the products formed during oxidative H2S metabolism, and thiosulfate has been used extensively and safely to treat calcific uremic arteriopathy in dialysis patients. Yet despite its significance, fundamental questions regarding how thiosulfate and H2S interact during redox signaling remain unanswered. In the present study, we examined the effect of exogenous thiosulfate on hypoxia-induced H2S metabolite bioavailability in human umbilical vein endothelial cells (HUVECs). Under hypoxic conditions, we observed a decrease of GSH and GSSG levels in HUVECs at 0.5 and 4 h as well as decreased free H2S and acid-labile sulfide and increased bound sulfide at all time points. Treatment with exogenous thiosulfate significantly decreased the ratio of GSH/GSSG to total sulfide of HUVECs under 0.5 h of hypoxia but significantly increased this ratio in HUVECs under 4 h of hypoxia. These responses reveal that thiosulfate has different effects at low and high doses and under different O2 tensions. In addition, treatment with thiosulfate also diminished VEGF-induced cystathionine-γ-lyase expression and reduced VEGF-induced HUVEC proliferation under both normoxic and hypoxic conditions. These results indicate that thiosulfate can modulate H2S metabolites and signaling under various culture conditions that impact angiogenic activity. Thus, thiosulfate may serve as a unique sulfide donor to modulate endothelial responses under pathophysiological conditions involving angiogenesis.NEW & NOTEWORTHY This report provides new evidence that different levels of exogenous thiosulfate dynamically change discrete sulfide biochemical metabolite bioavailability in endothelial cells under normoxia or hypoxia, acting in a slow manner to modulate sulfide metabolites. Moreover, our findings also reveal that thiosulfate surprisingly inhibits VEGF-dependent endothelial cell proliferation associated with a reduction in cystathionine-γ-lyase protein levels.

Nicotinamide nucleotide transhydrogenase activity impacts mitochondrial redox balance and the development of hypertension in mice.

Oxidant stress contributes to the initiation and progression of hypertension (HTN) by enhancing endothelial dysfunction and/or causing perturbations in nitric oxide homeostasis. Differences in mitochondrial function may augment this process and provide insight into why age of onset and clinical outcomes differ among individuals from distinct ethnic groups. We have previously demonstrated that variation in normal mitochondrial function and oxidant production exists in endothelial cells from individuals of Caucasian and African-American ethnicity and that this variation contributes to endothelial dysfunction. To model these distinct mitochondrial redox phenotypes, we used C57Bl/6N (6N) and C57Bl/6J (6J) mice that also display unique mitochondrial functional properties due to the differential expression nicotinamide nucleotide transhydrogenase (NNT). We demonstrate that the absence of NNT in 6J cells led to distinct mitochondrial bioenergetic profiles and a pro-oxidative mitochondrial phenotype characterized by increased superoxide production and reduced glutathione peroxidase activity. Interestingly, we found that 6J animals have significantly higher systolic blood pressure compared to 6N animals, and this difference is exacerbated by angiotensin II treatment. The changes in pressure were accompanied by both mitochondrial and vascular dysfunction revealed by impaired respiratory control ratios and endothelial-dependent vessel dilation. All end points could be significantly ameliorated by treatment with the mitochondria-targeted superoxide dismutase mimetic MitoTEMPO demonstrating a critical role for the production of mitochondrial reactive oxygen species in the development of HTN in these animals. Taken together, these data indicate that the absence of NNT leads to variation in mitochondrial function and contributes to a unique mitochondrial redox phenotype that influences susceptibility to HTN by contributing to endothelial and vascular dysfunction.

Hydrogen sulfide metabolism regulates endothelial solute barrier function.

Hydrogen sulfide (H2S) is an important gaseous signaling molecule in the cardiovascular system. In addition to free H2S, H2S can be oxidized to polysulfide which can be biologically active. Since the impact of H2S on endothelial solute barrier function is not known, we sought to determine whether H2S and its various metabolites affect endothelial permeability. In vitro permeability was evaluated using albumin flux and transendothelial electrical resistance. Different H2S donors were used to examine the effects of exogenous H2S. To evaluate the role of endogenous H2S, mouse aortic endothelial cells (MAECs) were isolated from wild type mice and mice lacking cystathionine γ-lyase (CSE), a predominant source of H2S in endothelial cells. In vivo permeability was evaluated using the Miles assay. We observed that polysulfide donors induced rapid albumin flux across endothelium. Comparatively, free sulfide donors increased permeability only with higher concentrations and at later time points. Increased solute permeability was associated with disruption of endothelial junction proteins claudin 5 and VE-cadherin, along with enhanced actin stress fiber formation. Importantly, sulfide donors that increase permeability elicited a preferential increase in polysulfide levels within endothelium. Similarly, CSE deficient MAECs showed enhanced solute barrier function along with reduced endogenous bound sulfane sulfur. CSE siRNA knockdown also enhanced endothelial junction structures with increased claudin 5 protein expression. In vivo, CSE genetic deficiency significantly blunted VEGF induced hyperpermeability revealing an important role of the enzyme for barrier function. In summary, endothelial solute permeability is critically regulated via exogenous and endogenous sulfide bioavailability with a prominent role of polysulfides.

Self-Immolative Thiocarbamates Provide Access to Triggered H2S Donors and Analyte Replacement Fluorescent Probes.

Hydrogen sulfide (H2S) is an important biological signaling molecule, and chemical tools for H2S delivery and detection have emerged as important investigative methods. Key challenges in these fields include developing donors that are triggered to release H2S in response to stimuli and developing probes that do not irreversibly consume H2S. Here we report a new strategy for H2S donation based on self-immolation of benzyl thiocarbamates to release carbonyl sulfide, which is rapidly converted to H2S by carbonic anhydrase. We leverage this chemistry to develop easily modifiable donors that can be triggered to release H2S. We also demonstrate that this approach can be coupled with common H2S-sensing motifs to generate scaffolds which, upon reaction with H2S, generate a fluorescence response and also release caged H2S, thus addressing challenges of analyte homeostasis in reaction-based probes.

Cystathionine γ-lyase regulates arteriogenesis through NO-dependent monocyte recruitment.

AIMS: Hydrogen sulfide (H2S) is a vasoactive gasotransmitter that is endogenously produced in the vasculature by the enzyme cystathionine γ-lyase (CSE). However, the importance of CSE activity and local H2S generation for ischaemic vascular remodelling remains completely unknown. In this study, we examine the hypothesis that CSE critically regulates ischaemic vascular remodelling involving H2S-dependent mononuclear cell regulation of arteriogenesis. METHODS AND RESULTS: Arteriogenesis including mature vessel density, collateral formation, blood flow, and SPY angiographic blush rate were determined in wild-type (WT) and CSE knockout (KO) mice at different time points following femoral artery ligation (FAL). The role of endogenous H2S in regulation of IL-16 expression and subsequent recruitment of monocytes, and expression of VEGF and bFGF in ischaemic tissues, were determined along with endothelial progenitor cell (CD34/Flk1) formation and function. FAL of WT mice significantly increased CSE activity, expression and endogenous H2S generation in ischaemic tissues, and monocyte infiltration, which was absent in CSE-deficient mice. Treatment of CSE KO mice with the polysulfide donor diallyl trisulfide restored ischaemic vascular remodelling, monocyte infiltration, and cytokine expression. Importantly, exogenous H2S therapy restored nitric oxide (NO) bioavailability in CSE KO mice that was responsible for monocyte recruitment and arteriogenesis. CONCLUSION: Endogenous CSE/H2S regulates ischaemic vascular remodelling mediated during hind limb ischaemia through NO-dependent monocyte recruitment and cytokine induction revealing a previously unknown mechanism of arteriogenesis.

Nitrite anion therapy protects against chronic ischemic tissue injury in db/db diabetic mice in a NO/VEGF-dependent manner.

Nitrite anion has been demonstrated to be a prodrug of nitric oxide (NO) with positive effects on tissue ischemia/reperfusion injury, cytoprotection, and vasodilation. However, effects of nitrite anion therapy for ischemic tissue vascular remodeling during diabetes remain unknown. We examined whether sodium nitrite therapy altered ischemic revascularization in BKS-Lepr(db/db) mice subjected to permanent unilateral femoral artery ligation. Sodium nitrite therapy completely restored ischemic hind limb blood flow compared with nitrate or PBS therapy. Importantly, delayed nitrite therapy 5 days after ischemia restored ischemic limb blood flow in aged diabetic mice. Restoration of blood flow was associated with increases in ischemic tissue angiogenesis activity and cell proliferation. Moreover, nitrite but not nitrate therapy significantly prevented ischemia-mediated tissue necrosis in aged mice. Nitrite therapy significantly increased ischemic tissue vascular endothelial growth factor (VEGF) protein expression that was essential for nitrite-mediated reperfusion of ischemic hind limbs. Nitrite significantly increased ischemic tissue NO bioavailability along with concomitant reduction of superoxide formation. Lastly, nitrite treatment also significantly stimulated hypoxic endothelial cell proliferation and migration in the presence of high glucose in an NO/VEGF-dependent manner. These results demonstrate that nitrite therapy effectively stimulates ischemic tissue vascular remodeling in the setting of metabolic dysfunction that may be clinically useful.