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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., Competing Interests: Declaration of competing interest C.G.K., G.K·K., S.A., and X.S. have a provisional patent on CSE phosphorylation mutants and uses., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Hydrogen sulfide (H 2 S) has emerged as a gaseous signalling molecule with crucial implications for cardiovascular health. H 2 S is involved in many biological functions, including interactions with nitric oxide, activation of molecular signalling cascades, post-translational modifications and redox regulation. Various preclinical and clinical studies have shown that H 2 S and its synthesizing enzymes - cystathionine γ-lyase, cystathionine β-synthase and 3-mercaptosulfotransferase - can protect against cardiovascular pathologies, including arrhythmias, atherosclerosis, heart failure, myocardial infarction and ischaemia-reperfusion injury. The bioavailability of H 2 S and its metabolites, such as hydropersulfides and polysulfides, is substantially reduced in cardiovascular disease and has been associated with single-nucleotide polymorphisms in H 2 S synthesis enzymes. In this Review, we highlight the role of H 2 S, its synthesizing enzymes and metabolites, their roles in the cardiovascular system, and their involvement in cardiovascular disease and associated pathologies. We also discuss the latest clinical findings from the field and outline areas for future study., (© 2022. Springer Nature Limited.)

Sulfides and persulfides/polysulfides (R-S n -R', n > 2; R-S n -H, n > 1) are endogenously produced metabolites that are abundant in mammalian and human cells and tissues. The most typical persulfides that are widely distributed among different organisms include various reactive persulfides-low-molecular-weight thiol compounds such as cysteine hydropersulfide, glutathione hydropersulfide, and glutathione trisulfide as well as protein-bound thiols. These species are generally more redox-active than are other simple thiols and disulfides. Although hydrogen sulfide (H 2 S) has been suggested for years to be a small signaling molecule, it is intimately linked biochemically to persulfides and may actually be more relevant as a marker of functionally active persulfides. Reactive persulfides can act as powerful antioxidants and redox signaling species and are involved in energy metabolism. Recent evidence revealed that cysteinyl-tRNA synthetases (CARSs) act as the principal cysteine persulfide synthases in mammals and contribute significantly to endogenous persulfide/polysulfide production, in addition to being associated with a battery of enzymes including cystathionine β-synthase, cystathionine γ-lyase, and 3-mercaptopyruvate sulfurtransferase, which have been described as H 2 S-producing enzymes. The reactive sulfur metabolites including persulfides/polysulfides derived from CARS2, a mitochondrial isoform of CARS, also mediate not only mitochondrial biogenesis and bioenergetics but also anti-inflammatory and immunomodulatory functions. The physiological roles of persulfides, their biosynthetic pathways, and their pathophysiology in various diseases are not fully understood, however. Developing basic and high precision techniques and methods for the detection, characterization, and quantitation of sulfides and persulfides is therefore of great importance so as to thoroughly understand and clarify the exact functions and roles of these species in cells and in vivo., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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 (H 2 S), a signaling molecule important in vascular homeostasis, as a biomarker of cardiovascular disease. We hypothesize that plasma H 2 S and its metabolites also relate to vascular and cognitive dysfunction in ADRD. We used analytical biochemical methods to measure plasma H 2 S metabolites and MRI to evaluate indicators of microvascular disease in ADRD. Levels of total H 2 S and specific metabolites were increased in ADRD versus controls. Cognition and microvascular disease indices were correlated with H 2 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 H 2 S is dysregulated in dementia, providing a potential biomarker for diagnosis and intervention., (© 2021 The Authors. Alzheimer's & Dementia published by Wiley Periodicals LLC on behalf of Alzheimer's Association.)

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 (H 2 S) are gasotransmitters that regulate key cardiovascular functions., Methods: Blood samples were obtained from 68 COVID-19 patients and 33 controls and NO and H 2 S metabolites were assessed. H 2 S 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 H 2 S 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 H 2 S levels in the patients with COVID-19 infection., Results: Significantly reduced NO and H 2 S 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 H 2 S levels compared to COVID-19 patients who survived. Receiver-operating characteristic analysis of NO and H 2 S 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 H 2 S levels in COVID-19 patients., Conclusion: These observations provide the first insight into the role of NO and H 2 S 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., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

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 (H 2 S), 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 H 2 S in plasma and increases of H 2 S levels in the liver of squirrels during torpor compared to levels during arousal and summer. The in vivo changes of liver H 2 S levels correspond with low activity of the mitochondrial H 2 S oxidizing enzyme sulfide:quinone oxidoreductase (SQR) during torpor. Taken together, these results suggest that during torpor, H 2 S 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, H 2 S 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., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The mammalian brain is highly vulnerable to oxygen deprivation, yet the mechanism underlying the brain's sensitivity to hypoxia is incompletely understood. Hypoxia induces accumulation of hydrogen sulfide, a gas that inhibits mitochondrial respiration. Here, we show that, in mice, rats, and naturally hypoxia-tolerant ground squirrels, the sensitivity of the brain to hypoxia is inversely related to the levels of sulfide:quinone oxidoreductase (SQOR) and the capacity to catabolize sulfide. Silencing SQOR increased the sensitivity of the brain to hypoxia, whereas neuron-specific SQOR expression prevented hypoxia-induced sulfide accumulation, bioenergetic failure, and ischemic brain injury. Excluding SQOR from mitochondria increased sensitivity to hypoxia not only in the brain but also in heart and liver. Pharmacological scavenging of sulfide maintained mitochondrial respiration in hypoxic neurons and made mice resistant to hypoxia. These results illuminate the critical role of sulfide catabolism in energy homeostasis during hypoxia and identify a therapeutic target for ischemic brain injury.

Recent evidence has revealed that exposing cells to exogenous H 2 S or inhibiting cellular H 2 S synthesis can modulate cell cycle checkpoints, DNA damage and repair, and the expression of proteins involved in the maintenance of genomic stability, all suggesting that H 2 S plays an important role in the DNA damage response (DDR). Here we review the role of H 2 S in the DRR and maintenance of genomic stability. Treatment of various cell types with pharmacologic H 2 S donors or cellular H 2 S synthesis inhibitors modulate the G 1 checkpoint, inhibition of DNA synthesis, and cause p21, and p53 induction. Moreover, in some cell models H 2 S exposure induces PARP-1 and g-H2AX foci formation, increases PCNA, CHK2, Ku70, Ku80, and DNA polymerase-d protein expression, and maintains mitochondrial genomic stability. Our group has also revealed that H 2 S bioavailability and the ATR kinase regulate each other with ATR inhibition lowering cellular H 2 S concentrations, whereas intracellular H 2 S concentrations regulate ATR kinase activity via ATR serine 435 phosphorylation. In summary, these findings have many implications for the DDR, for cancer chemotherapy, and fundamental biochemical metabolic pathways involving H 2 S., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

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 (H 2 S), 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 H 2 S 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 H 2 S 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 H 2 S 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 H 2 S 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 H 2 S bioavailability in regulating electrical remodeling and susceptibility to AF., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

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 (H 2 S/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 H 2 S, 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 NO 2 - 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 H 2 S. 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., Competing Interests: Declaration of competing interest C.G.K. has provisional patents regarding nitric oxide/nitrite and hydrogen sulfide chemistry and is a co-founder of Innolyzer LLC. X.S. has intellectual property regarding hydrogen sulfide chemistry., (Copyright © 2020. Published by Elsevier B.V.)

Hydrogen sulfide has emerged as an important gaseous signaling molecule and a regulator of critical biological processes. However, the physiological significance of hydrogen sulfide metabolites such as persulfides, polysulfides, and other reactive sulfur species (RSS) has only recently been appreciated. Emerging evidence suggests that these RSS molecules may have similar or divergent regulatory roles compared with hydrogen sulfide in various biological activities. However, the chemical nature of persulfides and polysulfides is complex and remains poorly understood within cardiovascular and other pathophysiological conditions. Recent reports suggest that RSS can be produced endogenously, with different forms having unique chemical properties and biological implications involving diverse cellular responses such as protein biosynthesis, cell-cell barrier functions, and mitochondrial bioenergetics. Enzymes of the transsulfuration pathway, CBS (cystathionine beta-synthase) and CSE (cystathionine gamma-lyase), may also produce RSS metabolites besides hydrogen sulfide. Moreover, CARSs (cysteinyl-tRNA synthetase) are also able to generate protein persulfides via cysteine persulfide (CysSSH) incorporation into nascently formed polypeptides suggesting a new biologically relevant amino acid. This brief review discusses the biochemical nature and potential roles of RSS, associated oxidative stress redox signaling, and future research opportunities in cardiovascular disease.

Glioblastoma (GBM) has a poor prognosis despite intensive treatment with surgery and chemoradiotherapy. Previous studies using dose-escalated radiotherapy have demonstrated improved survival; however, increased rates of radionecrosis have limited its use. Development of radiosensitizers could improve patient outcome. In the present study, we report the use of sodium sulfide (Na 2 S), a hydrogen sulfide (H 2 S) donor, to selectively kill GBM cells (T98G and U87) while sparing normal human cerebral microvascular endothelial cells (hCMEC/D3). Na 2 S also decreased mitochondrial respiration, increased oxidative stress and induced γH2AX foci and oxidative base damage in GBM cells. Since Na 2 S did not significantly alter T98G capacity to perform non-homologous end-joining or base excision repair, it is possible that GBM cell killing could be attributed to increased damage induction due to enhanced reactive oxygen species production. Interestingly, Na 2 S enhanced mitochondrial respiration, produced a more reducing environment and did not induce high levels of DNA damage in hCMEC/D3. Taken together, this data suggests involvement of mitochondrial respiration in Na 2 S toxicity in GBM cells. The fact that survival of LN-18 GBM cells lacking mitochondrial DNA (ρ 0 ) was not altered by Na 2 S whereas the survival of LN-18 ρ + cells was compromised supports this conclusion. When cells were treated with Na 2 S and photon or proton radiation, GBM cell killing was enhanced, which opens the possibility of H 2 S being a radiosensitizer. Therefore, this study provides the first evidence that H 2 S donors could be used in GBM therapy to potentiate radiation-induced killing., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Hydrogen sulfide (H 2 S) controls numerous physiological responses. To understand its proposed role in metabolic suppression, we measured free H 2 S and bound sulfane sulfur (BSS) in tissues of the freshwater turtle Trachemys scripta elegans , a species undergoing strong metabolic suppression when cold and anoxic. In warm normoxic turtles, free H 2 S 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 H 2 S-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 H 2 S reservoir. Cold acclimation caused significant changes in free and bound H 2 S 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., Competing Interests: Competing interestsThe authors declare no competing or financial interests. C.G.K. has intellectual property regarding sulfide measurement technologies and a commercial interest in Innolyzer Labs, LLC, Shreveport, LA 71103, USA., (© 2019. Published by The Company of Biologists Ltd.)

Ischemic vascular remodeling occurs in response to stenosis or arterial occlusion leading to a change in blood flow and tissue perfusion. Altered blood flow elicits a cascade of molecular and cellular physiological responses leading to vascular remodeling of the macro- and micro-circulation. Although cellular mechanisms of vascular remodeling such as arteriogenesis and angiogenesis have been studied, therapeutic approaches in these areas have had limited success due to the complexity and heterogeneous constellation of molecular signaling events regulating these processes. Understanding central molecular players of vascular remodeling should lead to a deeper understanding of this response and aid in the development of novel therapeutic strategies. Hydrogen sulfide (H 2 S) and nitric oxide (NO) are gaseous signaling molecules that are critically involved in regulating fundamental biochemical and molecular responses necessary for vascular growth and remodeling. This review examines how NO and H 2 S regulate pathophysiological mechanisms of angiogenesis and arteriogenesis, along with important chemical and experimental considerations revealed thus far. The importance of NO and H 2 S bioavailability, their synthesis enzymes and cofactors, and genetic variations associated with cardiovascular risk factors suggest that they serve as pivotal regulators of vascular remodeling responses. © 2019 American Physiological Society. Compr Physiol 9:1213-1247, 2019., (Copyright © 2019 American Physiological Society. All rights reserved.)

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 (H 2 S), an endogenous gasotransmitter, has been found to regulate multiple cellular processes through complex redox reactions under similar cell stress environments. Three enzymes synthesize H 2 S: cystathionine-β-synthase, cystathionine γ-lyase, and 3-mercaptopyruvate sulfurtransferase. Since H 2 S can under some conditions cause DNA damage, we hypothesized that ATR activity may regulate cellular H 2 S concentrations and H 2 S-syntheszing enzymes. Here we show that human colorectal cancer cells carrying biallelic knock-in hypomorphic ATR mutations have lower cellular H 2 S concentrations than do syngeneic ATR wild-type cells, and all three H 2 S-synthesizing enzymes show lower protein expression in the ATR hypomorphic mutant cells. Additionally, ATR serine 428 phosphorylation is altered by H 2 S donor and H 2 S synthesis enzyme inhibition, while the oxidative-stress induced phosphorylation of the ATR-regulated protein CHK1 on serine 345 is increased by H 2 S synthesis enzyme inhibition. Lastly, inhibition of H 2 S 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 H 2 S., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background/aim: Hydrogen sulfide (H 2 S) and the enzymes that synthesize it, cystathionine-b-synthase, cystathionine γ-lyase, and 3-mercaptopyruvate, are increased in different human malignancies. Due to its short half-life, H 2 S concentrations have not been directly measured in a human malignancy. Here we directly measured in vivo H 2 S levels within oral squamous cell carcinoma (OSCC)., Patients and Methods: Punch biopsies of OSCC and benign mucosae from 15 patients were analyzed by HPLC, western blotting, and tissue microarray analyses., Results: H 2 S concentrations were significantly higher in OSCC compared to adjacent benign oral mucosae. Western blot and tissue microarray studies revealed significantly increased cystathionine-b-synthase, cystathionine γ-lyase, and 3-mercaptopyruvate, phopho-Stat3, mitoNEET, hTERT, and MAPK protein levels in OSCC., Conclusion: H 2 S concentrations and the enzymes that synthesize it are significantly increased in OSCC. Here, for the first time H 2 S concentrations within a living human malignancy were measured and compared to adjacent counterpart benign tissue., (Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Insufficient hydrogen sulfide (H 2 S) has been implicated in Type 2 diabetic mellitus (T2DM) and hyperhomocysteinemia (HHcy)-related cardiovascular complications. We investigated the role of H 2 S in T2DM and HHcy-induced endothelial dysfunction in small mesenteric artery (SMA) of db/db mice fed a high methionine (HM) diet. HM diet (8 weeks) induced HHcy in both T2DM db/db mice and non-diabetic db/+ mice (total plasma Hcy: 48.4 and 31.3 µM, respectively), and aggravated the impaired endothelium-derived hyperpolarization factor (EDHF)-induced endothelium-dependent relaxation to acetylcholine (ACh), determined by the presence of eNOS inhibitor N(ω)-nitro-L-arginine methyl ester (L-NAME) and prostacyclin (PGI 2 ) inhibitor indomethacin (INDO), in SMA from db/db mice but not that from db/+ mice. A non-selective Ca 2+ -active potassium channel (K Ca ) opener NS309 rescued T2DM/HHcy-impaired EDHF-mediated vascular relaxation to ACh. EDHF-induced relaxation to ACh was inhibited by a non-selective K Ca blocker TEA and intermediate-conductance K Ca blocker (IK Ca ) Tram-34, but not by small-conductance K Ca (SK Ca ) blocker Apamin. HHcy potentiated the reduction of free sulfide, H 2 S and cystathionine γ-lyase protein, which converts L-cysteine to H 2 S, in SMA of db/db mice. Importantly, a stable H 2 S donor DATS diminished the enhanced O 2 - production in SMAs and lung endothelial cells of T2DM/HHcy mice. Antioxidant PEG-SOD and DATS improved T2DM/HHcy impaired relaxation to ACh. Moreover, HHcy increased hyperglycemia-induced IK Ca tyrosine nitration in human micro-vascular endothelial cells. EDHF-induced vascular relaxation to L-cysteine was not altered, whereas such relaxation to NaHS was potentiated by HHcy in SMA of db/db mice which was abolished by ATP-sensitive potassium channel blocker Glycolamide but not by K Ca blockers., Conclusions: Intermediate HHcy potentiated H 2 S reduction via CSE-downregulation in microvasculature of T2DM mice. H 2 S is justified as an EDHF. Insufficient H 2 S impaired EDHF-induced vascular relaxation via oxidative stress and IK Ca inactivation in T2DM/HHcy mice. H 2 S therapy may be beneficial for prevention and treatment of micro-vascular complications in patients with T2DM and HHcy., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

The chemical biology of thiols (RSH, e.g., cysteine and cysteine-containing proteins/peptides) has been a topic of extreme interest for many decades due to their reported roles in protein structure/folding, redox signaling, metal ligation, cellular protection, and enzymology. While many of the studies on thiol/sulfur biochemistry have focused on thiols, relatively ignored have been hydropersulfides (RSSH) and higher order polysulfur species (RSS n H, RSS n R, n > 1). Recent and provocative work has alluded to the prevalence and likely physiological importance of RSSH and related RSS n H. RSSH of cysteine (Cys-SSH) has been found to be prevalent in mammalian systems along with Cys-SSH-containing proteins. The RSSH functionality has not been examined to the extent of other biologically relevant sulfur derivatives (e.g., sulfenic acids, disulfides, etc.), whose roles in cell signaling are strongly indicated. The recent finding of Cys-SSH biosynthesis and translational incorporation into proteins is an unequivocal indication of its fundamental importance and necessitates a more profound look into the physiology of RSSH. In this Review, we discuss the currently reported chemical biology of RSSH (and related species) as a prelude to discussing their possible physiological roles., (© 2018 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Hydrogen sulfide (H 2 S) 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 H 2 S 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 H 2 S 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 H 2 S. 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 H 2 S bioavailability were not observed in African Americans, although H 2 S bioavailability was significantly lower overall in this ethnic group compared to Caucasians. We also performed SNP analysis of H 2 S 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 H 2 S bioavailability was found to be predictive for cardiovascular disease in Caucasian subjects as determined by receiver operator characteristic analysis. These findings reveal that plasma H 2 S 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., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Aging is associated with replacement of normal kidney parenchyma by fibrosis. Because hydrogen sulfide (H 2 S) ameliorates kidney fibrosis in disease models, we examined its status in the aging kidney. In the first study, we examined kidney cortical H 2 S metabolism and signaling pathways related to synthesis of proteins including matrix proteins in young and old male C57BL/6 mice. In old mice, increase in renal cortical content of matrix protein involved in fibrosis was associated with decreased H 2 S generation and AMPK activity, and activation of insulin receptor (IR)/IRS-2-Akt-mTORC1-mRNA translation signaling axis that can lead to increase in protein synthesis. In the second study, we randomized 18-19 month-old male C57BL/6 mice to receive 30 μmol/L sodium hydrosulfide (NaHS) in drinking water vs. water alone (control) for 5 months. Administration of NaHS increased plasma free sulfide levels. NaHS inhibited the increase in kidney cortical content of matrix proteins involved in fibrosis and ameliorated glomerulosclerosis. NaHS restored AMPK activity and inhibited activation of IR/IRS-2-Akt-mTORC1-mRNA translation axis. NaHS inhibited age-related increase in kidney cortical content of p21, IL-1β, and IL-6, components of the senescence-associated secretory phenotype. NaHS abolished increase in urinary albumin excretion seen in control mice and reduced serum cystatin C levels suggesting improved glomerular clearance function. We conclude that aging-induced changes in the kidney are associated with H 2 S deficiency. Administration of H 2 S ameliorates aging-induced kidney changes probably by inhibiting signaling pathways leading to matrix protein synthesis.

Significance: Hydrogen sulfide (H 2 S) metabolism leads to the formation of oxidized sulfide species, including polysulfide, persulfide, and others. Evidence is emerging that many biological effects of H 2 S may indeed be due to polysulfide and persulfide activation of signaling pathways and reactivity with discrete small molecules. Recent Advances: Exogenous oxidized sulfide species, including polysulfides, are more reactive than H 2 S with a wide range of molecules. Importantly, endogenous polysulfide and persulfide formation has been reported to occur via transsulfuration enzymes, cystathionine γ-lyase (CSE) and cystathionine β-synthase (CBS)., Critical Issues: In light of the recent understanding of oxidized sulfide metabolite formation and reactivity, comparatively few studies have been reported comparing cellular biological and in vivo effects of H 2 S donors versus polysulfide and persulfide donors. Likewise, it is equally unclear when, how, and to what extent persulfide and polysulfide formation occurs in vivo under pathophysiological conditions., Future Directions: Additional studies regarding persulfide and polysulfide formation and molecular reactions are needed in nearly all aspects of biology to better understand how sulfide metabolites contribute to key chemical biology reactions involved in cardiovascular health and immune responses. Antioxid. Redox Signal. 27, 634-653.

Recent reports have revealed that hydrogen sulfide (H 2 S) exerts critical actions to promote cardiovascular homeostasis and health. Thiosulfate is one of the products formed during oxidative H 2 S 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 H 2 S interact during redox signaling remain unanswered. In the present study, we examined the effect of exogenous thiosulfate on hypoxia-induced H 2 S 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 H 2 S 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 O 2 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 H 2 S 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., (Copyright © 2017 the American Physiological Society.)

Hydrogen sulfide (H 2 S) is an important gaseous signaling molecule in the cardiovascular system. In addition to free H 2 S, H 2 S can be oxidized to polysulfide which can be biologically active. Since the impact of H 2 S on endothelial solute barrier function is not known, we sought to determine whether H 2 S and its various metabolites affect endothelial permeability. In vitro permeability was evaluated using albumin flux and transendothelial electrical resistance. Different H 2 S donors were used to examine the effects of exogenous H 2 S. To evaluate the role of endogenous H 2 S, mouse aortic endothelial cells (MAECs) were isolated from wild type mice and mice lacking cystathionine γ-lyase (CSE), a predominant source of H 2 S 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., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

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.

Blockage or restriction of blood flow through conduit arteries results in tissue ischemia downstream of the disturbed area. Local tissues can adapt to this challenge by stimulating vascular remodeling through angiogenesis and arteriogenesis thereby restoring blood perfusion and removal of wastes. Multiple molecular mechanisms of vascular remodeling during ischemia have been identified and extensively studied. However, therapeutic benefits from these findings and insights are limited due to the complexity of various signaling networks and a lack of understanding central metabolic regulators governing these responses. The gasotransmitters NO and H2 S have emerged as master regulators that influence multiple molecular targets necessary for ischemic vascular remodeling. In this review, we discuss how NO and H2 S are individually regulated under ischemia, what their roles are in angiogenesis and arteriogenesis, and how their interaction controls ischemic vascular remodeling., (© 2015 John Wiley & Sons Ltd.)

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., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2015. For permissions please email: journals.permissions@oup.com.)

Nitric oxide (NO) and hydrogen sulfide (H2S) are gasotransmitter molecules important in numerous physiological and pathological processes. Although these molecules were first known as environmental toxicants, it is now evident that that they are intricately involved in diverse cellular functions with impact on numerous physiological and pathogenic processes. NO and H2S share some common characteristics but also have unique chemical properties that suggest potential complementary interactions between the two in affecting cellular biochemistry and metabolism. Central among these is the interactions between NO, H2S, and thiols that constitute new ways to regulate protein function, signaling, and cellular responses. In this review, we discuss fundamental biochemical principals, molecular functions, measurement methods, and the pathophysiological relevance of NO and H2S., (Copyright © 2015 the American Physiological Society.)

We determined the role of endogenous hydrogen sulfide (H2S) in cerebral vasodilation/hyperemia and early BBB disruption following ischemic stroke. A cranial window was prepared over the left frontal, parietal and temporal cortex in mice. Transient focal cerebral Ischemia was induced by directly ligating the middle cerebral artery (MCA) for two hours. Regional vascular response and cerebral blood flow (CBF) during ischemia and reperfusion were measured in real time. Early BBB disruption was assessed by Evans Blue (EB) and sodium fluorescein (Na-F) extravasation at 3 hours of reperfusion. Topical treatment with DL-propargylglycine (PAG, an inhibitor for cystathionine γ-lyase (CSE)) and aspartate (ASP, inhibitor for cysteine aminotransferase/3-mercaptopyruvate sulfurtransferase (CAT/3-MST)), but not O-(Carboxymethyl)hydroxylamine hemihydrochloride (CHH, an inhibitor for cystathionine β-synthase (CBS)), abolished postischemic cerebral vasodilation/hyperemia and prevented EB and Na-F extravasation. CSE knockout (CSE-/-) reduced postischemic cerebral vasodilation/hyperemia but only inhibited Na-F extravasation. An upregulated CBS was found in cerebral cortex of CSE-/- mice. Topical treatment with CHH didn't further alter postischemic cerebral vasodilation/hyperemia, but prevented EB extravasation in CSE-/- mice. In addition, L-cysteine-induced hydrogen sulfide (H2S) production similarly increased in ischemic side cerebral cortex of control and CSE-/- mice. Our findings suggest that endogenous production of H2S by CSE and CAT/3-MST during reperfusion may be involved in postischemic cerebral vasodilation/hyperemia and play an important role in early BBB disruption following transient focal cerebral ischemia.

The gasotransmitter hydrogen sulfide (H2S) is known as an important regulator in several physiological and pathological responses. Among the challenges facing the field is the accurate and reliable measurement of hydrogen sulfide bioavailability. We have reported an approach to discretely measure sulfide and sulfide pools using the monobromobimane (MBB) method coupled with reversed phase high-performance liquid chromatography (RP-HPLC). The method involves the derivatization of sulfide with excess MBB under precise reaction conditions at room temperature to form sulfide dibimane (SDB). The resultant fluorescent SDB is analyzed by RP-HPLC using fluorescence detection with the limit of detection for SDB (2 nM). Care must be taken to avoid conditions that may confound H2S measurement with this method. Overall, RP-HPLC with fluorescence detection of SDB is a useful and powerful tool to measure biological sulfide levels., (© 2015 Elsevier Inc. All rights reserved.)

Nitric oxide (NO) and hydrogen sulfide (H2S) are two major gaseous signaling molecules that regulate diverse physiological functions. Recent publications indicate the regulatory role of H2S on NO metabolism. In this chapter, we discuss the latest findings on H2S-NO interactions through formation of novel chemical derivatives and experimental approaches to study these adducts. This chapter also addresses potential H2S interference on various NO detection techniques, along with precautions for analyzing biological samples from various sources. This information will facilitate critical evaluation and clearer insight into H2S regulation of NO signaling and its influence on various physiological functions., (© 2015 Elsevier Inc. All rights reserved.)

Accurate measurement of hydrogen sulfide bioavailability remains a technical challenge due to numerous issues involving sample processing, detection methods used, and actual biochemical products measured. Our group and others have reported that reverse phase HPLC detection of sulfide dibimane (SDB) product from the reaction of H2S/HS(-) with monobromobimane allows for analytical detection of hydrogen sulfide bioavailability in free and other biochemical forms. However, it remains unclear whether possible interfering contaminants may contribute to HPLC SDB peak readings that may result in inaccurate measurements of bioavailable sulfide. In this study, we critically compared hydrogen sulfide dependent SDB detection using reverse phase HPLC (RP-HPLC) versus quantitative SRM electrospray ionization mass spectrometry (ESI/MS) to obtain greater clarity into the validity of the reverse phase HPLC method for analytical measurement of hydrogen sulfide. Using an LCQ-Deca ion-trap mass spectrometer, SDB was identified by ESI/MS positive ion mode, and quantified by selected reaction monitoring (SRM) using hydrocortisone as an internal standard. Collision induced dissociation (CID) parameters were optimized at MS2 level for SDB and hydrocortisone. ESI/MS detection of SDB standard was found to be a log order more sensitive than RP-HPLC with a lower limit of 0.25 nM. Direct comparison of tissue and plasma SDB levels using RP-HPLC and ESI/MS methods revealed comparable sulfide levels in plasma, aorta, heart, lung and brain. Together, these data confirm the use of SDB as valid indicator of H2S bioavailability and highlights differences between analytical detection methods., (Copyright © 2014. Published by Elsevier Inc.)

During winter hibernation, brown bears (Ursus arctos) lie in dens for half a year without eating while their basal metabolism is largely suppressed. To understand the underlying mechanisms of metabolic depression in hibernation, we measured type and content of blood metabolites of two ubiquitous inhibitors of mitochondrial respiration, hydrogen sulfide (H2S) and nitric oxide (NO), in winter-hibernating and summer-active free-ranging Scandinavian brown bears. We found that levels of sulfide metabolites were overall similar in summer-active and hibernating bears but their composition in the plasma differed significantly, with a decrease in bound sulfane sulfur in hibernation. High levels of unbound free sulfide correlated with high levels of cysteine (Cys) and with low levels of bound sulfane sulfur, indicating that during hibernation H2S, in addition to being formed enzymatically from the substrate Cys, may also be regenerated from its oxidation products, including thiosulfate and polysulfides. In the absence of any dietary intake, this shift in the mode of H2S synthesis would help preserve free Cys for synthesis of glutathione (GSH), a major antioxidant found at high levels in the red blood cells of hibernating bears. In contrast, circulating nitrite and erythrocytic S-nitrosation of glyceraldehyde-3-phosphate dehydrogenase, taken as markers of NO metabolism, did not change appreciably. Our findings reveal that remodeling of H2S metabolism and enhanced intracellular GSH levels are hallmarks of the aerobic metabolic suppression of hibernating bears., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Hydrogen sulfide (H2S) is the most recent endogenous gasotransmitter that has been reported to serve many physiological and pathological functions in different tissues. Studies over the past decade have revealed that H2S can be synthesized through numerous pathways and its bioavailability regulated through its conversion into different biochemical forms. H2S exerts its biological effects in various manners including redox regulation of protein and small molecular weight thiols, polysulfides, thiosulfate/sulfite, iron-sulfur cluster proteins, and anti-oxidant properties that affect multiple cellular and molecular responses. However, precise measurement of H2S bioavailability and its associated biochemical and pathophysiological roles remains less well understood. In this review, we discuss recent understanding of H2S chemical biology, its relationship to tissue pathophysiological responses and possible therapeutic uses., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background: Hydrogen sulfide (H2S) has been implicated in regulating cardiovascular pathophysiology in experimental models. However, there is a paucity of information regarding the levels of H2S in health and cardiovascular disease. In this study we examine the levels of H2S in patients with cardiovascular disease as well as bioavailability of nitric oxide and inflammatory indicators., Methods and Results: Patients over the age of 40 undergoing coronary or peripheral angiography were enrolled in the study. Ankle brachial index (ABI) measurement, measurement of plasma-free H2S and total nitric oxide (NO), thrombospondin-1 (TSP-1), Interleukin-6 (IL-6), and soluble intercellular adhesion molecule-1 (sICAM-1) levels were performed. Patients with either coronary artery disease alone (n = 66), peripheral arterial disease (PAD) alone (n = 13), or any vascular disease (n = 140) had higher plasma-free H2S levels compared to patients without vascular disease (n = 53). Plasma-free H2S did not distinguish between disease in different vascular beds; however, total NO levels were significantly reduced in PAD patients and the ratio of plasma free H2S to NO was significantly greater in patients with PAD. Lastly, plasma IL-6, ICAM-1, and TSP-1 levels did not correlate with H2S or NO bioavailability in either vascular disease condition., Conclusions: Findings reported in this study reveal that plasma-free H2S levels are significantly elevated in vascular disease and identify a novel inverse relationship with NO bioavailability in patients with peripheral arterial disease.

Background: Hydrogen sulfide (H2S) has been shown to induce angiogenesis in in vitro models and to promote vessel growth in the setting of hindlimb ischemia. The goal of the present study was to determine the therapeutic potential of a stable, long-acting H2S donor, diallyl trisulfide, in a model of pressure-overload heart failure and to assess the effects of chronic H2S therapy on myocardial vascular density and angiogenesis., Methods and Results: Transverse aortic constriction was performed in mice (C57BL/6J; 8-10 weeks of age). Mice received either vehicle or diallyl trisulfide (200 µg/kg) starting 24 hours after transverse aortic constriction and were followed up for 12 weeks using echocardiography. H2S therapy with diallyl trisulfide improved left ventricular remodeling and preserved left ventricular function in the setting of transverse aortic constriction. H2S therapy increased the expression of the proangiogenic factor, vascular endothelial cell growth factor, and decreased the angiogenesis inhibitor, angiostatin. Further studies revealed that H2S therapy increased the expression of the proliferation marker, Ki67, as well as increased the phosphorylation of endothelial NO synthase and the bioavailability of NO. Importantly, these changes were associated with an increase in vascular density within the H2S-treated hearts., Conclusions: These results suggest that H2S therapy attenuates left ventricular remodeling and dysfunction in the setting of heart failure by creating a proangiogenic environment for the growth of new vessels.

Hydrogen sulfide (H2S), generated through various endogenous enzymatic and nonenzymatic pathways, is emerging as a regulator of physiological and pathological events throughout the body. Bacteria in the gastrointestinal tract also produce significant amounts of H2S that regulates microflora growth and virulence responses. However, the impact of the microbiota on host global H2S bioavailability and metabolism remains unknown. To address this question, we examined H2S bioavailability in its various forms (free, acid labile, or bound sulfane sulfur), cystathionine γ-lyase (CSE) activity, and cysteine levels in tissues from germ-free versus conventionally housed mice. Free H2S levels were significantly reduced in plasma and gastrointestinal tissues of germ-free mice. Bound sulfane sulfur levels were decreased by 50-80% in germ-free mouse plasma and adipose and lung tissues. Tissue CSE activity was significantly reduced in many organs from germ-free mice, whereas tissue cysteine levels were significantly elevated compared to conventional mice. These data reveal that the microbiota profoundly regulates systemic bioavailability and metabolism of H2S., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Nitric oxide (NO) and hydrogen sulfide (H2S) have emerged as dominant redox regulators of numerous aspects of cellular and physiological functions within several organ systems included cardiovascular, immune and neurological tissues. Recent studies have begun to reveal that these two gaseous molecules may have redundant or overlapping pathophysiological functions often involving similar molecular targets. However, it remains less clear when and how NO and H2S may interact under biological and disease processes. In this graphical review, we discuss the current understanding of NO and H2S interactions and how they may functionally influence each other and what this may mean for biology and mechanisms of disease.

Background: Hydrogen sulfide (H(2)S) therapy is recognized as a modulator of vascular function during tissue ischemia with the notion of potential interactions of nitric oxide (NO) metabolism. However, little is known about specific biochemical mechanisms or the importance of H(2)S activation of NO metabolism during ischemic tissue vascular remodeling. The goal of this study was to determine the effect of H(2)S on NO metabolism during chronic tissue ischemia and subsequent effects on ischemic vascular remodeling responses., Methods and Results: The unilateral, permanent femoral artery ligation model of hind-limb ischemia was performed in C57BL/6J wild-type and endothelial NO synthase-knockout mice to evaluate exogenous H(2)S effects on NO bioavailability and ischemic revascularization. We found that H(2)S selectively restored chronic ischemic tissue function and viability by enhancing NO production involving both endothelial NO synthase and nitrite reduction mechanisms. Importantly, H(2)S increased ischemic tissue xanthine oxidase activity, hind-limb blood flow, and angiogenesis, which were blunted by the xanthine oxidase inhibitor febuxostat. H(2)S treatment increased ischemic tissue and endothelial cell hypoxia-inducible factor-1α expression and activity and vascular endothelial growth factor protein expression and function in a NO-dependent manner that was required for ischemic vascular remodeling., Conclusions: These data demonstrate that H(2)S differentially regulates NO metabolism during chronic tissue ischemia, highlighting novel biochemical pathways to increase NO bioavailability for ischemic vascular remodeling.

Hydrogen sulfide (H₂S) is a ubiquitous gaseous signaling molecule that plays a vital role in numerous cellular functions and has become the focus of many research endeavors, including pharmacotherapeutic manipulation. Among the challenges facing the field is the accurate measurement of biologically active H₂S. We have recently reported that the typically used methylene blue method and its associated results are invalid and do not measure bona fide H₂S. The complexity of analytical H₂S measurement reflects the fact that hydrogen sulfide is a volatile gas and exists in the body in various forms, including a free form, an acid-labile pool, and bound as sulfane sulfur. Here we describe a new protocol to discretely measure specific H₂S pools using the monobromobimane method coupled with RP-HPLC. This new protocol involves selective liberation, trapping, and derivatization of H₂S. Acid-labile H₂S is released by incubating the sample in an acidic solution (pH 2.6) of 100 mM phosphate buffer with 0.1mM diethylenetriaminepentaacetic acid (DTPA), in an enclosed system to contain volatilized H₂S. Volatilized H₂S is then trapped in 100 mM Tris-HCl (pH 9.5, 0.1 mM DTPA) and then reacted with excess monobromobimane. In a separate aliquot, the contribution of the bound sulfane sulfur pool was measured by incubating the sample with 1 mM TCEP (tris(2-carboxyethyl)phosphine hydrochloride), a reducing agent, to reduce disulfide bonds, in 100 mM phosphate buffer (pH 2.6, 0.1 mM DTPA), and H₂S measurement was performed in a manner analogous to the one described above. The acid-labile pool was determined by subtracting the free hydrogen sulfide value from the value obtained by the acid-liberation protocol. The bound sulfane sulfur pool was determined by subtracting the H₂S measurement from the acid-liberation protocol alone compared to that of TCEP plus acidic conditions. In summary, our new method allows very sensitive and accurate measurement of the three primary biological pools of H₂S, including free, acid-labile, and bound sulfane sulfur, in various biological specimens., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The gasotransmitter hydrogen sulfide is known to regulate multiple cellular functions during normal and pathophysiological states. However, a paucity of concise information exists regarding quantitative amounts of hydrogen sulfide involved in physiological and pathological responses. This is primarily due to disagreement among various methods employed to measure free hydrogen sulfide. In this article, we describe a very sensitive method of measuring the presence of H₂S in plasma down to nanomolar levels, using monobromobimane (MBB). The current standard assay using methylene blue provides erroneous results that do not actually measure H₂S. The method presented herein involves derivatization of sulfide with excess MBB in 100 mM Tris-HCl buffer (pH 9.5, 0.1 mM DTPA) for 30 min in 1% oxygen at room temperature. The fluorescent product sulfide-dibimane (SDB) is analyzed by RP-HPLC using an eclipse XDB-C18 (4.6 × 250 mm) column with gradient elution by 0.1% (v/v) trifluoroacetic acid in acetonitrile. The limit of detection for sulfide-dibimane is 2 nM and the SDB product is very stable over time, allowing batch storage and analysis. In summary, our MBB method is suitable for sensitive quantitative measurement of free hydrogen sulfide in multiple biological samples such as plasma, tissue and cell culture lysates, or media., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Rationale: The recent emergence of hydrogen sulfide (H(2)S) as a potent cardioprotective signaling molecule necessitates the elucidation of its cytoprotective mechanisms., Objective: The present study evaluated potential mechanisms of H(2)S-mediated cardioprotection using an in vivo model of pharmacological preconditioning., Methods and Results: H(2)S (100 microg/kg) or vehicle was administered to mice via an intravenous injection 24 hours before myocardial ischemia. Treated and untreated mice were then subjected to 45 minutes of myocardial ischemia followed by reperfusion for up to 24 hours, during which time the extent of myocardial infarction was evaluated, circulating troponin I levels were measured, and the degree of oxidative stress was evaluated. In separate studies, myocardial tissue was collected from treated and untreated mice during the early (30 minutes and 2 hours) and late (24 hours) preconditioning periods to evaluate potential cellular targets of H(2)S. Initial studies revealed that H(2)S provided profound protection against ischemic injury as evidenced by significant decreases in infarct size, circulating troponin I levels, and oxidative stress. During the early preconditioning period, H(2)S increased the nuclear localization of Nrf2, a transcription factor that regulates the gene expression of a number of antioxidants and increased the phosphorylation of protein kinase Cepsilon and STAT-3. During the late preconditioning period, H(2)S increased the expression of antioxidants (heme oxygenase-1 and thioredoxin 1), increased the expression of heat shock protein 90, heat shock protein 70, Bcl-2, Bcl-xL, and cyclooxygenase-2 and also inactivated the proapoptogen Bad., Conclusions: These results reveal that the cardioprotective effects of H(2)S are mediated in large part by a combination of antioxidant and antiapoptotic signaling.