Physiological roles of hydrogen sulfide in mammalian cells, tissues, and organs.

Over the last two decades, hydrogen sulfide (H2S) has emerged as an endogenous regulator of a broad range of physiological functions. H2S belongs to the class of molecules known as gasotransmitters, which typically include nitric oxide (NO) and carbon monoxide (CO). Three enzymes are recognized as endogenous sources of H2S in various cells and tissues: cystathionine γ-lyase (CSE), cystathionine ß-synthase (CBS), and 3-mercaptopyruvate sulfurtransferase (3-MST). The present article reviews the regulation of these enzymes as well as the pathways of their enzymatic and nonenzymatic degradation and elimination. The multiple interactions of H2S with other labile endogenous molecules (e.g., NO) and reactive oxygen species are also outlined. Next, the various biological targets and signaling pathways are outlined, with special reference to H2S or oxidative posttranscriptional modification (persulfidation or sulfhydration) of proteins and the effect of H2S on various channels and intracellular second messenger pathways, the regulation of gene transcription and translation, and the regulation of cellular bioenergetics and metabolism. The pharmacological and molecular tools currently available to study H2S physiology are also reviewed, including their utility and limitations. In subsequent sections, the role of H2S in the regulation of various physiological and cellular functions is reviewed, including the regulation of membrane potential, endo- and exocytosis, regulation of various cell organelles (endoplasmic reticulum, Golgi, mitochondria), regulation of cell movement, cell cycle, cell differentiation, and physiological aspects of regulated cell death. Next, the physiological roles of H2S in various cell types and organ systems are overviewed, including the role of H2S in red blood cells, immune cells, the central and peripheral nervous systems (with focus on neuronal transmission, learning, and memory formation), and regulation of vascular function (including angiogenesis as well as its specialized roles in the cerebrovascular, renal, and pulmonary vascular beds) and the role of H2S in the regulation of special senses, vision, hearing, taste and smell, and pain-sensing. Finally, the roles of H2S in the regulation of various organ functions (lung, heart, liver, kidney, urogenital organs, reproductive system, bone and cartilage, skeletal muscle, and endocrine organs) are presented, with a focus on physiology (including physiological aging) but also extending to some common pathophysiological conditions. From these data, a wide array of significant roles of H2S in the physiological regulation of all organ functions emerges and the characteristic bell-shaped biphasic effects of H2S are highlighted. In addition, key pathophysiological aspects, debated areas, and future research and translational areas are identified.

Hydrogen Sulfide Modulates Endothelial-Mesenchymal Transition in Heart Failure.

BACKGROUND: Hydrogen sulfide is a critical endogenous signaling molecule that exerts protective effects in the setting of heart failure. Cystathionine γ-lyase (CSE), 1 of 3 hydrogen-sulfide-producing enzyme, is predominantly localized in the vascular endothelium. The interaction between the endothelial CSE-hydrogen sulfide axis and endothelial-mesenchymal transition, an important pathological process contributing to the formation of fibrosis, has yet to be investigated. METHODS: Endothelial-cell-specific CSE knockout and Endothelial cell-CSE overexpressing mice were subjected to transverse aortic constriction to induce heart failure with reduced ejection fraction. Cardiac function, vascular reactivity, and treadmill exercise capacity were measured to determine the severity of heart failure. Histological and gene expression analyses were performed to investigate changes in cardiac fibrosis and the activation of endothelial-mesenchymal transition. RESULTS: Endothelial-cell-specific CSE knockout mice exhibited increased endothelial-mesenchymal transition and reduced nitric oxide bioavailability in the myocardium, which was associated with increased cardiac fibrosis, impaired cardiac and vascular function, and worsened exercise performance. In contrast, genetic overexpression of CSE in endothelial cells led to increased myocardial nitric oxide, decreased endothelial-mesenchymal transition and cardiac fibrosis, preserved cardiac and endothelial function, and improved exercise capacity. CONCLUSIONS: Our data demonstrate that endothelial CSE modulates endothelial-mesenchymal transition and ameliorate the severity of pressure-overload-induced heart failure, in part, through nitric oxide-related mechanisms. These data further suggest that endothelium-derived hydrogen sulfide is a potential therapeutic for the treatment of heart failure with reduced ejection fraction.

Disrupted Binding of Cystathionine γ-Lyase to p53 Promotes Endothelial Senescence.

BACKGROUND: Advanced age is unequivocally linked to the development of cardiovascular disease; however, the mechanisms resulting in reduced endothelial cell regeneration remain poorly understood. Here, we investigated novel mechanisms involved in endothelial cell senescence that impact endothelial cell transcription and vascular repair after injury. METHODS: Native endothelial cells were isolated from young (20±3.4 years) and aged (80±2.3 years) individuals and subjected to molecular analyses to assess global transcriptional and metabolic changes. In vitro studies were conducted using primary human and murine endothelial cells. A murine aortic re-endothelialization model was used to examine endothelial cell regenerative capacity in vivo. RESULTS: RNA sequencing of native endothelial cells revealed that aging resulted in p53-mediated reprogramming to express senescence-associated genes and suppress glycolysis. Reduced glucose uptake and ATP contributed to attenuated assembly of the telomerase complex, which was required for endothelial cell proliferation. Enhanced p53 activity in aging was linked to its acetylation on K120 due to enhanced activity of the acetyltransferase MOZ (monocytic leukemic zinc finger). Mechanistically, p53 acetylation and translocation were, at least partially, attributed to the loss of the vasoprotective enzyme, CSE (cystathionine γ-lyase). CSE physically anchored p53 in the cytosol to prevent its nuclear translocation and CSE absence inhibited AKT (Protein kinase B)-mediated MOZ phosphorylation, which in turn increased MOZ activity and subsequently p53 acetylation. In mice, the endothelial cell-specific deletion of CSE activated p53, induced premature endothelial senescence, and arrested vascular repair after injury. In contrast, the adeno-associated virus 9-mediated re-expression of an active CSE mutant retained p53 in the cytosol, maintained endothelial glucose metabolism and proliferation, and prevented endothelial cell senescence. Adenoviral overexpression of CSE in native endothelial cells from aged individuals maintained low p53 activity and reactivated telomerase to revert endothelial cell senescence. CONCLUSIONS: Aging-associated impairment of vascular repair is partly determined by the vasoprotective enzyme CSE.

Host cystathionine-γ lyase derived hydrogen sulfide protects against Pseudomonas aeruginosa sepsis.

Hydrogen sulfide (H2S) has recently been recognized as a novel gaseous transmitter with several anti-inflammatory properties. The role of host- derived H2S in infections by Pseudomonas aeruginosa was investigated in clinical and mouse models. H2S concentrations and survival was assessed in septic patients with lung infection. Animal experiments using a model of severe systemic multidrug-resistant P. aeruginosa infection were performed using mice with a constitutive knock-out of cystathionine-γ lyase (Cse) gene (Cse-/-) and wild-type mice with a physiological expression (Cse+/+). Experiments were repeated in mice after a) treatment with cyclophosphamide; b) bone marrow transplantation (BMT) from a Cse+/+ donor; c) treatment with H2S synthesis inhibitor aminooxyacetic acid (ΑΟΑΑ) or propargylglycine (PAG) and d) H2S donor sodium thiosulfate (STS) or GYY3147. Bacterial loads and myeloperoxidase activity were measured in tissue samples. The expression of quorum sensing genes (QS) was determined in vivo and in vitro. Cytokine concentration was measured in serum and incubated splenocytes. Patients survivors at day 28 had significantly higher serum H2S compared to non-survivors. A cut- off point of 5.3 µΜ discriminated survivors with sensitivity 92.3%. Mortality after 28 days was 30.9% and 93.7% in patients with H2S higher and less than 5.3 µΜ (p = 7 x 10-6). In mice expression of Cse and application of STS afforded protection against infection with multidrug-resistant P. aeruginosa. Cyclophosphamide pretreatment eliminated the survival benefit of Cse+/+ mice, whereas BMT increased the survival of Cse-/- mice. Cse-/- mice had increased pathogen loads compared to Cse+/+ mice. Phagocytic activity of leukocytes from Cse-/- mice was reduced but was restored after H2S supplementation. An H2S dependent down- regulation of quorum sensing genes of P.aeruginosa could be demonstrated in vivo and in vitro. Endogenous H2S is a potential independent parameter correlating with the outcome of P. aeruginosa. H2S provides resistance to infection by MDR bacterial pathogens.

Mapping the Endothelial Cell S-Sulfhydrome Highlights the Crucial Role of Integrin Sulfhydration in Vascular Function.

BACKGROUND: In vascular endothelial cells, cysteine metabolism by the cystathionine γ lyase (CSE), generates hydrogen sulfide-related sulfane sulfur compounds (H2Sn), that exert their biological actions via cysteine S-sulfhydration of target proteins. This study set out to map the "S-sulfhydrome" (ie, the spectrum of proteins targeted by H2Sn) in human endothelial cells. METHODS: Liquid chromatography with tandem mass spectrometry was used to identify S-sulfhydrated cysteines in endothelial cell proteins and ß3 integrin intraprotein disulfide bond rearrangement. Functional studies included endothelial cell adhesion, shear stress-induced cell alignment, blood pressure measurements, and flow-induced vasodilatation in endothelial cell-specific CSE knockout mice and in a small collective of patients with endothelial dysfunction. RESULTS: Three paired sample sets were compared: (1) native human endothelial cells isolated from plaque-free mesenteric arteries (CSE activity high) and plaque-containing carotid arteries (CSE activity low); (2) cultured human endothelial cells kept under static conditions or exposed to fluid shear stress to decrease CSE expression; and (3) cultured endothelial cells exposed to shear stress to decrease CSE expression and treated with solvent or the slow-releasing H2Sn donor, SG1002. The endothelial cell "S-sulfhydrome" consisted of 3446 individual cysteine residues in 1591 proteins. The most altered family of proteins were the integrins and focusing on ß3 integrin in detail we found that S-sulfhydration affected intraprotein disulfide bond formation and was required for the maintenance of an extended-open conformation of the ß leg. ß3 integrin S-sulfhydration was required for endothelial cell mechanotransduction in vitro as well as flow-induced dilatation in murine mesenteric arteries. In cultured cells, the loss of S-sulfhydration impaired interactions between ß3 integrin and Gα13 (guanine nucleotide-binding protein subunit α 13), resulting in the constitutive activation of RhoA (ras homolog family member A) and impaired flow-induced endothelial cell realignment. In humans with atherosclerosis, endothelial function correlated with low H2Sn generation, impaired flow-induced dilatation, and failure to detect ß3 integrin S-sulfhydration, all of which were rescued after the administration of an H2Sn supplement. CONCLUSIONS: Vascular disease is associated with marked changes in the S-sulfhydration of endothelial cell proteins involved in mediating responses to flow. Short-term H2Sn supplementation improved vascular reactivity in humans highlighting the potential of interfering with this pathway to treat vascular disease.

The role of mitochondrial reactive oxygen species, NO and H2 S in ischaemia/reperfusion injury and cardioprotection.

Redox signalling in mitochondria plays an important role in myocardial ischaemia/reperfusion (I/R) injury and in cardioprotection. Reactive oxygen and nitrogen species (ROS/RNS) modify cellular structures and functions by means of covalent changes in proteins including among others S-nitros(yl)ation by nitric oxide (NO) and its derivatives, and S-sulphydration by hydrogen sulphide (H2 S). Many enzymes are involved in the mitochondrial formation and handling of ROS, NO and H2 S under physiological and pathological conditions. In particular, the balance between formation and removal of reactive species is impaired during I/R favouring their accumulation. Therefore, various interventions aimed at decreasing mitochondrial ROS accumulation have been developed and have shown cardioprotective effects in experimental settings. However, ROS, NO and H2 S play also a role in endogenous cardioprotection, as in the case of ischaemic pre-conditioning, so that preventing their increase might hamper self-defence mechanisms. The aim of the present review was to provide a critical analysis of formation and role of reactive species, NO and H2 S in mitochondria, with a special emphasis on mechanisms of injury and protection that determine the fate of hearts subjected to I/R. The elucidation of the signalling pathways of ROS, NO and H2 S is likely to reveal novel molecular targets for cardioprotection that could be modulated by pharmacological agents to prevent I/R injury.

Cystathionine γ Lyase Sulfhydrates the RNA Binding Protein Human Antigen R to Preserve Endothelial Cell Function and Delay Atherogenesis.

BACKGROUND: Hydrogen sulfide (H2S), generated by cystathionine γ lyase (CSE), is an important endogenous regulator of vascular function. The aim of the present study was to investigate the control and consequences of CSE activity in endothelial cells under physiological and proatherogenic conditions. METHODS: Endothelial cell CSE knockout mice were generated, and lung endothelial cells were studied in vitro (gene expression, protein sulfhydration, and monocyte adhesion). Mice were crossed onto the apolipoprotein E-deficient background, and atherogenesis (partial carotid artery ligation) was monitored over 21 days. CSE expression, H2S bioavailability, and amino acid profiling were also performed with human material. RESULTS: The endothelial cell-specific deletion of CSE selectively increased the expression of CD62E and elevated monocyte adherence in the absence of an inflammatory stimulus. Mechanistically, CD62E mRNA was more stable in endothelial cells from CSE-deficient mice, an effect attributed to the attenuated sulfhydration and dimerization of the RNA-binding protein human antigen R. CSE expression was upregulated in mice after partial carotid artery ligation and in atheromas from human subjects. Despite the increase in CSE protein, circulating and intraplaque H2S levels were reduced, a phenomenon that could be attributed to the serine phosphorylation (on Ser377) and inhibition of the enzyme, most likely resulting from increased interleukin-1ß. Consistent with the loss of H2S, human antigen R sulfhydration was attenuated in atherosclerosis and resulted in the stabilization of human antigen R-target mRNAs, for example, CD62E and cathepsin S, both of which are linked to endothelial cell activation and atherosclerosis. The deletion of CSE from endothelial cells was associated with the accelerated development of endothelial dysfunction and atherosclerosis, effects that were reversed on treatment with a polysulfide donor. Finally, in mice and humans, plasma levels of the CSE substrate l-cystathionine negatively correlated with vascular reactivity and H2S levels, indicating its potential use as a biomarker for vascular disease. CONCLUSIONS: The constitutive S-sulfhydration of human antigen R (on Cys13) by CSE-derived H2S prevents its homodimerization and activity, which attenuates the expression of target proteins such as CD62E and cathepsin S. However, as a consequence of vascular inflammation, the beneficial actions of CSE-derived H2S are lost owing to the phosphorylation and inhibition of the enzyme.

Hydrogen sulfide: An endogenous regulator of the immune system.

Hydrogen sulfide (H2S) is now recognized as an endogenous signaling gasotransmitter in mammals. It is produced by mammalian cells and tissues by various enzymes - predominantly cystathionine ß-synthase (CBS), cystathionine γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST) - but part of the H2S is produced by the intestinal microbiota (colonic H2S-producing bacteria). Here we summarize the available information on the production and functional role of H2S in the various cell types typically associated with innate immunity (neutrophils, macrophages, dendritic cells, natural killer cells, mast cells, basophils, eosinophils) and adaptive immunity (T and B lymphocytes) under normal conditions and as it relates to the development of various inflammatory and immune diseases. Special attention is paid to the physiological and the pathophysiological aspects of the oral cavity and the colon, where the immune cells and the parenchymal cells are exposed to a special "H2S environment" due to bacterial H2S production. H2S has many cellular and molecular targets. Immune cells are "surrounded" by a "cloud" of H2S, as a result of endogenous H2S production and exogenous production from the surrounding parenchymal cells, which, in turn, importantly regulates their viability and function. Downregulation of endogenous H2S producing enzymes in various diseases, or genetic defects in H2S biosynthetic enzyme systems either lead to the development of spontaneous autoimmune disease or accelerate the onset and worsen the severity of various immune-mediated diseases (e.g. autoimmune rheumatoid arthritis or asthma). Low, regulated amounts of H2S, when therapeutically delivered by small molecule donors, improve the function of various immune cells, and protect them against dysfunction induced by various noxious stimuli (e.g. reactive oxygen species or oxidized LDL). These effects of H2S contribute to the maintenance of immune functions, can stimulate antimicrobial defenses and can exert anti-inflammatory therapeutic effects in various diseases.

International Union of Basic and Clinical Pharmacology. CII: Pharmacological Modulation of H2S Levels: H2S Donors and H2S Biosynthesis Inhibitors.

Over the last decade, hydrogen sulfide (H2S) has emerged as an important endogenous gasotransmitter in mammalian cells and tissues. Similar to the previously characterized gasotransmitters nitric oxide and carbon monoxide, H2S is produced by various enzymatic reactions and regulates a host of physiologic and pathophysiological processes in various cells and tissues. H2S levels are decreased in a number of conditions (e.g., diabetes mellitus, ischemia, and aging) and are increased in other states (e.g., inflammation, critical illness, and cancer). Over the last decades, multiple approaches have been identified for the therapeutic exploitation of H2S, either based on H2S donation or inhibition of H2S biosynthesis. H2S donation can be achieved through the inhalation of H2S gas and/or the parenteral or enteral administration of so-called fast-releasing H2S donors (salts of H2S such as NaHS and Na2S) or slow-releasing H2S donors (GYY4137 being the prototypical compound used in hundreds of studies in vitro and in vivo). Recent work also identifies various donors with regulated H2S release profiles, including oxidant-triggered donors, pH-dependent donors, esterase-activated donors, and organelle-targeted (e.g., mitochondrial) compounds. There are also approaches where existing, clinically approved drugs of various classes (e.g., nonsteroidal anti-inflammatories) are coupled with H2S-donating groups (the most advanced compound in clinical trials is ATB-346, an H2S-donating derivative of the non-steroidal anti-inflammatory compound naproxen). For pharmacological inhibition of H2S synthesis, there are now several small molecule compounds targeting each of the three H2S-producing enzymes cystathionine-ß-synthase (CBS), cystathionine-γ-lyase, and 3-mercaptopyruvate sulfurtransferase. Although many of these compounds have their limitations (potency, selectivity), these molecules, especially in combination with genetic approaches, can be instrumental for the delineation of the biologic processes involving endogenous H2S production. Moreover, some of these compounds (e.g., cell-permeable prodrugs of the CBS inhibitor aminooxyacetate, or benserazide, a potentially repurposable CBS inhibitor) may serve as starting points for future clinical translation. The present article overviews the currently known H2S donors and H2S biosynthesis inhibitors, delineates their mode of action, and offers examples for their biologic effects and potential therapeutic utility.

Endothelial-Tumor Cell Interaction in Brain and CNS Malignancies.

Glioblastoma and other brain or CNS malignancies (like neuroblastoma and medulloblastoma) are difficult to treat and are characterized by excessive vascularization that favors further tumor growth. Since the mean overall survival of these types of diseases is low, the finding of new therapeutic approaches is imperative. In this review, we discuss the importance of the interaction between the endothelium and the tumor cells in brain and CNS malignancies. The different mechanisms of formation of new vessels that supply the tumor with nutrients are discussed. We also describe how the tumor cells (TC) alter the endothelial cell (EC) physiology in a way that favors tumorigenesis. In particular, mechanisms of EC-TC interaction are described such as (a) communication using secreted growth factors (i.e., VEGF, TGF-ß), (b) intercellular communication through gap junctions (i.e., Cx43), and (c) indirect interaction via intermediate cell types (pericytes, astrocytes, neurons, and immune cells). At the signaling level, we outline the role of important mediators, like the gasotransmitter nitric oxide and different types of reactive oxygen species and the systems producing them. Finally, we briefly discuss the current antiangiogenic therapies used against brain and CNS tumors and the potential of new pharmacological interventions that target the EC-TC interaction.

Screening of Heteroaromatic Scaffolds against Cystathionine Beta-Synthase Enables Identification of Substituted Pyrazolo[3,4-c]Pyridines as Potent and Selective Orthosteric Inhibitors.

Cystathionine ß-synthase (CBS) is a key enzyme in the production of the signaling molecule hydrogen sulfide, deregulation of which is known to contribute to a range of serious pathological states. Involvement of hydrogen sulfide in pathways of paramount importance for cellular homeostasis renders CBS a promising drug target. An in-house focused library of heteroaromatic compounds was screened for CBS modulators by the methylene blue assay and a pyrazolopyridine derivative with a promising CBS inhibitory potential was discovered. The compound activity was readily comparable to the most potent CBS inhibitor currently known, aminoacetic acid, while a promising specificity over the related cystathionine γ-lyase was identified. To rule out any possibility that the inhibitor may bind the enzyme regulatory domain due to its high structural similarity with cofactor s-adenosylmethionine, differential scanning fluorimetry was employed. A sub-scaffold search guided follow-up screening of related compounds, providing preliminary structure-activity relationships with respect to requisites for efficient CBS inhibition by this group of heterocycles. Subsequently, a hypothesis regarding the exact binding mode of the inhibitor was devised on the basis of the available structure-activity relationships (SAR) and a deep neural networks analysis and further supported by induced-fit docking calculations.

Hydrogen sulfide inhibits Kir2 and Kir3 channels by decreasing sensitivity to the phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2).

Inwardly rectifying potassium (Kir) channels establish and regulate the resting membrane potential of excitable cells in the heart, brain, and other peripheral tissues. Phosphatidylinositol 4,5-bisphosphate (PIP2) is a key direct activator of ion channels, including Kir channels. The gasotransmitter carbon monoxide has been shown to regulate Kir channel activity by altering channel-PIP2 interactions. Here, we tested in two cellular models the effects and mechanism of action of another gasotransmitter, hydrogen sulfide (H2S), thought to play a key role in cellular responses under ischemic conditions. Direct administration of sodium hydrogen sulfide as an exogenous H2S source and expression of cystathionine γ-lyase, a key enzyme that produces endogenous H2S in specific brain tissues, resulted in comparable current inhibition of several Kir2 and Kir3 channels. This effect resulted from changes in channel-gating kinetics rather than in conductance or cell-surface localization. The extent of H2S regulation depended on the strength of the channel-PIP2 interactions. H2S regulation was attenuated when channel-PIP2 interactions were strengthened and was increased when channel-PIP2 interactions were weakened by depleting PIP2 levels. These H2S effects required specific cytoplasmic cysteine residues in Kir3.2 channels. Mutation of these residues abolished H2S inhibition, and reintroduction of specific cysteine residues back into the background of the cytoplasmic cysteine-lacking mutant rescued H2S inhibition. Molecular dynamics simulation experiments provided mechanistic insights into how potential sulfhydration of specific cysteine residues could lead to changes in channel-PIP2 interactions and channel gating.

Comparison of the effects of e-cigarette vapor with cigarette smoke on lung function and inflammation in mice.

Electronic cigarettes (e-cigs) are advertised as a less harmful nicotine delivery system or as a new smoking cessation tool. We aimed to assess the in vivo effects of e-cig vapor in the lung and to compare them to those of cigarette smoke (CS). We exposed C57BL/6 mice for either 3 days or 4 wk to ambient air, CS, or e-cig vapor containing 1) propylene glycol/vegetable glycerol (PG:VG-Sol; 1:1), 2) PG:VG with nicotine (G:VG-N), or 3) PG:VG with nicotine and flavor (PG:VG-N+F) and determined oxidative stress, inflammation, and pulmonary mechanics. E-cig vapors, especially PG:VG-N+F, increased bronchoalveolar lavage fluid (BALF) cellularity, Muc5ac production, as well as BALF and lung oxidative stress markers at least comparably and in many cases more than CS. BALF protein content at both time points studied was only elevated in the PG:VG-N+F group. After 3 days, PG:VG-Sol altered tissue elasticity, static compliance, and airway resistance, whereas after 4 wk CS was the only treatment adversely affecting these parameters. Airway hyperresponsiveness in response to methacholine was increased similarly in the CS and PG:VG-N+F groups. Our findings suggest that exposure to e-cig vapor can trigger inflammatory responses and adversely affect respiratory system mechanics. In many cases, the added flavor in e-cigs exacerbated the detrimental effects of e-cig vapor. We conclude that both e-cig vaping and conventional cigarette smoking negatively impact lung biology.

Mercaptopyruvate acts as endogenous vasodilator independently of 3-mercaptopyruvate sulfurtransferase activity.

Hydrogen sulfide (H2S) is produced by the action of cystathionine-ß-synthase (CBS), cystathionine-γ-lyase (CSE) or 3-mercaptopyruvate sulfurtransferase (3-MST). 3-MST converts 3-mercaptopyruvate (MPT) to H2S and pyruvate. H2S is recognized as an endogenous gaseous mediator with multiple regulatory roles in mammalian cells and organisms. In the present study we demonstrate that MPT, the endogenous substrate of 3-MST, acts also as endogenous H2S donor. Colorimetric, amperometric and fluorescence based assays demonstrated that MPT releases H2S in vitro in an enzyme-independent manner. A functional study was performed on aortic rings harvested from C57BL/6 (WT) or 3-MST-knockout (3-MST-/-) mice with and without endothelium. MPT relaxed mouse aortic rings in endothelium-independent manner and at the same extent in both WT and 3-MST-/- mice. N5-(1-Iminoethyl)-l-ornithine dihydrochloride (L-NIO, an inhibitor of endothelial nitric oxide synthase) as well as 1H-[1,2,4]oxadiazolo [4,3-a]quinoxalin-1-one (ODQ, a soluble guanylyl cyclase inhibitor) did not affect MPT relaxant action. Conversely, hemoglobin (as H2S scavenger), as well as glybenclamide (an ATP-dependent potassium channel blocker) markedly reduced MPT-induced relaxation. The functional data clearly confirmed a non enzymatic vascular effect of MPT. In conclusion, MPT acts also as an endogenous H2S donor and not only as 3-MST substrate. MPT could, thus, be further investigated as a means to increase H2S in conditions where H2S bioavailability is reduced such as hypertension, coronary artery disease, diabetes or urogenital tract disease.

Reduced adipose tissue H2S in obesity.

Hydrogen sulfide (H2S) is an endogenously produced signaling molecule synthesized by cystathionine γ-lyase (CSE), cystathionine ß-synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (3-MST). Given that H2S exerts significant effects on bioenergetics and metabolism, the goal of the current study was to determine the expression of H2S-producing enzymes in adipose tissues in models of obesity and metabolic disruption. Mice fed a western diet expressed lower mRNA levels of all three enzymes in epididymal fat (EWAT), while only CSE and 3-MST were reduced in brown adipose tissue (BAT). At the protein level 3-MST was reduced in all fat depots studied. Using db/db mice, a genetic model of obesity, we found that CSE, CBS and 3-MST mRNA were reduced in white fat, while only CSE was reduced in BAT. CBS and CSE protein levels were suppressed in all three fat depots. In a model of age-related weight gain, no reduction in the mRNA of any of the enzymes was noted. Smaller amounts of 3-MST protein were found in EWAT, while both CSE and 3-MST were reduced in BAT. Tissue levels of H2S were lower in WAT in HFD mice; both WAT and BAT contained lower H2S amounts in db/db animals. Taken together, our data suggest that obesity is associated with a decreased expression of H2S-synthesizing enzymes and reduced H2S levels in adipose tissues of mice. We propose that the reduction in H2S may contribute to the metabolic response associated with obesity. Further work is needed to determine whether restoring H2S levels may have a beneficial effect on obesity-associated metabolic alterations.

Vascular biology of hydrogen sulfide.

Hydrogen sulfide (H2S) is a ubiquitous signaling molecule with important functions in many mammalian organs and systems. Observations in the 1990s ascribed physiological actions to H2S in the nervous system, proposing that this gasotransmitter acts as a neuromodulator. Soon after that, the vasodilating properties of H2S were demonstrated. In the past decade, H2S was shown to exert a multitude of physiological effects in the vessel wall. H2S is produced by vascular cells and exhibits antioxidant, antiapoptotic, anti-inflammatory, and vasoactive properties. In this concise review, we have focused on the impact of H2S on vascular structure and function with an emphasis on angiogenesis, vascular tone, vascular permeability and atherosclerosis. H2S reduces arterial blood pressure, limits atheromatous plaque formation, and promotes vascularization of ischemic tissues. Although the beneficial properties of H2S are well established, mechanistic insights into the molecular pathways implicated in disease prevention and treatment remain largely unexplored. Unraveling the targets and downstream effectors of H2S in the vessel wall in the context of disease will aid in translation of preclinical observations. In addition, acute regulation of H2S production is still poorly understood and additional work delineating the pathways regulating the enzymes that produce H2S will allow pharmacological manipulation of this pathway. As the field continues to grow, we expect that H2S-related compounds will find their way into clinical trials for diseases affecting the blood vessels.

Hydrogen Sulfide Preserves Endothelial Nitric Oxide Synthase Function by Inhibiting Proline-Rich Kinase 2: Implications for Cardiomyocyte Survival and Cardioprotection.

Hydrogen sulfide (H2S) exhibits beneficial effects in the cardiovascular system, many of which depend on nitric oxide (NO). Proline-rich tyrosine kinase 2 (PYK2), a redox-sensitive tyrosine kinase, directly phosphorylates and inhibits endothelial NO synthase (eNOS). We investigated the ability of H2S to relieve PYK2-mediated eNOS inhibition and evaluated the importance of the H2S/PYK2/eNOS axis on cardiomyocyte injury in vitro and in vivo. Exposure of H9c2 cardiomyocytes to H2O2 or pharmacologic inhibition of H2S production increased PYK2 (Y402) and eNOS (Y656) phosphorylation. These effects were blocked by treatment with Na2S or by overexpression of cystathionine γ-lyase (CSE). In addition, PYK2 overexpression reduced eNOS activity in a H2S-reversible manner. The viability of cardiomyocytes exposed to Η2Ο2 was reduced and declined further after the inhibition of H2S production. PYK2 downregulation, l-cysteine supplementation, or CSE overexpression alleviated the effects of H2O2 on H9c2 cardiomyocyte survival. Moreover, H2S promoted PYK2 sulfhydration and inhibited its activity. In vivo, H2S administration reduced reactive oxygen species levels, as well as PYK2 (Y402) and eNOS (Y656) phosphorylation. Pharmacologic blockade of PYK2 or inhibition of PYK2 activation by Na2S reduced myocardial infarct size in mice. Coadministration of a PYK2 inhibitor and Na2S did not result in additive effects on infarct size. We conclude that H2S relieves the inhibitory effect of PYK2 on eNOS, allowing the latter to produce greater amounts of NO, thereby affording cardioprotection. Our results unravel the existence of a novel H2S-NO interaction and identify PYK2 as a crucial target for the protective effects of H2S under conditions of oxidative stress.

Cystathionine-beta-synthase inhibition for colon cancer: Enhancement of the efficacy of aminooxyacetic acid via the prodrug approach.

Colon cancer cells contain high levels of cystathionine-beta-synthase (CBS). Its product, hydrogen sulfide (H2S) promotes the growth and proliferation of colorectal tumor cells. In order to improve the antitumor efficacy of the prototypical CBS inhibitor aminooxyacetic acid (AOAA), we have designed and synthesized YD0171, a methyl ester derivative of AOAA. The antiproliferative effect of YD0171 exceeded the antiproliferative potency of AOAA in HCT116 human colon cancer cells. The esterase inhibitor paraoxon prevented the cellular inhibition of CBS activity by YD0171. YD0171 suppressed mitochondrial respiration and glycolytic function and induced G0/G1 arrest, but did not induce tumor cell apoptosis or necrosis. Metabolomic analysis in HCT116 cells showed that YD0171 affects multiple pathways of cell metabolism. The efficacy of YD0171 as an inhibitor of tumor growth was also tested in nude mice bearing subcutaneous HCT116 cancer cell xenografts. Animals were treated via subcutaneous injection of vehicle, AOAA (1, 3 or 9 mg/kg/day) or YD0171 (0.1, 0.5 or 1 mg/kg/day) for 3 weeks. Tumor growth was significantly reduced by 9 mg/kg/day AOAA, but not at the lower doses. YD0171 was more potent: tumor volume was significantly inhibited at 0.5 and 1 mg/kg/day. Thus, the in vivo efficacy of YD0171 is 9-times higher than that of AOAA. YD0171 (1 mg/kg/day) attenuated tumor growth and metastasis formation in the intracecal HCT116 tumor model. YD0171 (3 mg/kg/day) also reduced tumor growth in patient-derived tumor xenograft (PDTX) bearing athymic mice. YD0171 (3 mg/kg/day) induced the regression of established HCT116 tumors in vivo. A 5-day safety study in mice demonstrated that YD0171 at 20 mg/kg/day (given in two divided doses) does not increase plasma markers of organ injury, nor does it induce histological alterations in the liver or kidney. YD0171 caused a slight elevation in plasma homocysteine levels. In conclusion, the prodrug approach improves the pharmacological profile of AOAA; YD0171 represents a prototype for CBS inhibitory anticancer prodrugs. By targeting colorectal cancer bioenergetics, an emerging important hallmark of cancer, the approach exemplified herein may offer direct translational opportunities.

Exposure to cigarette smoke abrogates the beneficial effect of ischemic postconditioning.

Cigarette smoking is one of the risk factors for coronary artery disease. Although conditioning decreases infarct size in hearts from healthy animals, comorbidities may render it ineffective. We investigated the effects of cigarette smoke (CS) exposure on intracellular myocardial signaling, infarct size after ischemia-reperfusion, and the potential interference with ischemic conditioning. Exposure of mice to CS increased blood pressure, caused cardiac hypertrophy, and upregulated the nitric oxide synthatse (NOS)/soluble guanylate cyclase (sGC)/cGMP pathway. To test the effect of CS exposure on the endogenous cardioprotective mechanisms, mice were subjected to regional myocardial ischemia and reperfusion with no further intervention or application of preconditioning (PreC) or postconditioning (PostC). Exposure to CS did not increase the infarction compared with the room air (RA)-exposed group. PreC was beneficial for both CS and RA vs. nonconditioned animals. PostC was effective only in RA animals, while the infarct size-limiting effect was not preserved in the CS group. Differences in oxidative stress markers, Akt, and endothelial NOS phosphorylation and cGMP levels were observed between RA and CS groups subjected to PostC. In conclusion, exposure to CS does not per se increase infarct size. The beneficial effect of ischemic PreC is preserved in mice exposed to CS, as it does not affect the cardioprotective signaling; in contrast, PostC fails to protect CS-exposed mice due to impaired activation of the Akt/eNOS/cGMP axis that occurs in parallel to enhanced oxidative stress.

Cardioprotection by H2S Donors: Nitric Oxide-Dependent and ­Independent Mechanisms.

Hydrogen sulfide (H2S) is a signaling molecule with protective effects in the cardiovascular system. To harness the therapeutic potential of H2S, a number of donors have been developed. The present study compares the cardioprotective actions of representative H2S donors from different classes and studies their mechanisms of action in myocardial injury in vitro and in vivo. Exposure of cardiomyocytes to H2O2 led to significant cytotoxicity, which was inhibited by sodium sulfide (Na2S), thiovaline (TV), GYY4137 [morpholin-4-ium 4 methoxyphenyl(morpholino) phosphinodithioate], and AP39 [(10-oxo-10-(4-(3-thioxo-3H-1,2-dithiol5yl)phenoxy)decyl) triphenylphospho-nium bromide]. Inhibition of nitric oxide (NO) synthesis prevented the cytoprotective effects of Na2S and TV, but not GYY4137 and AP39, against H2O2-induced cardiomyocyte injury. Mice subjected to left anterior descending coronary ligation were protected from ischemia-reperfusion injury by the H2S donors tested. Inhibition of nitric oxide synthase (NOS) in vivo blocked only the beneficial effect of Na2S. Moreover, Na2S, but not AP39, administration enhanced the phosphorylation of endothelial NOS and vasodilator-associated phosphoprotein. Both Na2S and AP39 reduced infarct size in mice lacking cyclophilin-D (CypD), a modulator of the mitochondrial permeability transition pore (PTP). Nevertheless, only AP39 displayed a direct effect on mitochondria by increasing the mitochondrial Ca(2+) retention capacity, which is evidence of decreased propensity to undergo permeability transition. We conclude that although all the H2S donors we tested limited infarct size, the pathways involved were not conserved. Na2S had no direct effects on PTP opening, and its action was nitric oxide dependent. In contrast, the cardioprotection exhibited by AP39 could result from a direct inhibitory effect on PTP acting at a site different than CypD.