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Background Mesenchymal stem cells (MSC) hold great promise for treating cardiovascular disease. Recently, we genetically modified MSCs with high mobility group box 1 (HMGB1), and these cells demonstrated high mobility by efficient migrating and homing to target neointima. The possible mechanism was investigated in the current study. Methods Rat MSCs were transfected with lentivirus containing HMGB1 cDNA to yield MSC-H cell line stably overexpressing HMGB1. The MSC-C cells which were transfected with empty lentivirus served as negative control, and the differentially expressed genes were analyzed by microarray. The cell mobility was determined by transwell migration assay. Intracellular free calcium and the expression of Cav3.2 T-type calcium channel (CACNA1H) were assayed to analyze activity of CACNA1H-mediated calcium influx. H2S production and γ-cystathionase expression were examined to assess the activity of γ-cystathionase/H2S signaling. The interaction of HMGB1 with γ-cystathionase in MSC-H cells was analyzed by co-immunoprecipitation. Luciferase reporter assay was performed to determine whether the promoter activity of γ-cystathionase was regulated by interaction of β-catenin and TCF/LEF binding site. Intercellular cAMP, PKA activity, phosphorylation of β-catenin, and GSK3β were investigated to reveal cAMP/PKA mediated β-catenin activation. Result Microarray analysis revealed that differentially expressed genes were enriched in cAMP signaling and calcium signaling. CACNA1H was upregulated to increase intracellular free calcium and MSC-H cell migration. Blockage of CACNA1H by ABT-639 significantly reduced intracellular free calcium and cell migration. The γ-cystathionase/H2S signaling was responsible for CACNA1H activation. H2S production was increased with high expression of γ-cystathionase in MSC-H cells, which was blocked by γ-cystathionase inhibitor DL-propargylglycine. Upregulation of γ-cystathionase was not attributed to interaction with HMGB1 overexpressed in MSC-H cells although γ-cystathionase was suggested to co-immunoprecipitate with oxidized HMGB1. Bioinformatics analysis identified a conserved TCF/LEF binding site in the promoter of γ-cystathionase gene. Luciferase reporter assay confirmed that the promoter had positive response to β-catenin which was activated in MSC-H cells. Finally, cAMP/PKA was activated to phosphorylate β-catenin at Ser657 and GSK3β, enabling persisting activation of Wnt/β-catenin signaling in MSC-H cells. Conclusion Our study revealed that modification of MSCs with HMGB1 promoted CACNA1H-mediated calcium influx via PKA/β-catenin/γ-cystathionase pathway. This was a plausible mechanism for high mobility of MSC-H cell line.

Hydrogen sulphide (H2S), a newly identified gasotransmitter, can be endogenously produced by cystathionine gamma-lyase (CSE) in the cardiovascular system. This study investigated the role of the CSE/H2S system on lipid overload-induced lipotoxicity and cardiac senescence. Lipid overload in rat cardiomyocyte cells (H9C2) promoted intracellular accumulation of lipid, oxidative stress, mitochondrial dysfunctions, lipid peroxidation and inhibited cell viability, all of which could be reversed by exogenously applied H2S. Further data revealed that H2S protected H9C2 cells from lipid overload-induced senescence by altering the expressions of lipid metabolism-related genes and inhibiting cellular acetyl-CoA and global protein acetylation. Enhancement of protein acetylation abolished the protective role of H2S on cardiac senescence. In vivo, knockout of the CSE gene strengthened cardiac lipid accumulation, protein acetylation, and cellular ageing in high fat diet-fed mice. Taken together, the CSE/H2S system is capable of maintaining lipid homeostasis and cellular senescence in heart cells under lipid overload.

Purpose S-propargyl-cysteine (SPRC), an excellent endogenous hydrogen sulfide (H2S) donor, could elevate H2S levels via the cystathionine γ-lyase (CSE)/H2S pathway both in vitro and in vivo. However, the immediate release of H2S in vivo and daily administration of SPRC potentially limited its clinical use. Methods To solve the fore-mentioned problem, in this study, the dendritic mesoporous silica nanoparticles (DMSN) was firstly prepared, and a sustained H2S delivery system consisted of SPRC and DMSN (SPRC@DMSN) was then constructed. Their release profiles, both in vitro and in vivo, were investigated, and their therapeutical effect toward adjuvant-induced arthritis (AIA) rats was also studied. Results The spherical morphology of DMSN could be observed under scanning Electron Microscope (SEM), and the transmission electron microscope (TEM) images showed a central-radiational pore channel structure of DMSN. DMSN showed excellent SPRC loading capacity and attaining a sustained releasing ability than SPRC both in vitro and in vivo, and the prolonged SPRC releasing could further promote the release of H2S in a sustained manner through CSE/H2S pathway both in vitro and in vivo. Importantly, the SPRC@DMSN showed promising anti-inflammation effect against AIA in rats was also observed. Conclusions A sustained H2S releasing donor consisting of SPRC and DMSN was constructed in this study, and this sustained H2S releasing donor might be of good use for the treatment of AIA.

Triple-negative breast cancer (TNBC) is a high-risk subtype of breast cancer with high capacity for metastasis and lacking of therapeutic targets. Our previous studies indicated that cystathionine γ-lyase (CSE) may be a new target related to the recurrence or metastasis of TNBC. Downregulation of CSE could inhibit the growth and metastasis of TNBC. The purpose of this study was to investigate the activity of the novel CSE inhibitor I194496 against TNBC in vivo and in vitro. The anticancer activity of I194496 in vitro were detected by MTS, EdU, and transwell assays. Methylene blue assay was used to determine the H2S level. Western blot was performed to analyze the expression of related pathway proteins. Xenograft tumors in nude mice were used to analyze the anticancer activity of I194496 in vivo. I194496 exerted potent inhibitory effects than l-propargylglycine (PAG, an existing CSE inhibitor) on human TNBC cells and possessed lower toxicity in normal breast epithelial Hs578Bst cells. I194496 reduced the activity and expression of CSE protein and the release of H2S in human TNBC cells. Meanwhile, the protein levels of PI3K, Akt, phospho (p)-Akt, Ras, Raf, p-ERK, p-Anxa2, STAT3, p-STAT3, VEGF, FAK, and Paxillin were decreased in human TNBC cells administrated with I194496. Furthermore, I194496 showed more stronger inhibitory effects on human TNBC xenograft tumors in nude mice. I194496 could inhibit the growth of human TNBC cells via the dual targeting PI3K/Akt and Ras/Raf/ERK pathway and suppress the metastasis of human TNBC cells via down-regulating Anxa2/STAT3 and VEGF/FAK/Paxillin signaling pathways. CSE inhibitor I194496 might become a novel and potential agent in the treatment of TNBC.

Background: In vascular endothelial cells, cysteine metabolism by the cystathionine γ lyase (CSE), generates hydrogen sulfide–related sulfane sulfur compounds (H 2 S n ), 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 H 2 S n ) in human endothelial cells. Methods: Liquid chromatography with tandem mass spectrometry was used to identify S -sulfhydrated cysteines in endothelial cell proteins and β3 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 H 2 S n 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 β3 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 β leg. β3 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 β3 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 H 2 S n generation, impaired flow-induced dilatation, and failure to detect β3 integrin S -sulfhydration, all of which were rescued after the administration of an H 2 S n 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 H 2 S n supplementation improved vascular reactivity in humans highlighting the potential of interfering with this pathway to treat vascular disease.

The present study was performed to investigate whether H2S could restore the diurnal variation in cardiac function of aging mice and explore the potential mechanisms. We found that ejection fraction (EF) and fractional shortening (FS) in 3-month-old mice exhibited diurnal variations over a 24-hour period. However, the diurnal variations were disrupted in 18-month-old mice, and there was a decline in EF and FS. In addition, the plasma malondialdehyde (MDA) levels were increased, and H2S concentrations and superoxide dismutase (SOD) activities were decreased in 18-month-old mice. Then, CSE KO mice were used to determine if there was a relationship between endogenous H2S and diurnal variations in EF and FS. There was no difference in 12-hour averaged EF and FS between dark and light periods in CSE KO mice accompanying increased MDA levels and decreased SOD activities in plasma, indicating that deficiency of endogenous H2S blunted diurnal variations of cardiac function. To determine whether oxidative stress disrupted the diurnal variations in cardiac function, D-galactose-induced subacute aging mice were employed. After 3-month D-gal treatment, both 12-hour averaged EF and FS in dark or light periods were decreased; meanwhile, there was no difference in 12-hour averaged EF and FS between dark and light periods. After 3-month NaHS treatment in the D-gal group, the plasma MDA levels were decreased and SOD activities were increased. The EF and FS were lower during the 12-hour light period than those during the 12-hour dark period which was fit to sine curves in the D-gal+NaHS group. Identical findings were also observed in 18-month-old mice. In conclusion, our studies revealed that the disrupted diurnal variation in cardiac function was associated with increased oxidative stress and decreased H2S levels in aging mice. H2S could restore the diurnal variation in cardiac function of aging mice by reducing oxidative stress.

The development and utilization of inorganic material biosynthesis have evolved into single macromolecular systems. A putative cystathionine γ-lyase of bacteria Stenotrophomonas maltophilia (smCSE) is a newly identified biomolecule that enables the synthesis of nanomaterials. Due to the lack of structural information, the mechanism of smCSE biosynthesis remains unclear. Herein, we obtain two atomic-resolution smCSE-form X-ray structures and confirm that the conformational changes of Tyr108 and Lys206 within the enzyme active sites are critical for the protein-driven synthesis of metal sulfide quantum dots (QDs). The structural stability of tetramer and the specificity of surface amino acids are the basis for smCSE to synthesize quantum dots. The size of QD products can be regulated by predesigned amino acids and the morphology can be controlled through proteolytic treatments. The growth rate is enhanced by the stabilization of a flexible loop in the active site, as shown by the X-ray structure of the engineered protein which fused with a dodecapeptide. We further prove that the smCSE-driven route can be applied to the general synthesis of other metal sulfide nanoparticles. These results provide a better understanding of the mechanism of QD biosynthesis and a new perspective on the control of this biosynthesis by protein modification.

Hydrogen sulfide (H2S) is a cytoprotective redox-active metabolite that signals through protein persulfidation (R-SSnH). Despite the known importance of persulfidation, tissue-specific persulfidome profiles and their associated functions are not well characterized, specifically under conditions and interventions known to modulate H2S production. We hypothesize that dietary restriction (DR), which increases lifespan and can boost H2S production, expands tissue-specific persulfidomes. Here, we find protein persulfidation enriched in liver, kidney, muscle, and brain but decreased in heart of young and aged male mice under two forms of DR, with DR promoting persulfidation in numerous metabolic and aging-related pathways. Mice lacking cystathionine γ-lyase (CGL) have overall decreased tissue protein persulfidation numbers and fail to functionally augment persulfidomes in response to DR, predominantly in kidney, muscle, and brain. Here, we define tissue- and CGL-dependent persulfidomes and how diet transforms their makeup, underscoring the breadth for DR and H2S to impact biological processes and organismal health., Dietary restriction (DR) can increase protein persulfidation but the tissue specificity of this process is not well understood. Here, the authors compare organ-specific protein persulfidomes in young and aged mice under DR, and show that DR-dependent persulfidome changes depend on cystathionine γ-lyase.

An elevated plasma homocysteine level is an independent risk factor for cardiovascular diseases, neurological disorders, and pregnancy complications. We recently demonstrated partial lactation failure in cystathionine γ-lyase-deficient (Cth-/-) dam mice and their defective oxytocin responses in peripheral tissues: uterine (ex vivo) and mammary gland (in vivo). We reasoned that elevated levels of circulatory homocysteine in Cth-/- dam mice counteract with oxytocin-dependent milk ejection from the mammary gland. Based on our observation that those mice displayed normal maternal behaviors against their pups and adult Cth-/- male mice exhibited normal social behaviors against adult wild-type female mice, both of which are regulated by oxytocin in the central nervous system (CNS), we conducted the present study to investigate the amino acid profiles, including total homocysteine, in both blood and cerebrospinal fluid (CSF) of wild-type and Cth-/- female mice before pregnancy and at day 1 of lactation (L1). Serum levels of total homocysteine in wild-type and Cth-/- L1 dam mice were 9.44 and 188 µmol/L, respectively, whereas their CSF levels were below 0.21 (limit of quantification) and 3.62 µmol/L, respectively. Their CSF/serum level ratio was the lowest (1/51.9) among all 20 proteinogenic amino acids, sulfur-containing amino acids, and citrulline/ornithine in Cth-/- mice. Therefore, we hypothesize that the blood-brain barrier protects the CNS from high levels of circulatory homocysteine in Cth-/- dam mice, thereby conferring normal oxytocin-dependent maternal behaviors.

Hydrogen sulfide (H2 S), which has been identified as the third gaseous signaling molecule after nitric oxide (NO) and carbon monoxide (CO), plays an important role in maintaining homeostasis in the cardiovascular system. Endogenous H2 S is produced mainly by three endogenous enzymes: cystathionine β-synthase, cystathionine γ-lyase, and 3-mercaptopyruvate sulfur transferase. Numerous studies have shown that H2 S has a significant protective role in myocardial ischemia. The mechanisms by which H2 S affords cardioprotection include the antifibrotic and antiapoptotic effects, regulation of ion channels, protection of mitochondria, reduction of oxidative stress and inflammatory response, regulation of microRNA expression, and promotion of angiogenesis. Amplification of NO- and CO-mediated signaling through crosstalk between H2 S, NO, and CO may also contribute to the cardioprotective effect. Exogenous H2 S donors are expected to become effective drugs for the treatment of cardiovascular diseases. This review article focuses on the protective mechanisms and potential therapeutic applications of H2 S in myocardial ischemia.

Context Dysregulated immune hemostasis occurs in unexplained recurrent spontaneous abortion (URSA). Synthesized by cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), hydrogen sulfide (H2S) promotes regulatory T-cell differentiation and regulates immune hemostasis; yet, its role in URSA is elusive. Objective To determine if H2S plays a role in early pregnancy and if dysregulated H2S signaling results in recurrent spontaneous abortion. Design First trimester placenta villi and decidua were collected from normal and URSA pregnancies. Protein expression was examined by immunohistochemistry and immunoblotting. Human trophoblast HTR8/SVneo and JEG3 cells were treated with H2S donors; HTR8/SVneo cells were transfected with CBS ribonucleic acid interference (RNAi) or complementary deoxyribonucleic acid. Cell migration and invasion were determined by transwell assays; trophoblast transcriptomes were determined by RNA sequencing (RNA-seq). Wild-type, CBS-deficient, and CBA/J × DBA/2 mice were treated with CBS and CSE inhibitors or H2S donors to determine the role of H2S in early pregnancy in vivo. Results CBS and CSE proteins showed cell-specific expressions, but only CBS decreased in the villous cytotrophoblast in URSA versus normal participants. H2S donors promoted migration and invasion and MMP-2 and VEGF expression in human placenta trophoblast cells that contain SV40 viral deoxyribonucleic acid sequences (HTR8/SVneo) and human placenta trophoblast cells (JEG3 cells), similar to forced CBS expression in HTR8/SVneo cells. The CBS-responsive transcriptomes in HTR8/SVneo cells contained differentially regulated genes (ie, interleukin-1 receptor and prostaglandin-endoperoxide synthase 2) that are associated with nuclear factor-κB-mediated inflammatory response. In vivo, dysregulated CBS/H2S signaling significantly increased embryonic resorption and decidual T-helper 1/T-helper 2 imbalance in mice, which was partially rescued by H2S donors. Conclusion CBS/H2S signaling maintains early pregnancy, possibly via regulating maternal-fetal interface immune hemostasis, offering opportunities for H2S-based immunotherapies for URSA.

Background Acute pancreatitis (AP), an inflammatory condition of pancreas, destructs the exocrine cells by releasing various pro-inflammatory cytokines that activates the stellate cells. However, the underlying molecular mechanism remains unclear. The present study investigated the role of retinoblastoma (Rb), hydrogen sulphide and nuclear factor-κB (NF-κB) in the regulation of exocrine cell proliferation under inflammatory condition. Methods The randomly grouped male swiss mice were administered with 6 consecutive hourly i.p injections of caerulein to induce AP. Palbociclib (PD) (25 mg/kg body weight), a CDK4/6 inhibitor, was administered 1 h after the first cerulein injection intraperitoneally to block the RB pathway by inhibiting the activity of the CDK4/6 complexes and DL propargylglycine (PAG) which blocks the endogenous H2S production. Results Pharmacological inhibition of CDK4/6 and H2S significantly improved pancreas and lung histopathological changes, decreased serum amylase level, both lung and pancreas myeloperoxidase (MPO) activity, TNFα expression and elevated IL10 expression. Furthermore, inhibition of RB pathway reduced cerulein-induced H2S level by reducing the expression of cystathionine gamma lyase (CSE) and NF-κB activation in pancreas and lungs. Also, blocking the RB signalling reduced the α-SMA expression in pancreas preventing the risk for pancreatic fibrosis. Whereas administration of H2S inhibitor PAG resulted in a decrease in CDK4/6-Rb expression in cerulein-induced AP. Conclusion These results reveal a novel link between H2S/RB/NF-κB pathways, in AP and provide insight into possible mechanism that can be targeted in prevention of inflammation to cancer development.

Among amniotes, reptiles and mammals are differently adapted to terrestrial life. It is generally appreciated that terrestrialization required adaptive changes of vertebrate metabolism, particularly in the mode of nitrogen excretion. However, the current paradigm is that metabolic adaptation to life on land did not involve synthesis of enzymatic pathways de novo, but rather repurposing of existing ones. Here, by comparing the inventory of pyridoxal 5'-phosphate-dependent enzymes in different amniotes, we identify in silico a pathway for sulfur metabolism present in chick embryos but not in mammals. Cysteine lyase contains haem and pyridoxal 5'-phosphate co-factors and converts cysteine and sulfite into cysteic acid and hydrogen sulfide, respectively. A specific cysteic acid decarboxylase produces taurine, while hydrogen sulfide is recycled into cysteine by cystathionine beta-synthase. This reaction sequence enables the formation of sulfonated amino acids during embryo development in the egg at no cost of reduced sulfur. The pathway originated around 300 million years ago in a proto-reptile by cystathionine beta-synthase duplication, cysteine lyase neofunctionalization and cysteic acid decarboxylase co-option. Our findings indicate that adaptation to terrestrial life involved innovations in metabolic pathways, and reveal the molecular mechanisms by which such innovations arose in amniote evolution. Invasion of land required changes of vertebrate metabolism. Here, the authors report a pathway for sulfur metabolism present in chick embryos but not in mammals, which originated around 300 million years ago in a proto-reptile.

The phytohormone abscisic acid (ABA) plays pivotal roles in triggering stomatal closure and facilitating adaptation of plants to drought stress. Hydrogen sulfide (H2S), a small signaling gas molecule, is involved in ABA-dependent stomatal closure. However, how H2S regulates ABA signaling remains largely unclear. Here, we show that ABA induces the production of H2S catalyzed by L-CYSTEINE DESULFHYDRASE1 (DES1) in guard cells, and H2S in turn positively regulates ABA signaling through persulfidation of Open Stomata 1 (OST1)/SNF1-RELATED PROTEIN KINASE2.6 (SnRK2.6). Two cysteine (Cys) sites, Cys131 and Cys137, which are exposed on the surface of SnRK2.6 and close to the activation loop, were identified to be persulfidated, which promotes the activity of SnRK2.6 and its interaction with ABA response element-binding factor 2 (ABF2), a transcription factor acting downstream of ABA signaling. When Cys131, Cys137, or both residues in SnRK2.6 were substituted with serine (S), H2S-induced SnRK2.6 activity and SnRK2.6–ABF2 interaction were partially (SnRK2.6C131S and SnRK2.6C137S) or completely (SnRK2.6C131SC137S) compromised. Introduction of SnRK2.6C131S, SnRK2.6C137S, or SnRK2.6C131SC137S into the ost1-3 mutant could not rescue the mutant phenotype: less sensitivity to ABA- and H2S-induced stomatal closure and Ca2+ influx as well as increased water loss and decreased drought tolerance. Taken together, our study reveals a novel post-translational regulatory mechanism of ABA signaling whereby H2S persulfidates SnRK2.6 to promote ABA signaling and ABA-induced stomatal closure.

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.

Hydrogen sulfide (H(2)S), a plant gasotransmitter, functions in the plant response to cadmium (Cd) stress, implying a role for cysteine desulfhydrase in producing H(2)S in this process. Whether d-CYSTEINE DESULFHYDRASE (DCD) acts in the plant Cd response remains to be identified, and if it does, how DCD is regulated in this process is also unknown. Here, we report that DCD-mediated H(2)S production enhances plant Cd tolerance in Arabidopsis (Arabidopsis thaliana). When subjected to Cd stress, a dcd mutant accumulated more Cd and reactive oxygen species and showed increased Cd sensitivity, whereas transgenic lines overexpressing DCD had decreased Cd and reactive oxygen species levels and were more tolerant to Cd stress compared with wild-type plants. Furthermore, the expression of DCD was stimulated by Cd stress, and this up-regulation was mediated by a Cd-induced transcription factor, WRKY13, which bound to the DCD promoter. Consistently, the higher Cd sensitivity of the wrky13-3 mutant was rescued by the overexpression of DCD. Together, our results demonstrate that Cd-induced WRKY13 activates DCD expression to increase the production of H(2)S, leading to higher Cd tolerance in plants.

Significance Tuberculosis (TB) is responsible for millions of deaths each year and several billion people are latently infected with Mycobacterium tuberculosis (Mtb). Mtb modulates host factors, such as endogenous gaseous signalling molecules, to persist in humans for decades. H2S has diverse biological functions, including modulation of immunity and cellular respiration. However, the role of H2S in TB is unclear. We found that mice deficient in H2S production are more resistant to Mtb infection than WT mice. Upon infection, Mtb increases host H2S, which suppresses central carbon metabolism and increases inflammation. Distribution of H2S-producing enzymes in human TB lungs showed that H2S is produced at the site of infection. These findings identify glycolysis and H2S-producing enzymes as targets for TB host-directed therapies., The ubiquitous gasotransmitter hydrogen sulfide (H2S) has been recognized to play a crucial role in human health. Using cystathionine γ-lyase (CSE)-deficient mice, we demonstrate an unexpected role of H2S in Mycobacterium tuberculosis (Mtb) pathogenesis. We showed that Mtb-infected CSE−/− mice survive longer than WT mice, and support reduced pathology and lower bacterial burdens in the lung, spleen, and liver. Similarly, in vitro Mtb infection of macrophages resulted in reduced colony forming units in CSE−/− cells. Chemical complementation of infected WT and CSE−/− macrophages using the slow H2S releaser GYY3147 and the CSE inhibitor DL-propargylglycine demonstrated that H2S is the effector molecule regulating Mtb survival in macrophages. Furthermore, we demonstrate that CSE promotes an excessive innate immune response, suppresses the adaptive immune response, and reduces circulating IL-1β, IL-6, TNF-α, and IFN-γ levels in response to Mtb infection. Notably, Mtb infected CSE−/− macrophages show increased flux through glycolysis and the pentose phosphate pathway, thereby establishing a critical link between H2S and central metabolism. Our data suggest that excessive H2S produced by the infected WT mice reduce HIF-1α levels, thereby suppressing glycolysis and production of IL-1β, IL-6, and IL-12, and increasing bacterial burden. Clinical relevance was demonstrated by the spatial distribution of H2S-producing enzymes in human necrotic, nonnecrotic, and cavitary pulmonary tuberculosis (TB) lesions. In summary, CSE exacerbates TB pathogenesis by altering immunometabolism in mice and inhibiting CSE or modulating glycolysis are potential targets for host-directed TB control.

Transforming growth factor-β1 (TGF-β1) occurs at high levels at damage sites of vascular endothelial cell layers and regulates the functions of vascular endothelial cells. Reactive sulfur species (RSS), such as cysteine persulfide, glutathione persulfide, and hydrogen persulfide, are cytoprotective factors against electrophiles such as reactive oxygen species and heavy metals. Previously, we reported that sodium trisulfide, a sulfane sulfur donor, promotes vascular endothelial cell proliferation. The objective of the present study was to clarify the regulation and significance of RSS synthesis in vascular endothelial cells after exposure to TGF-β1. Bovine aortic endothelial cells in a culture system were treated with TGF-β1 to assess the expression of intracellular RSS, the effect of RSS on cell proliferation in the presence of TGF-β1, induction of RSS-producing enzymes by TGF-β1, and intracellular signal pathways that mediate this induction. The results suggest that TGF-β1 increased intracellular RSS levels to modulate its inhibitory effect on proliferation. The increased production of RSS, probably high-molecular-mass RSS, was due to the induction of cystathionine γ-lyase and cystathionine β-synthase, which are RSS-producing enzymes, and the induction was mediated by the ALK5-Smad2/3/4 and ALK5-Smad2/3-ATF4 pathways in vascular endothelial cells. TGF-β1 regulates vascular endothelial cell functions such as proliferation and fibrinolytic activity, intracellular high-molecular-mass RSS, which are increased by TGF-β1, may modulate the regulation activity in vascular endothelial cells.

Our main objective was to investigate the effect of chronic administration of hydrogen sulphide donor (sodium hydrosulphide) on the expression of intercellular adhesion molecule-1 (ICAM-1) and concentration of nuclear factor-kappa B (NF-kB) in a renal ischemia-reperfusion injury (IRI) model of WKY and L-nitro-arginine-methyl-ester (L-NAME)-induced hypertensive rats. Sodium hydrosulphide (NaHS) was administered intraperitoneally (i.p.) for 35 days while cystathionine gamma lyase (CSE) inhibitor dL-propargylglycine (PAG) was administered at a single dose of 50 mg/kg. Animals were anesthetised using sodium pentobarbitone (60 mg/kg) and then prepared to induce renal ischemia by clamping the left renal artery for 30 min followed by 3 h of reperfusion. Pre-treatment with NaHS improved the renal functional parameters in both WKY and L-NAME-induced hypertensive rats along with reduction of blood pressure in hypertensive groups. Oxidative stress markers like malondialdehyde (MDA), total superoxide dismutase (T-SOD) and glutathione (GSH) were also improved by NaHS treatment following renal IRI. Levels of ICAM-1 and NF-kB concentration were reduced by chronic treatment with NaHS and increased by PAG administration after renal IRI in plasma and kidney. Treatment with NaHS improved tubular morphology and glomerulus hypertrophy. Pre-treatment with NaHS reduced the degree of renal IRI by potentiating its antioxidant and anti-inflammatory mechanism, as evidenced by decreased NF-kB concentration and downregulation of ICAM-1 expression.

Duchenne muscular dystrophy (DMD) is the most frequent X chromosome-linked disease caused by mutations in the gene encoding for dystrophin, leading to progressive and unstoppable degeneration of skeletal muscle tissues. Despite recent advances in the understanding of the molecular processes involved in the pathogenesis of DMD, there is still no cure. In this study, we aim at investigating the potential involvement of the transsulfuration pathway (TSP), and its by-end product namely hydrogen sulfide (H2S), in primary human myoblasts isolated from DMD donors and skeletal muscles of dystrophic (mdx) mice. In myoblasts of DMD donors, we demonstrate that the expression of key genes regulating the H2S production and TSP activity, including cystathionine γ lyase (CSE), cystathionine beta-synthase (CBS), 3 mercaptopyruvate sulfurtransferase (3-MST), cysteine dioxygenase (CDO), cysteine sulfonic acid decarboxylase (CSAD), glutathione synthase (GS) and γ -glutamylcysteine synthetase (γ-GCS) is reduced. Starting from these findings, using Nuclear Magnetic Resonance (NMR) and quantitative Polymerase Chain Reaction (qPCR) we show that the levels of TSP-related metabolites such as methionine, glycine, glutathione, glutamate and taurine, as well as the expression levels of the aforementioned TSP related genes, are significantly reduced in skeletal muscles of mdx mice compared to healthy controls, at both an early (7 weeks) and overt (17 weeks) stage of the disease. Importantly, the treatment with sodium hydrosulfide (NaHS), a commonly used H2S donor, fully recovers the impaired locomotor activity in both 7 and 17 old mdx mice. This is an effect attributable to the reduced expression of pro-inflammatory markers and restoration of autophagy in skeletal muscle tissues. In conclusion, our study uncovers a defective TSP pathway activity in DMD and highlights the role of H2S-donors for novel and safe adjuvant therapy to treat symptoms of DMD.

Objective-To investigate cystathionine β synthase (CBS)/hydrogen sulfide (H2S) signaling in multiple myeloma (MM) patients and to identify its effect on the proliferation of U266 cells. Methods-Bone marrow samples of 19 MM patients and 23 healthy donors were collected. qRT-PCR was performed to measure the mRNA expression levels of H2S synthases, cystathionine β synthase, and cystathionine γ lyase. ELISA assays quantified the amount of H2S produced by the two enzymes CBS and CSE. CCK-8 experiment was used to investigate the influence of the CBS inhibitor amino oxyacetic acid and the CSE inhibitor propargylglycine on the proliferation of U266 cells. Flow cytometry and western blotting were performed to determine the effects of AOAA, PAG, and NaHS on cell cycle distribution as well as Caspase-3 and Bcl-2 expression. Results-Patients with MM had higher level of CBS compared with healthy donors. AOAA significantly inhibited cell proliferation in both a time and concentration dependent characteristic, whereas PAG does not. After 24 hours of treatment, AOAA significantly elevated the G0/G1 phase proportion of cells, and reduced the cell distribution in both S and G2/M phases, while NaHS accelerated cell cycle progression by reducing the relative number of cells in G0/G1 phase and increasing the proportion of cells in the G2/M phase. Moreover, AOAA abolished the impact of NaHS on cell cycle progression of U266 cells. AOAA treatment also led to a significant decrease in Bcl-2 expression and dramatic increase in Caspase-3 expression, though NaHS reversed these effects. Conclusion-CBS/H2S system might have a certain effect on the proliferation and apoptosis of MM cells.

Previous studies have shown that during severe acute pancreatitis (SAP) attacks, hydrogen sulfide (H2S) is released in the colon. However, the roles played by H2S in regulating enteric nerves remain unclear. In this study, we examined the association between SAP-induced H2S release and loss of intestinal motility, and also explored the relevant mechanism in enteric nerve cells. A rat SAP model was constructed and enteric nerve cells were prepared. Intestinal mobility was evaluated by measuring the number of bowel movements at indicated time points and by performing intestinal propulsion tests. The production of inflammatory cytokines during a SAP attack was quantified by ELISA, and the levels of cystathionine-γ-lyase (CSE) and cystathionine-β-synthase (CBS) were examined by immunohistochemistry and western blot analysis. In vivo studies showed that PI3K/Akt/Sp1 signaling in enteric nerve cells was blocked, confirming the mechanism of endogenous H2S formation by western blot analysis and immunofluorescence. Our results also showed that rats with SAP symptoms had reduced intestinal motility. Furthermore, PI3K/Akt/Sp1 signaling was triggered and CSE expression was up-regulated, and these changes were associated with H2S formation in the colon. In addition, propargylglycine reduced the levels of inflammatory cytokines and suppressed the release of H2S. Enteric nerve cells that were incubated with LY294002 and transfected with a Sp1-knockdown vector displayed decreased levels of CSE production, which led to a decrease in H2S production. These results suggest that SAP symptoms suppressed the intestinal motility of rats via the release of H2S in enteric nerve cells, which was dependent on the inflammation-induced PI3K/Akt/Sp1 signaling pathway.

H2S causes vasorelaxation however there is considerable heterogeneity in the reported pharmacological mechanism of this effect. This study examines the contribution of endogenously released H2S in the regulation of vascular tone and the mechanism of H2S-induced vasorelaxation in small resistance-like arteries. Mesenteric arteries from C57 and eNOS-/- mice were mounted in myographs to record isometric force. Vasorelaxation responses to NaHS were examined in the presence of various inhibitors of vasorelaxation pathways. Expression and activity of the H2S-producing enzyme, cystathionine-γ-lyase (CSE), were also examined. CSE was expressed in vascular smooth muscle and perivascular adipose cells from mouse mesenteric artery. The substrate for CSE, L-cysteine, caused a modest vasorelaxation (35%) in arteries from C57 mice and poor vasorelaxation (10%) in arteries from eNOS-/- mice that was sensitive to the CSE inhibitor DL-propargylglycine. The fast H2S donor, NaHS, elicited a full and biphasic vasorelaxation response in mesenteric arteries (EC50 (1) 8.7 μM, EC50 (2) 0.6 mM), which was significantly inhibited in eNOS-/- vessels (P < 0.05), unaffected by endothelial removal, or blockers at any point in the NO via soluble guanylate cyclase and cGMP (NO-sGC-cGMP) vasorelaxation pathway. Vasorelaxation to NaHS was significantly inhibited by blocking K+ channels of the KCa and KV subtypes and the Cl-/HCO3- exchanger (P < 0.05). Further experiments showed that NaHS can significantly inhibit voltage-gated Ca2+ channel function (P < 0.05). The vasorelaxant effect of H2S in small resistance-like arteries is complex, involving eNOS, K+ channels, Cl-/HCO3- exchanger, and voltage-gated Ca2+ channels. CSE is present in the smooth muscle and periadventitial adipose tissue of these resistance-like vessels and can be activated to cause modest vasorelaxation under these in vitro conditions.

Hepatic fibrosis is caused by chronic inflammation and characterized as the excessive accumulation of extra cellular matrix (ECM) by activated hepatic stellate cells (HSCs). Gasotransmitters like NO and CO are known to modulate inflammation and fibrosis, however, little is known about the role of the gasotransmitter hydrogen sulfide (H2S) in liver fibrogenesis and stellate cell activation. Endogenous H2S is produced by the enzymes cystathionine beta-synthase (CBS), cystathionine gamma-lyase (CTH) and 3-mercaptopyruvate sulfur transferase (MPST) [1]. The aim of this study was to elucidate the role of endogenously produced and/or exogenously administered H2S on rat hepatic stellate cell activation and fibrogenesis. Primary rat HSCs were culture-activated for 7 days and treated with different H2S releasing donors (slow releasing donor GYY4137, fast releasing donor NaHS) or inhibitors of the H2S producing enzymes CTH and CBS (DL-PAG, AOAA). The main message of our study is that mRNA and protein expression level of H2S synthesizing enzymes are low in HSCs compared to hepatocytes and Kupffer cells. However, H2S promotes hepatic stellate cell activation. This conclusion is based on the fact that production of H2S and mRNA and protein expression of its producing enzyme CTH are increased during hepatic stellate cell activation. Furthermore, exogenous H2S increased HSC proliferation while inhibitors of endogenous H2S production reduce proliferation and fibrotic makers of HSCs. The effect of H2S on stellate cell activation correlated with increased cellular bioenergetics. Our results indicate that the H2S generation in hepatic stellate cells is a target for anti-fibrotic intervention and that systemic interventions with H2S should take into account cell-specific effects of H2S.

We investigated the effects of sulforaphane (SFN), an isothiocyanate from cruciferous vegetables, in the regulation of cerebral blood flow using cranial windows in newborn pigs. SFN administered topically (10 µM–1 mM) or systemically (0.4 mg/kg ip) caused immediate and sustained dilation of pial arterioles concomitantly with elevated H2S in periarachnoid cortical cerebrospinal fluid. H2S is a potent vasodilator of cerebral arterioles. SFN is not a H2S donor but it acts via stimulating H2S generation in the brain catalyzed by cystathionine γ-lyase (CSE) and cystathionine β-synthase (CBS). CSE/CBS inhibitors propargylglycine, β-cyano-L-alanine, and aminooxyacetic acid blocked brain H2S generation and cerebral vasodilation caused by SFN. The SFN-elicited vasodilation requires activation of potassium channels in cerebral arterioles. The inhibitors of KATP and BK channels glibenclamide, paxilline, and iberiotoxin blocked the vasodilator effects of topical and systemic SFN, supporting the concept that H2S is the mediator of the vasodilator properties of SFN in cerebral circulation. Overall, we provide first evidence that SFN is a brain permeable compound that increases cerebral blood flow via a non-genomic mechanism that is mediated via activation of CSE/CBS-catalyzed H2S formation in neurovascular cells followed by H2S-induced activation of KATP and BK channels in arteriolar smooth muscle.

Endogenous hydrogen sulfide mediates anti-aging benefits of dietary restriction (DR). However, it is unclear how H2S production is regulated by pathways related to DR. Due to the importance of mTORC1 pathway in DR, we investigated the effects of Sch9, a yeast homolog of mammalian S6K1 and a major substrate of mTORC1 on H2S production in yeast Saccharomyces cerevisiae. We found that inhibition of the mTORC1-Sch9 pathway by SCH9 deletion, rapamycin or myriocin treatment resulted in a dramatic decrease in H2S production. Although deficiency of SCH9 did not alter the intracellular level of methionine, the intracellular level of cysteine increased in Δsch9 cells. The expression of CYS3 and CYS4, two transsulfuration pathway genes encoding cystathionine gamma-lyase (CGL) and cystathionine beta-synthase (CBS), were also decreased under mTORC1-Sch9 inhibition. Overexpression of CYS3 or CYS4 in Δsch9 cells or WT cells treated with rapamycin rescued the deficiency of H2S production. Finally, we also observed a reduction in H2S production and lowering of both mRNA and protein levels of CGL and CBS in cultured human cells treated with rapamycin to reduce mTORC1 pathway activity. Thus, our findings reveal a probably conserved mechanism in which H2S production by the transsulfuration pathway is regulated by mTORC1-Sch9 signaling.

The dysregulation of pulmonary arterial vasoactive mediators or excessive proliferation of pulmonary arterial smooth muscle cells (PASMCs) might result in contraction or remodeling of pulmonary blood vessels, leading to related lung diseases. Recent studies suggest that hydrogen sulfide (H2S), a gaseous vasodilator generated in the blood vessels by the enzymes cystathionine γ-lyase (CSE) and cystathionine-β-synthase (CBS), could induce the vasodilation, thus improving contraction or remodeling-induced lung diseases. In this study, we hypothesized that PASMCs could produce H2S and relax the pulmonary artery, and its mechanism is related to CSE, CBS, and TRPV4 channels by affecting both the excessive proliferation and pulmonary vasoconstriction in PASMCs. We found that the sildenafil treatment could remarkably promote H2S production and control the proliferation in PASMCs; meanwhile, the protein levels of CSE and CBS and the intracellular concentration of calcium could also be increased by sildenafil. Moreover, the effects of sildenafil could be reversed by a CBS inhibitor or a CSE inhibitor, indicating that sildenafil could affect CSE and CBS to modulate the production of H2S and the proliferation in rat PASMCs. Together, we demonstrated a new mechanism for sildenafil to modulate the synthesis of H2S and cell proliferation in PASMCs by affecting CSE and CBS. TRPV4-dependent Ca events and BMP4 may also be involved.

Hydrogen sulfide (H2S), a multifunctional gasotransmitter, participates in a wide range of cellular signal transduction and pathophysiological processes. Cystathionine gamma-lyase (CSE) acts as a major H2S-generating enzyme in peripheral organs and tissues. As a cysteine-rich and heavy metal-binding protein, metallothionein-1 (MT-1) is known to protect cells from various environmental stresses. Here we demonstrated that exposure of cadmium (Cd) induced oxidative stress, depleted intracellular thiols, and stimulated apoptotic cell death in mouse myoblast cells. CSE expression and H2S production were significantly enhanced by Cd treatment. NaHS, a well-known H2S donor, at physiologically relevant concentration significantly alleviated Cd-induced damage in both myoblasts and mouse skeletal muscles. In contrast, down-regulation of CSE/H2S system deteriorated Cd-stimulated oxidative stress and cell death. Exposure of the cells to Cd lead to increased expressions of metal regulatory transcription factor 1 and MT-1, while siRNA-mediated MT-1 knockdown alleviated Cd-induced CSE expression and caused more oxidative stress and cell death. In addition, H2S post-translationally modified MT-1 by S-sulfhydration and stabilized zinc-protein complex. Taken together, these data suggest that CSE/H2S system would protect myoblasts and skeletal muscles from Cd-induced damage by S-sufhydrating MT-1.

BACKGROUND To explore the protective effects of Shexiang Tongxin Dropping Pill (STP) in improving peripheral microvascular dysfunction in mice and to explore the involved mechanism. MATERIAL AND METHODS A peripheral microvascular dysfunction model was established by combined myocardial infarction (MI) and lipopolysaccharide (LPS) injection in mice. Then, the mice were randomized into a model group (n=10) or an STP group (n=10), which were treated with normal saline and STP, respectively. The cremaster muscle microvascular blood flow velocity and numbers of leukocytes adherent to the venular wall were evaluated before and after drug intervention. We assessed the expression of adhesion molecule CD11b and related transcript factor FOXO1 in leukocytes, cystathionine-γ-lyase (CSE) mRNA expression in the cremaster muscle, and mitochondrial DNA copy numbers. RESULTS Compared with those of control mice, the cremaster microvascular blood flow velocity, cremaster CSE expression, and mitochondrial DNA copy number in mice from the model group were significantly lower and leukocyte adhesion and CD11b and FOXO1 expression were significantly higher. Intervention with STP could significantly increase the cremaster microvascular flow velocity (0.480±0.010 mm/s vs. 0.075±0.005 mm/s), mRNA expression of cremaster CSE, and mitochondrial DNA copy number, but it inhibited leukocyte adhesion and decreased leukocyte CD11b and FOXO1 expression. CONCLUSIONS STP significantly improved peripheral microcirculation, in which increased CSE expression might be the underlying mechanism.

Purpose. This study investigates whether functionality and/or expression changes of transient receptor potential vanilloid 1 (TRPV1) and transient receptor potential ankyrin 1 (TRPA1) channels, oxidative stress, and hydrogen sulfide (H2S) are involved in the bladder dysfunction from an insulin-resistant obese Zucker rat (OZR). Materials and Methods. Detrusor smooth muscle (DSM) samples from the OZR and their respective controls, a lean Zucker rat (LZR), were processed for immunohistochemistry for studying the expression of TRPA1 and TRPV1 and the H2S synthase cystathionine beta-synthase (CBS) and cysthathionine-γ-lyase (CSE). Isometric force recordings to assess the effects of TRPA1 agonists and antagonists on DSM contractility and measurement of oxidative stress and H2S production were also performed. Results. Neuronal TRPA1 expression was increased in the OZR bladder. Electrical field stimulation- (EFS-) elicited contraction was reduced in the OZR bladder. In both LZR and OZR, TRPA1 activation failed to modify DSM basal tension but enhanced EFS contraction; this response is inhibited by the TRPA1 blockade. In the OZR bladder, reactive oxygen species, malondialdehyde, and protein carbonyl contents were increased and antioxidant enzyme activities (superoxide dismutase, catalase, GR, and GPx) were diminished. CSE expression and CSE-generated H2S production were also reduced in the OZR. Both TRPV1 and CBS expressions were not changed in the OZR. Conclusions. These results suggest that an increased expression and functionality of TRPA1, an augmented oxidative stress, and a downregulation of the CSE/H2S pathway are involved in the impairment of nerve-evoked DSM contraction from the OZR.

Responses of marine ectotherms to variable environmental temperature often entails maintanence of cellular homeostasis and physiological function through temperature compensation and physiological changes. We investigated the physiological response to thermal stress by examining proteomic changes in the marine kelp forest gastropod and emerging fisheries species Kellet's whelk (Kelletia kelletii) across a naturally-existing thermal gradient that ranges from a warmer-water site inside the species' native range and extends to the northern, cold-water edge of the range. We hypothesized that abundance of cellular stress response and energy metabolism proteins would increase with decreasing temperature in support of cold-compensation. Our exploratory proteomic analysis of whelk gill tissue (N = 6 whelks) from each of the four California Channel Island sites revealed protein abundance changes related to the cytoskeleton, energy metabolism/oxidative stress, and cell signaling. The changes did not correlate consistently with temperature. Nonetheless, whelks from the coldest island site showed increased abundance of energy metabolism and oxidative stress proteins, possibly suggesting oxidative damage of lipid membranes that is ameliorated by antioxidants and may aid in their cold stress response. Similarly, our exploratory analysis revealed abundances of cell signaling proteins that were higher at the coldest site compared to the warmest site, possibly indicating an importance for cell signaling regulation in relatively cooler environments. This study provides protein targets for future studies related to thermal effects in marine animals and may contribute to understanding the physiological response of marine organisms to future ocean conditions.

Endogenous gaseous transmitters (nitric oxide, carbon monoxide, and hydrogen sulphide) form a special neuromodulation system mediating the development and modification of nerve centers. Here, we examined the localization of key gaseous transmitter enzymes: cystathionine β-synthetase (CBS), cystathionine γ-lyase (CSE), heme oxygenase 2 (HO-2), and constitutive NO synthase (nNOS) in the fetal human retina at different stages of development. The number of CBS- and CSE-positive photoreceptors and intermediate retinal neurons was high in trimester I and gradually decreased to the end of trimester III. The number of HO-2-positive cells followed the same trend. The number of nNOS-positive intermediate retinal neurons and neurons within the ganglion cell layer showed the opposite dynamics with the peak in trimester III. The results are interpreted in terms of the role of gaseous transmitters in retinogenesis and cytoprotection.

Low-dose alcohol consumption (LAC) has been shown to suppress post-ischemic inflammation and alleviate cerebral ischemia/reperfusion (I/R) injury. Cystathionine γ-Lyase (CSE) is one of the enzymes that endogenously produce hydrogen sulfide (H(2)S), which has an anti-inflammatory property at low concentration. We determined the potential role of CSE in the protective effect of LAC. Male C57BL/6J mice were divided into two groups, an ethanol group and a control group, and gavage fed with 0.7 g/kg/day ethanol or volume-matched water once a day for 8 weeks. Transient focal cerebral ischemia was induced by unilateral middle cerebral artery occlusion (MCAO) for 90 minutes. CSE inhibitors were intraperitoneally given 30 minutes prior to the ischemia. Cerebral I/R injury, H(2)S production, adhesion molecules, IL-1 receptor accessory protein (IL-1RAcP), IL-1β, microglial activation, and neutrophil infiltration were evaluated at 24 hours of reperfusion. Eight-week ethanol feeding upregulated CSE in the cerebral cortex and reduced cerebral I/R injury. Moreover, ethanol increased post-ischemic H(2)S production and alleviated the post-ischemic inflammatory response (expression of adhesion molecules, IL-1RAcP, IL-1β, microglial activation, and neutrophil infiltration) in the peri-infarct cerebral cortex. Both inhibitors of CSE, DL-Proparglyglycine (PAG) and β-cyano-L-alanine (BCA), abolished the protective effect of ethanol on cerebral I/R injury. In addition, PAG attenuated the inhibitory effect of ethanol on the post-ischemic inflammation. Thus, LAC may protect against cerebral I/R injury by suppressing post-ischemic inflammation via an upregulated CSE.

BACKGROUND: Environmental tobacco smoke (ETS) is a known risk factor for severe respiratory syncytial virus (RSV) infections, yet the mechanisms of ETS/RSV co-morbidity are largely unknown. Cystathionine γ-lyase regulates important physiological functions of the respiratory tract. METHODS: We used mice genetically deficient in the cystathionine γ-lyase enzyme (CSE), the major H2S-generating enzyme in the lung to determine the contribution of H2S to airway disease in response to side-stream tobacco smoke (TS), and to TS /RSV co-exposure. RESULTS: Following a two-week period of exposure to TS, CSE-deficient mice (KO) showed a dramatic increase in airway hyperresponsiveness (AHR) to methacholine challenge, and greater airway cellular inflammation, compared to wild type (WT) mice. TS-exposed CSE KO mice that were subsequently infected with RSV exhibited more severe clinical disease, airway obstruction and AHR, enhanced viral replication and lung inflammation, compared to TS-exposed RSV-infected WT mice. TS-exposed RSV-infected CSE KO mice had also a significant increase in the number of neutrophils in bronchoalveolar lavage (BAL) fluid and increased levels of inflammatory cytokines and chemokines. CONCLUSION: This study demonstrates the critical contribution of the H2S-generating pathway to airway reactivity and disease following exposure to ETS alone or in combination with RSV infection.

Chronic exposure of the retina to light and high concentrations of polyunsaturated fatty acid in photoreceptor cells make this tissue susceptible to oxidative damage. As retinal degenerative diseases are associated with photoreceptor degeneration, the antioxidant activity of both hydrogen sulfide (H(2)S) and glutathione (GSH) may play an important role in ameliorating disease progression. H(2)S production is driven by cystathionine-γ-lyase (CSE) and cystathionine β–synthase (CBS), the key enzymes that also drive transsulfuration pathway (TSP) necessary for GSH production. As it is currently unclear whether localized production of either H(2)S or GSH contributes to retinal homeostasis, we undertook a comparative analysis of CBS and CSE expression in canine, non-human primates (NHP) and human retinas to determine if these antioxidants could play a regulatory role in age-related or disease-associated retinal degeneration. Retinas from normal dogs, NHPs and humans were used for the study. Laser capture microdissection (LCM) was performed to isolate individual layers of the canine retina and analyze CBS and CSE gene expression by qRT-PCR. Immunohistochemistry and western blotting were performed for CBS and CSE labeling and protein expression in dog, NHP, and human retina, respectively. Using qRT-PCR, western blot, and immunohistochemistry (IHC), we showed that CBS and CSE are expressed in the canine, NHP, and human retina. IHC results from canine retina demonstrated increased expression levels of CBS but not CSE with post-developmental aging. IHC results also showed non-overlapping localization of both proteins with CBS presenting in rods, amacrine, horizontal, and nerve fiber cell layers while CSE was expressed by RPE, cones and Mϋller cells. Finally, we demonstrated that these enzymes localized to all three layers of canine, NHP and human retina: photoreceptors, outer plexiform layer (OPL) and notably in the ganglion cells layer/nerve fiber layer (GCL/NFL). QRT-PCR performed using RNA extracted from tissues isolated from these cell layers using laser captured microdissection (LCM) confirmed that each of CBS and CSE are expressed equally in these three layers. Together, these findings reveal that CSE and CBS are expressed in the retina, thereby supporting further studies to determine the role of H(2)S and these proteins in oxidative stress and apoptosis in retinal degenerative diseases.

Cystathionine gamma-lyase (CSE)/hydrogen sulfide (H2S) plays a protective role in cardiovascular diseases including hypertension and ischemia/reperfusion (I/R) injury. This study was aimed to screen natural small molecule compounds that activate CSE activity and then evaluate its effect(s) on kidney I/R injury and hypertension. Applying computer molecular docking technology, we screened the natural small molecule compound norswertianolin (NW)-specific binding to CSE. Using the microscale thermophoresis technology, we confirmed that the Leu68 site was the essential hydrogen bond site of NW binding to CSE. NW supplementation significantly increased CSE expression and its activity for H2S generation both in vivo and in vitro. In the model of acute and long-term kidney I/R injury, NW pretreatment dramatically attenuated kidney damage, associated with decreasing blood urea nitrogen (BUN), serum creatinine (Cr) level, reactive oxygen species (ROS) production, and cleaved caspase 3 expression. In spontaneously hypertensive rats (SHRs), NW treatment also lowered blood pressure, the media/lumen ratio of the femoral artery, and the mRNA level of inflammatory cytokines. In conclusion, NW acts as a novel small molecular chemical compound CSE agonist, directly binding to CSE, heightening CSE generation–H2S activity, and then alleviating kidney I/R injury and hypertension. NW has a potential therapeutic merit for cardiovascular diseases.

Since the first description of hydrogen sulfide (H2S) as a toxic gas in 1713 by Bernardino Ramazzini, most studies on H2S have concentrated on its toxicity. In 1989, Warenycia et al. demonstrated the existence of endogenous H2S in the brain, suggesting that H2S may have physiological roles. In 1996, we demonstrated that hydrogen sulfide (H2S) is a potential signaling molecule, which can be produced by cystathionine β-synthase (CBS) to modify neurotransmission in the brain. Subsequently, we showed that H2S relaxes vascular smooth muscle in synergy with nitric oxide (NO) and that cystathionine γ-lyase (CSE) is another producing enzyme. This study also opened up a new research area of a crosstalk between H2S and NO. The cytoprotective effect, anti-inflammatory activity, energy formation, and oxygen sensing by H2S have been subsequently demonstrated. Two additional pathways for the production of H2S with 3-mercaptopyruvate sulfurtransferase (3MST) from l- and d-cysteine have been identified. We also discovered that hydrogen polysulfides (H2Sn, n ≥ 2) are potential signaling molecules produced by 3MST. H2Sn regulate the activity of ion channels and enzymes, as well as even the growth of tumors. S-Sulfuration (S-sulfhydration) proposed by Snyder is the main mechanism for H2S/H2Sn underlying regulation of the activity of target proteins. This mini review focuses on the key findings on H2S/H2Sn signaling during the first 25 years.

Hypertension and age are key risk factors for cardiovascular morbidity and mortality. Hydrogen sulfide (H2S), a gaseous transmitter, contributes significantly to regulating arterial blood pressure and aging processes. This study evaluated the effects of hypertension and aging on the hepatic metabolism of sulfur-containing compounds, the activity of the enzymes involved in sulfur homeostasis, and the liver’s ability to generate H2S. Livers isolated from 16- and 60-week-old normotensive Wistar Kyoto rats (WKY) and Spontaneously Hypertensive Rats (SHR) were used to evaluate gene expression using RT-PCR, and the activity of enzymes participating in H2S metabolism, including thiosulfate sulfurtransferase (rhodanese, TST), cystathionine gamma-lyase (CTH), and 3-mercaptopyruvate sulfurtransferase (MPST). The levels of cysteine, cystine, reduced and oxidized glutathione were measured using RP-HPLC. SHR livers from both age groups showed a higher capacity to generate H2S than livers from WKY. The gene expression and activity of enzymes involved in sulfur metabolism differed between WKY and SHR, and between the age groups. For example, 16-week-old SHR had significantly higher activity of TST than 16-week-old WKY. Furthermore, differences between younger and older WKY rats in the expression and/or activity of TST and MPST were present. In conclusion, our study shows that arterial hypertension and aging affect hepatic sulfur metabolism and H2S production in rats. These findings pave the way for interventional studies evaluating a potential causal relation between liver sulfur metabolism, hypertension and aging.

During pregnancy, estrogen (E2) stimulates uterine artery blood flow (UBF) by enhancing nitric oxide (NO)-dependent vasodilation. Cystathionine γ-lyase (CSE) promotes vascular NO signaling by producing hydrogen sulfide (H2S) and by maintaining the ratio of reduced-to-oxidized intracellular glutathione (GSH/GSSG) through l-cysteine production. Because redox homeostasis can influence NO signaling, we hypothesized that CSE mediates E2 stimulation of UBF by modulating local intracellular cysteine metabolism and GSH/GSSG levels to promote redox homeostasis. Using non-pregnant ovariectomized WT and CSE-null (CSE KO) mice, we performed micro-ultrasound of mouse uterine and renal arteries to assess changes in blood flow upon exogenous E2 stimulation. We quantified serum and uterine artery NO metabolites (NOx), serum amino acids, and uterine and renal artery GSH/GSSG. WT and CSE KO mice exhibited similar baseline uterine and renal blood flow. Unlike WT, CSE KO mice did not exhibit expected E2 stimulation of UBF. Renal blood flow was E2-insensitive for both genotypes. While serum and uterine artery NOx were similar between genotypes at baseline, E2 decreased NOx in CSE KO serum. Cysteine was also lower in CSE KO serum, while citrulline and homocysteine levels were elevated. E2 and CSE deletion additively decreased GSH/GSSG in uterine arteries. In contrast, renal artery GSH/GSSG was insensitive to E2 or CSE deletion. Together, these findings suggest that CSE maintenance of uterine artery GSH/GSSG facilitates nitrergic signaling in uterine arteries and is required for normal E2 stimulation of UBF. These data have implications for pregnancy pathophysiology and the selective hormone responses of specific vascular beds., Graphical abstract Image 1, Highlights • CSE-null mice exhibit abnormal estrogen augmentation of uterine artery blood flow. • Estrogen lowers uterine artery nitric oxide metabolites in CSE null mice. • CSE loss and estrogen additively impair uterine artery glutathione homeostasis. • Neither CSE loss nor estrogen influences renal artery blood flow or glutathione.

Hydrogen sulfide (H2S) exerts an anti-atherosclerotic effect and decreases foam cell formation. Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a key factor involved in foam cell formation. However, the association between H2S and Lp-PLA2 expression levels with respect to foam cell formation has not yet been elucidated. The present study investigated whether H2S can affect foam cell formation and potential signalling pathways via regulation of the expression and activity of Lp-PLA2. Using human monocytic THP-1 cells as a model system, it was observed that oxidized low-density lipoprotein (ox-LDL) not only upregulates the expression level and activity of Lp-PLA2, it also downregulates the expression level and activity of Cystathionine γ lyase. Exogenous supplementation of H2S decreased the expression and activity of Lp-PLA2 induced by ox-LDL. Moreover, ox-LDL induced the expression level and activity of Lp-PLA2 via activation of the p38MAPK signalling pathway. H2S blocked the expression levels and activity of Lp-PLA2 induced by ox-LDL via inhibition of the p38MAPK signalling pathway. Furthermore, H2S inhibited Lp-PLA2 activity by blocking the p38MAPK signaling pathway and significantly decreased lipid accumulation in ox-LDL-induced macrophages, as detected by Oil Red O staining. The results of the present study indicated that H2S inhibited ox-LDL-induced Lp-PLA2 expression levels and activity by blocking the p38MAPK signalling pathway, thereby improving foam cell formation. These findings may provide novel insights into the role of H2S intervention in the progression of atherosclerosis.

Cysteine is implicated in important biological processes. It is synthesized through two different pathways. Cystathionine β‐synthase and cystathionine γ‐lyase participate in the reverse transsulfuration pathway, while serine acetyltransferase and cysteine synthase function in the de novo pathway. Two evolutionarily related pyridoxal 5′‐phosphate‐dependent enzymes, cystathionine β‐synthase TtCBS1 (TTHERM_00558300) and cysteine synthase TtCSA1 (TTHERM_00239430), were identified from a freshwater protozoan Tetrahymena thermophila. TtCbs1 contained the N‐terminal heme binding domain, catalytic domain, and C‐terminal regulatory domain, whereas TtCsa1 consisted of two α/β domains. The catalytic core of the two enzymes is similar. TtCBS1 and TtCSA1 showed high expression levels in the vegetative growth stage and decreased during the sexual developmental stage. TtCbs1 and TtCsa1 were localized in the cytoplasm throughout different developmental stages. His‐TtCbs1 and His‐TtCsa1 were expressed and purified in vitro. TtCbs1 catalyzed the canonical reaction with the highest velocity and possessed serine sulfhydrylase activity. TtCsa1 showed cysteine synthase activity with high Kₘ for O‐acetylserine and low Kₘ for sulfide and also had serine sulfhydrylase activity toward serine. Both TtCbs1 and TtCsa1 catalyzed hydrogen sulfide producing. TtCBS1 knockdown and TtCSA1 knockout mutants affected cysteine and glutathione synthesis. TtCbs1 and TtCsa1 are involved in cysteine synthesis through two different pathways in T. thermophila.

BACKGROUND Accumulated evidence has suggested that hydrogen sulfide (H₂S) has a role in bone formation and bone tissue regeneration. However, it is unknown whether the H₂S content is associated with bone mineral density (BMD) in patients with osteopenia/osteoporosis. MATERIAL AND METHODS In the present study, we aimed to explore the changes of serum H₂S in osteopenia and osteoporosis patients. We analyzed femur expression of cystathionine ß synthase (CBS), cystathionine γ lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3-MST), which are key enzymes for generating H₂S. RESULTS Sixteen (16%) patients had osteopenia, 9 (9%) had osteoporosis, and 75 (75%) had normal BMD. In comparison with patients with normal BMD (controls), the serum levels of H₂S were unexpectedly increased in patients with osteopenia and osteoporosis. This increase was much higher in patients with osteoporosis than in those with osteopenia. Serum H₂S levels were negatively correlated with femoral BMD, but not lumbar BMD. Interestingly, the expression of CBS and CSE were downregulated in femur tissues in patients with osteoporosis, whereas the expression of 3-MST remained unchanged. Serum phosphorus levels, alkaline phosphatase, hemoglobin, and triglycerides were found to be closely associated with CBS and CSE scores in femur tissues. CONCLUSIONS Serum H₂S levels and femur CBS and CSE expression may be involved in osteoporosis pathogenesis.

Alzheimer’s disease (AD), the most common cause of dementia and neurodegeneration in the elderly, is characterized by deterioration of memory and executive and motor functions. Neuropathologic hallmarks of AD include neurofibrillary tangles (NFTs), paired helical filaments, and amyloid plaques. Mutations in the microtubule-associated protein Tau, a major component of the NFTs, cause its hyperphosphorylation in AD. We have shown that signaling by the gaseous molecule hydrogen sulfide (H(2)S) is dysregulated during aging. H(2)S signals via a posttranslational modification termed sulfhydration/persulfidation, which participates in diverse cellular processes. Here we show that cystathionine γ-lyase (CSE), the biosynthetic enzyme for H(2)S, binds wild type Tau, which enhances its catalytic activity. By contrast, CSE fails to bind Tau P301L, a mutant that is present in the 3xTg-AD mouse model of AD. We further show that CSE is depleted in 3xTg-AD mice as well as in human AD brains, and that H(2)S prevents hyperphosphorylation of Tau by sulfhydrating its kinase, glycogen synthase kinase 3β (GSK3β). Finally, we demonstrate that sulfhydration is diminished in AD, while administering the H(2)S donor sodium GYY4137 (NaGYY) to 3xTg-AD mice ameliorates motor and cognitive deficits in AD.

Background Hydrogen sulfide (H 2 S) is an important endogenous physiological signaling molecule and exerts protective properties in the cardiovascular system. Cystathionine γ‐lyase (CSE), 1 of 3 H 2 S producing enzyme, is predominantly localized in the vascular endothelium. However, the regulation of CSE in vascular endothelium remains incompletely understood. Methods and Results We generated inducible endothelial cell‐specific CSE overexpressed transgenic mice (EC‐CSE Tg) and endothelial cell‐specific CSE knockout mice (EC‐CSE KO), and investigated vascular function in isolated thoracic aorta, treadmill exercise capacity, and myocardial injury following ischemia‐reperfusion in these mice. Overexpression of CSE in endothelial cells resulted in increased circulating and myocardial H 2 S and NO, augmented endothelial‐dependent vasorelaxation response in thoracic aorta, improved exercise capacity, and reduced myocardial‐reperfusion injury. In contrast, genetic deletion of CSE in endothelial cells led to decreased circulating H 2 S and cardiac NO production, impaired endothelial dependent vasorelaxation response and reduced exercise capacity. However, myocardial‐reperfusion injury was not affected by genetic deletion of endothelial cell CSE. Conclusions CSE‐derived H 2 S production in endothelial cells is critical in maintaining endothelial function, exercise capacity, and protecting against myocardial ischemia/reperfusion injury. Our data suggest that the endothelial NO synthase—NO pathway is likely involved in the beneficial effects of overexpression of CSE in the endothelium.

Endothelial dysfunction is a hallmark of preeclampsia, a life-threatening complication of pregnancy characterised by hypertension and elevated soluble Fms-Like Tyrosine Kinase-1 (sFlt-1). Dysregulation of hydrogen sulfide (H2S) by inhibition of cystathionine γ-lyase (CSE) increases sFlt-1 and soluble endoglin (sEng) release. We explored whether compromise in CSE/H2S pathway is linked to dysregulation of the mitochondrial bioenergetics and oxidative status. We investigated whether these effects were linked to CSE-induced sFlt-1 and sEng production in endothelial cells. Here, we demonstrate that CSE/H2S pathway sustain endothelial mitochondrial bioenergetics and loss of CSE increases the production of mitochondrial-specific superoxide. As a compensatory effect, low CSE environment enhances the reliance on glycolysis. The mitochondrial-targeted H2S donor, AP39, suppressed the antiangiogenic response and restored the mitochondrial bioenergetics in endothelial cells. AP39 revealed that upregulation of sFlt-1, but not sEng, is independent of the mitochondrial H2S metabolising enzyme, SQR. These data provide new insights into the molecular mechanisms for antiangiogenic upregulation in a mitochondrial-driven environment. Targeting H2S to the mitochondria may be of therapeutic benefit in the prevention of endothelial dysfunction associated with preeclampsia.

In recent years, the gasotransmitter hydrogen sulphide (H2S), produced by the transsulphuration pathway, has been recognized as a biological mediator playing an important role under normal conditions and in various pathologies in both eukaryotes and prokaryotes. The transsulphuration pathway (TSP) includes the conversion of homocysteine to cysteine following the breakdown of methionine. In Drosophila melanogaster and other eukaryotes, H2S is produced by cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulphurtransferase (MST). In the experiments performed in this study, we were able to explore the CRISPR/Cas9 technique to obtain single and double deletions in homozygotes of these three major genes responsible for H2S production in Drosophila melanogaster. In most cases, the deletion of one studied gene does not result in the compensatory induction of two other genes responsible for H2S production. Transcriptomic studies demonstrated that the deletions of the above CBS and CSE genes alter genome expression to different degrees, with a more pronounced effect being exerted by deletion of the CBS gene. Furthermore, the double deletion of both CBS and CSE resulted in a cumulative effect on transcription in the resulting strains. Overall, we found that the obtained deletions affect numerous genes involved in various biological pathways. Specifically, genes involved in the oxidative reduction process, stress-response genes, housekeeping genes, and genes participating in olfactory and reproduction are among the most strongly affected. Furthermore, characteristic differences in the response to the deletions of the studied genes are apparently organ-specific and have clear-cut sex-specific characteristics. Single and double deletions of the three genes responsible for the production of H2S helped to elucidate new aspects of the biological significance of this vital physiological mediator., Graphical abstract Image 1

Altindirek, Didem/0000-0001-5068-2968; Armutak, Elif Ilkay/0000-0002-7359-6568 WOS:000574979000007 PubMed ID: 32871779 Background/Aim: This study was designed to provide further evidence for the interactions between hydrogen sulfide (H2S) and nitric oxide (NO) in ischemia/reperfiision (I/R) injury. Materials and Methods: Rat hearts were studied with the Langendorff technique using the H2S donor sodium hydrosulfide (NaHS, 40 mu M) and the cystathionine gamma-lyase (CTH or CSE) inhibitor DL-propargylglycine (PAG, 1 mM). NO synthase inhibitor L-NG-nitroarginine methyl ester (L-NAME, 30 mg/kg, 7 days) was administered before the isolation. The hearts were homogenized for biochemical and molecular analysis. Results: NaHS reversed I/R-induced cardiac performance impairment, increased tissue nitric oxide production and decreased tissue markers for cardiac injury, while L-NAME inhibited these effects. The expression of CTH was increased with PAG, which was suppressed by L-NAME. Conclusion: H2S and NO increase each other's production suggesting their interaction and cooperation in cardioprotection against I/R injury. Scientific Research Projects Coordination Unit of Istanbul UniversityIstanbul University [31508, 55736, 47109] This work was supported by Scientific Research Projects Coordination Unit of Istanbul University [grant numbers; 31508, 55736 and 47109].

Hydrogen sulfide (H2S) is an important gasotransmitter expressed in various tissues of the organism, including the eye. It is known that H2S is localized especially in the retina and corneal layers in bovine eye. The enzymes that mediate H2S synthesis are 3-mercaptopyruvate sulfurtransferase (3-MST), cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE). Herein, we aimed to investigate the concentration levels and distribution profiles of these enzymes in bovine retina and retinal artery. Enzyme levels were measured by ELISA and distribution were determined by immunofluorescence microscopic analysis. Much higher concentrations of CBS and CSE have been detected in the retinal artery compared to the retina. In both tissues, particulary 3-MST was found at the lowest level while, CSE was determined to be the most abundant enzyme among the others. CBS distribution was shown in both endothelial and smooth muscle layers, while CSE was seen especially in the endothelial layer of the retinal artery. In the retina, CBS and CSE were expressed in cone-basil cells and retinal ganglion cells, while CSE was also present in bipolar cells. Our results indicated that H2S is synthesized endogenously in ocular tissues. The widespread expression of H2S synthesizing enzymes in the retina and retinal artery of the bovine eye, which has anatomical similarities with the human eye, may suggest a protective role for H2S against retinal vascular diseases as well as a regulatory role in the retinal vascular tone.