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Extracellular matrix (ECM) is a non-cellular three-dimensional macromolecular network that not only provides mechanical support but also transduces essential molecular signals in organ functions. ECM is constantly remodeled to control tissue homeostasis, responsible for cell adhesion, cell migration, cell-to-cell communication, and cell differentiation, etc. The dysregulation of ECM components contributes to various diseases, including cardiovascular diseases, fibrosis, cancer, and neurodegenerative diseases, etc. Aberrant ECM remodeling is initiated by various stress, such as oxidative stress, inflammation, ischemia, and mechanical stress, etc. Hydrogen sulfide (H2S) is a gasotransmitter that exhibits a wide variety of cytoprotective and physiological functions through its anti-oxidative and anti-inflammatory actions. Amounting research shows that H2S can attenuate aberrant ECM remodeling. In this review, we discussed the implications and mechanisms of H2S in the regulation of ECM remodeling in cardiovascular diseases, and highlighted the potential of H2S in the prevention and treatment of cardiovascular diseases through attenuating adverse ECM remodeling.

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.

Hydrogen sulfide (H2 S) is a gasotransmitter that regulates both physiological and pathophysiological processes in mammalian cells. Recent studies have demonstrated that H2 S promotes aerobic energy production in the mitochondria in response to hypoxia, but its effect on anaerobic energy production has yet to be established. Glycolysis is the anaerobic process by which ATP is produced through the metabolism of glucose. Mammalian red blood cells (RBCs) extrude mitochondria and nucleus during erythropoiesis. These cells would serve as a unique model to observe the effect of H2 S on glycolysis-mediated energy production. The purpose of this study was to determine the effect of H2 S on glycolysis-mediated energy production in mitochondria-free mouse RBCs. Western blot analysis showed that the only H2 S-generating enzyme expressed in mouse RBCs is 3-mercaptopyruvate sulfurtransferase (MST). Supplement of the substrate for MST stimulated, but the inhibition of the same suppressed, the endogenous production of H2 S. Both exogenously administered H2 S salt and MST-derived endogenous H2 S stimulated glycolysis-mediated ATP production. The effect of NaHS on ATP levels was not affected by oxygenation status. On the contrary, hypoxia increased intracellular H2 S levels and MST activity in mouse RBCs. The mitochondria-targeted H2 S donor, AP39, did not affect ATP levels of mouse RBCs. NaHS at low concentrations (3-100 μM) increased ATP levels and decreased cell viability after 3 days of incubation in vitro. Higher NaHS concentrations (300-1000 μM) lowered ATP levels, but prolonged cell viability. H2 S may offer a cytoprotective effect in mammalian RBCs to maintain oxygen-independent energy production.

Hydrogen sulfide (H

The outbreak of COVID-19 pneumonia caused by a new coronavirus (severe acute respiratory syndrome coronavirus 2, SARS-CoV-2) is posing a global health emergency and has led to more than 380,000 deaths worldwide. The cell entry of SARS-CoV-2 depends on two host proteins angiotensin-converting enzyme 2 (ACE2) and transmembrane protease serine 2 (TMPRSS2). There is currently no vaccine available and also no effective drug for the treatment of COVID-19. Hydrogen sulfide (H2S) as a novel gasotransmitter has been shown to protect against lung damage via its anti-inflammation, antioxidative stress, antiviral, prosurvival, and antiaging effects. In light of the research advances on H2S signaling in biology and medicine, this review proposed H2S as a potential defense against COVID-19. It is suggested that H2S may block SARS-CoV-2 entry into host cells by interfering with ACE2 and TMPRSS2, inhibit SARS-CoV-2 replication by attenuating virus assembly/release, and protect SARS-CoV-2-induced lung damage by suppressing immune response and inflammation development. Preclinical studies and clinical trials with slow-releasing H2S donor(s) or the activators of endogenous H2S-generating enzymes should be considered as a preventative treatment or therapy for COVID-19.

Aims: The physiological and pathological importance of hydrogen sulfide (H2S) as a novel gasotransmitter has been widely recognized. Cystathionine gamma-lyase (CSE) is one of the major H2S-producing enzymes and it regulates diverse functions in connection with intracellular calcium (Ca2+). The aim of this study is to examine the role of H2S in Golgi stress-related cell injury and skeletal muscle disorders. Results: Golgi stressors (brefeldin A [BFA] and monensin) decreased the expression of GM130 and ATP2C1 (two markers of Golgi stress response), induced Golgi apparatus fragmentation, and caused a higher level of oxidative stress and cell apoptosis in mouse myoblast cells. In addition, Golgi stressors upregulated CSE expression and endogenous H2S generation, and exogenously applied H2S was able to protect but inhibition of CSE/H2S system deteriorated Golgi stress response. Activating transcription factor 4 (ATF4) acted as an upstream molecule to increase CSE expression on Golgi stress response. Mechanically, Golgi stressors induced intracellular level of Ca2+, and chelating cellular Ca2+ markedly attenuated Golgi stress response, indicating the key role of Ca2+ in initiating Golgi stress and cell apoptosis. Further, administration of either angiotensin II or BFA initiated Golgi stress response and induced skeletal muscle atrophy in mice, which was further deteriorated by CSE deficiency but rescued by exogenously applied sodium hydrosulfide (NaHS). Innovation and Conclusion: The activation of the CSE/H2S pathway and the decrease of intracellular Ca2+ are two cellular protective mechanisms against Golgi stress, and the CSE/H2S system would be a target for preventing skeletal muscle dysfunctions.

Hydrogen sulfide (H2 S) can be endogenously produced and belongs to the class of signaling molecules known as gasotransmitters. Cystathionine gamma-lyase (CSE)-derived H2 S is implicated in the regulation of cell differentiation and the aging process, but the involvements of the CSE/H2 S system in myogenesis upon aging and injury have not been explored. In this study, we demonstrated that CSE acts as a major H2 S-generating enzyme in skeletal muscles and is significantly down-regulated in aged skeletal muscles in mice. CSE deficiency exacerbated the age-dependent sarcopenia and cardiotoxin-induced injury/regeneration in mouse skeletal muscle, possibly attributed to inefficient myogenesis. In contrast, supplement of NaHS (an H2 S donor) induced the expressions of myogenic genes and promoted muscle regeneration in mice. In vitro, incubation of myoblast cells (C2C12) with H2 S promoted myogenesis, as evidenced by the inhibition of cell cycle progression and migration, altered expressions of myogenic markers, elongation of myoblasts, and formation of multinucleated myotubes. Myogenesis was also found to upregulate CSE expression, while blockage of CSE/H2 S signaling resulted in a suppression of myogenesis. Mechanically, H2 S significantly induced the heterodimer formation between MEF2c and MRF4 and promoted the binding of MEF2c/MRF4 to myogenin promoter. MEF2c was S-sulfhydrated at both cysteine 361 and 420 in the C-terminal transactivation domain, and blockage of MEF2c S-sulfhydration abolished the stimulatory role of H2 S on MEF2c/MRF4 heterodimer formation. These findings support an essential role for H2 S in maintaining myogenesis, presenting it as a potential candidate for the prevention of age-related sarcopenia and treatment of muscle injury.

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.

Hydrogen sulfide (H2S) is a gaseous signaling molecule that participates in multiple physiological processes in both animals and plants. Mitogen-activated protein kinase (MAPK) is important signaling molecule that links the growth and developmental signals and environment stimuli to cellular responses. In the current study we explored the relationship between H2S and MAPK in drought stress resistance in Arabidopsis. The quantitative real-time (qRT)-PCR, root tip bending experiment and stomatal aperture assay were used in this paper. Drought stress activated both H2S biosynthesis and gene expression of MAPKs. The increase in MAPK expression was depressed in lcd/des1, a double mutant of H2S synthesis. Then we selected MPK4 as our target and used mpk4 mutants for further studies. H2S was able to alleviate the drought stress in wild-type (WT) Arabidopsis but not in mpk4 mutants. Meanwhile, H2S-induced stomatal movement was impaired in mpk4 mutants. We then examined the role of H2S and MPK4 in stomatal movements in response to abscisic acid (ABA) and hydrogen peroxide (H2O2). ABA- and H2O2- mediated stomatal movements were impaired in lcd/des1 and mpk4 mutants, and H2S-induced stomatal closure was impaired in slac1–3 mutants. Our results suggested that MPK4 is important downstream of H2S in the drought stress response and in stomatal movement, and that the H2S-MPK4 cascade is involved in ABA-mediated stomatal movement to regulate the drought stress.

Nickel as a heavy metal is known to bring threat to human health, and nickel exposure is associated with changes in fibroblast activation which may contribute to its fibrotic properties. H2S has recently emerged as an important gasotransmitter involved in numerous cellular signal transduction and pathophysiological responses. Interaction of nickel and H2S on fibroblast cell activation has not been studied so far. Here, we showed that a lower dose of nickel (200 μM) induced the activation of human fibroblast cells, as evidenced by increased cell growth, migration and higher expressions of α-smooth muscle actin (αSMA) and fibronectin, while high dose of nickel (1 mM) inhibited cell viability. Nickel reduced intracellular thiol contents and stimulated oxidative stress. Nickel also repressed the mRNA and protein expression of cystathionine gamma-lyase (CSE, a H2S-generating gene) and blocked the endogenous production of H2S. Exogenously applied NaHS (a H2S donor) had no effect on nickel-induced cell viability but significantly attenuated nickel-stimulated cell migration and the expression of αSMA and fibronectin. In contrast, CSE deficiency worsened nickel-induced αSMA expression. Moreover, H2S incubation reversed nickel-stimulated TGFβ1/SMAD1 signal and blocked TGFβ1-initiated expressions of αSMA and fibronectin. Nickel inhibited the interaction of Sp1 with CSE promoter but strengthened the binding of Sp1 with TGFβ1 promoter, which was reversed by exogenously applied NaHS. These data reveal that H2S protects from nickel-stimulated fibroblast activation and CSE/H2S system can be a potential target for the treatment of tissue fibrosis induced by nickel.

Hydrogen sulfide (H2S) is a gasotransmitter along with nitric oxide and carbon oxide, which is involved in plant growth and development as well as biotic and abiotic stress resistance. In a previous study, we reported that mitogen-activated protein kinases, especially MPK4, are important downstream components of H2S involved in alleviating cold stress; however the underlying mechanism is unclear. In this study, we determined that the ability of H2S to alleviate cold stress is impaired in mpk4 mutants, but not in the upstream mek2 and crlk1 mutants. MPK4 was basically persulfidated, and NaHS (H2S donor) further increased the persulfidation level of MPK4. MEK2 was not persulfidated by H2S. NaHS treatments increased the MPK4 activity level nearly tenfold. The persulfidation signal of MPK4 did not disappear after eight cystein residues in MPK4 were site-mutated, respectively. Above all, our results suggested that H2S alleviates cold stress directly by persulfidating MPK4 and increasing the MPK4 kinase activity.

The role of endogenous hydrogen sulfide (H2S) as an antioxidant regulator has sparked interest in its function within inflammatory diseases. Cigarette and alcohol use are major causes of premature death, resulting from chronic oxidative stress and subsequent tissue damage. The activation of the Nrf2 antioxidant response by H2S suggests that this novel gasotransmitter may function to prevent or potentially reverse disease progression caused by cigarette smoking or alcohol use. The purpose of this study is to review the interrelationship between H2S signaling and cigarette smoking or alcohol drinking. Based on the databases of cellular, animal, and clinical studies from Pubmed using the keywords of H2S, smoking, and/or alcohol, this review article provides a comprehensive insight into disrupted H2S signaling by alcohol drinking and cigarette smoking-caused disorders. Major signaling and metabolic pathways involved in H2S-derived antioxidant and anti-inflammatory responses are further reviewed. H2S supplementation may prove to be an invaluable asset in treating or preventing diseases in those suffering from cigarette or alcohol addiction.

To determine the mediation of spermine on energy metabolism disorder and diabetic cardiomyopathy (DCM) development as well as the underlying mechanisms. An in vitro model of DCM was established by incubating primary cultured neonatal rat cardiomyocytes with high glucose (HG). Spermine content was assessed by RP-HPLC. The protein levels were detected by western blot. Mitochondrial functions were analyzed using the respiratory chain complex assay kit and immunofluorescence staining. The endogenous content of spermine was decreased in the HG group, and the protein levels of ornithine decarboxylase, respiratory chain complex (I–V), mitochondrial fusion-related protein (Mfn1, Mfn2), Cx43, N-cadherin, CaSR, and β-catenin (in cytomembrane) were also down-regulated by HG. In contrast, the protein levels of spermine-N1-acetyltransferase, gp78, Fis1, Drp1, and β-catenin were up-regulated by HG. Meanwhile, we observed that HG increased ubiquitination levels of Mfn1, Mfn2, and Cx43, decreased membrane potential (ΔΨm), and the opening of mitochondrial permeability transport pore (mPTP) followed by intracellular ATP leakage. The supplement of spermine or siRNA-mediated knockdown of gp78 significantly alleviated the detrimental effects of HG, while downregulation of CaSR aggravated the development of DCM. We further confirmed that the lower level of spermine by HG activates the gp78-ubiquitin-proteasome pathway via downregulation of CaSR protein level, which in turn damages mitochondrial gap junction intercellular communication and leads to reduced ATP level. The protective role of spermine on energy metabolism disorder is based on higher CaSR protein level and lower gp78 activation, pointing to the possibility that spermine can be a target for the prevention and treatment of DCM.

Recent advances in the biomedical importance of H2S have help us understand various cellular functions and pathophysiological processes from a new aspect. Specially, H2S has been demonstrated to play multiple roles in regulating cell behaviors, including cell survival, cell differentiation, cell senescence, cell hypertrophy, cell atrophy, cell metaplasia, and cell death, etc. H2S contributes to cell behavior changes via various mechanisms, such as histone modification, DNA methylation, non-coding RNA changes, DNA damage repair, transcription factor activity, and post-translational modification of proteins by S-sulfhydration, etc. In this review, we summarized the recent research progress on H2S signaling in control of cell behaviors and discussed the ways of H2S regulation of gene expressions. Given the key roles of H2S in both health and diseases, a better understanding of the regulation of H2S on cell behavior change and the underlying molecular mechanisms will help us to develop novel and more effective strategies for clinical therapy.

Hydrogen sulfide (H2S) is mostly produced by cystathionine-gamma-lyase (CSE) in vascular system and it inhibits the proliferation of vascular smooth muscle cells (SMCs). Insulin-like growth factor-1 (IGF-1), via its receptor (IGF-1R), exerts multiple physiological and pathophysiological effects on the vasculature, including stimulating SMC proliferation and migration, and inhibiting SMC apoptosis. Since H2S and IGF-1/IGF-1R have opposite effects on SMC proliferation, it becomes imperative to better understand the interaction of these two signaling mechanisms on SMC proliferation. SMCs isolated from small mesenteric arteries of CSE knockout (KO) and wild-type (WT) mice were used in the present study. The effects of IGF-1 and H2S on SMC proliferation were evaluated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and bromodeoxyuridine (BrdU) assays. Protein expression was determined by western blot, and H2S-induced protein S-sulfhydration was assessed with a modified biotin switch assay. We found that IGF-1 dose-dependently increased the proliferation of both WT-SMCs and KO-SMCs, and this effect was more significant in KO-SMCs. Supplement of sodium hydrosulfide (NaHS) inhibited IGF-1-induced cell proliferation, while this effect was abolished by blocking IGF-1/IGF-1R signaling with picropodophyllin (PPP) or knocking out of the expression of IGF-1R. H2S significantly down-regulates the expression of IGF-1R, stimulates IGF-1R S-sulfhydration, and attenuates the binding of IGF-1 with IGF-1R. This study provides novel insight on the involvement of IGF-1/IGF-1R in H2S-inhibited SMC proliferation and suggests H2S-based innovative treatment strategies for proliferative cardiovascular diseases such as atherosclerosis.

Hydrogen sulfide (H2S) is a novel gasotransmitter and acts as a multifunctional regulator in various cellular functions. Past studies have demonstrated a significant role of H2S and its generating enzyme cystathionine gamma-lyase (CSE) in the cardiovascular system. Lipopolysaccharide (LPS), a major pathogenic factor, is known to initiate the inflammatory immune response. The cross talk between LPS-induced inflammation and the CSE/H2S system in vascular cells has not yet been elucidated in detail. Here we showed that LPS decreased CSE mRNA and protein expression in human endothelial cells and blocked H2S production in mouse aorta tissues. Transfection of the cells with TLR4-specific siRNA knockdown TLR4 mRNA expression and abolished the inhibitory role of LPS on CSE expression. Higher dose of LPS (100μg/ml) decreased cell viability, which was reversed by exogenously applied H2S at physiologically relevant concentration (30μM). Lower dose of LPS (10μg/ml) had no effect on cell viability, but significantly induced inflammation gene expressions and cytokines secretion and stimulated cell hyper-permeability. H2S treatment prevented LPS-induced inflammation and hyper-permeability. Lower VE-cadherin expression in LPS-incubated cells would contribute to cell hyper-permeability, which was reversed by H2S co-incubation. In addition, H2S treatment blocked LPS-induced NFκB transactivation. We further validated that LPS-induced hyper-permeability was reversed by CSE overexpression but further deteriorated by CRISPR/Cas9-mediated knockout of CSE. In vivo, deficiency of CSE sensitized the mice to LPS-induced inflammation in vascular tissues. Take together, these data suggest that CSE/H2S system protects LPS-induced inflammation and cell hyper-permeability by blocking NFκB transactivation.

Hydrogen sulfide (H2S) is a gaseous signaling molecule that mediates physiological processes in animals and plants. In this study, we investigated the relationship of H2S and mitogen activated protein kinase (MAPK) under cold stress in Arabidopsis. H2S up-regulated MAPK expression levels and was involved in the cold stress-related upregulation of MAPK genes expression. We then chose MPK4 whose expression level was influenced the most by H2S as a target and found that H2S's ability to alleviate cold stress required MPK4. Both H2S and MPK4 regulated the expression levels of the cold response genes inducer of CBF expression 1 (ICE1), C-repeat-binding factors (CBF3), cold responsive 15A (COR15A) and cold responsive 15B (COR15B). H2S inhibited the opening of stomata under cold stress, which required the participation of MPK4. In conclusion, MPK4 is a downstream component of H2S-related cold-stress resistance, and H2S and MPK4 both regulated the cold response genes and stomatal movement to response the cold stress.

The physiological effects of the endogenously generated hydrogen sulfide (H2S) have been extensively studied in recent years. This review summarized the role of H2S in the origin of life and H2S metabolism in organisms from bacteria to vertebrates, examined the relationship between H2S and oxygen from an evolutionary perspective and emphasized the oxygen-dependent manner of H2S signaling in various physiological and pathological processes. H2S and oxygen are inextricably linked in various cellular functions. H2S is involved in aerobic respiration and stimulates oxidative phosphorylation and ATP production within the cell. Besides, H2S has protective effects on ischemia and reperfusion injury in several organs by acting as an oxygen sensor. Also, emerging evidence suggests the role of H2S is in an oxygen-dependent manner. All these findings indicate the subtle relationship between H2S and oxygen and further explain why H2S, a toxic molecule thriving in an anoxia environment several billion years ago, still affects homeostasis today despite the very low content in the body.

Signal transduction mediated by photoreceptors regulates many physiological processes during plant growth and development including seed germination, flowering and photosynthesis, which are also regulated by hydrogen sulfide (H2S). However, studies of the connection between the vital environmental factors – light and the significant endogenous gasotransmitter – H2S, is lacking. Here, the seedlings of foxtail millet were used to reveal the mechanism of light regulation in H2S generation. Results showed that seedling hypocotyl elongation was promoted by H2S, but inhibited by HA under dark or white light condition. H2S contents in hypocotyl increased at first under red, blue or white light then decreased, and the duration of increase under white light was longer than under red or blue light. The activity of cysteine desulfhydrases, which catalyse H2S generation, was increased by red light but decreased by blue and white light. The expressions of cysteine desulfhydrases coding genes LCD1 and LCD2 were promoted by red or white light, but inhibited by blue light. In contrast, DES gene was promoted by white light but inhibited by red or blue light. In addition, the activities of LCDs were regulated by the phosphorylation mediated by photoreceptors PHYB and CRY1/CRY2. Finally, there are two pathways of light regulating H2S production, including a rapid process that involves the modification of phosphorylation on LCDs protein mediated by photoreceptors directly or indirectly, as well as a slower process that involves in regulating the expressions of LCDs and DES genes. This discovery has potential value for the application of H2S in agricultural production protecting the crops from unsuited light condition.

Cardiac hypertrophy is defined as the enlargement of the cardiac myocytes, leading to improper nourishment and oxygen supply due to the increased functional demand. This increased stress on the cardiac system commonly leads to myocardial infarction, contributing to 85% of all cardiac-related deaths. Cystathionine gamma-lyase- (CSE-) derived H2S is a novel gasotransmitter and plays a critical role in the preservation of cardiac functions. Selenocysteine lyase (SCLY) has been identified to produce H2Se, the selenium homologue of H2S. Deficiency of selenium is often found in Keshan disease, a congestive cardiomyopathy. The interaction of H2S and H2Se in cardiac cell hypertrophy has not been explored. In this study, cell viability was evaluated with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Oxidative stress and cell size were observed through immunostaining. The expression of genes was determined by real-time PCR and western blot. Here, we demonstrated that incubation of rat cardiac cells (H9C2) with H2O2lead to increased oxidative stress and cell surface area, which were significantly attenuated by pretreatment of either H2S or H2Se. H2S incubation induced SCLY/H2Se signaling, which next caused higher expressions and activities of selenoproteins, including glutathione peroxidase and thioredoxin reductase. Furthermore, deficiency of CSE inhibited the expressions of SCLY and selenoprotein P in mouse heart tissues. We also found that both H2S and H2Se stimulated Nrf2-targeted downstream genes. These data suggests that H2S protects against cardiac hypertrophy through enhancement of a group of antioxidant proteins.

Hydrogen sulfide (H2S) is a novel gasotransmitter in both mammals and plants. H2S plays important roles in various plant developmental processes and stress responses. Leaf senescence is the last developmental stage and is a sequential degradation process that eventually leads to leaf death. A mutation of the H2S-producing enzyme-encoding gene L-cysteine desulfhydrase1 (DES1) leads to premature leaf senescence but the underlying mechanisms are not clear. In this present study, wild-type, DES1 defective mutant (des1) and over-expression (OE-DES1) Arabidopsis plants were used to investigate the underlying mechanism of H2S signaling in energy production and leaf senescence under drought stress. The des1 mutant was more sensitive to drought stress and displayed accelerated leaf senescence, while the leaves of OE-DES1 contained adequate chlorophyll levels, accompanied by significantly increased drought resistance. Under drought stress, the expression levels of ATPβ-1, -2, and -3 were significantly downregulated in des1 and significantly upregulated in OE-DES1, and ATPe showed the opposite trend. Senescence-associated gene (SAG) 12 correlated with age-dependent senescence and participated in the drought resistance of OE-DES1. SAG13, which was induced by environmental factors, responded positively to drought stress in des1 plants, while there was no significant difference in the SAG29 expression between des1 and OE-DES1. Using transmission electron microscopy, the mitochondria of des1 were severely damaged and bubbled in older leaves, while OE-DES1 had complete mitochondrial structures and a homogeneous matrix. Additionally, mitochondria isolated from OE-DES1 increased the H2S production rate, H2S content and ATPase activity level, as well as reduced swelling and lowered the ATP content in contrast with wild-type and des1 significantly. Therefore, at subcellular levels, H2S appeared to determine the ability of mitochondria to regulate energy production and protect against cellular aging, which subsequently delayed leaf senescence under drought-stress conditions in plants.

The gasotransmitter role of H2S in mammalian has been extensively studied, and cystathionine gamma-lyase (CSE) is the major H2S-producing enzyme in vascular system. Dysregulation of CSE/H2S system was found in various pathophysiological conditions. MicroRNA (miRNA) and short interfering RNA (siRNA) are known to inhibit gene expression by mRNA degradation and/or translation repression. The regulation of CSE expression by miRNA and siRNA has been reported recently, but the off-target effect of miRNA and the lower knockdown efficiency of siRNA have shadowed the application of these approaches. In the present study, we designed CSE-specific miRNAs based on the rules of miRNA–mRNA complementary and human CSE cDNA sequence. The CSE-specific miRNAs significantly inhibited CSE expression and H2S production, increased reactive oxygen species generation, and induced proliferation of human aorta smooth muscle cells (HASMCs). The designed CSE-specific miRNAs specifically targeted on CSE gene as evidenced by the direct inhibition of luciferase activity from reporter gene containing human CSE 3′-UTR sequence. The expression of other genes, such as estrogen receptor α, heme oxygenase 1, specificity protein 1, and 3-mercaptopyruvate sulfurtransferase, was not affected by the CSE-specific miRNAs. Compared with CSE-siRNAs, CSE-specific miRNAs displayed significantly higher efficacy in suppressing CSE expression and H2S production. miR-143, a highly expressed miRNA in vascular system, down-regulated the expressions of CSE as well as other genes, such as insulin-like growth factor binding protein 5 and kruppel-like factor 4. miR-143 suppressed H2S production but had no effect on HASMC proliferation. In conclusion, CSE-specific miRNAs designed in our study provide a highly effective research tool for regulating CSE expression and H2S production. These CSE-specific miRNAs have potential as being novel therapeutic agents for treating vascular disorders related to abnormal oxidative stress and SMC growth.

Hydrogen sulfide (H2S) has been recognized as an important gasotransmitter analogous to nitric oxide and carbon monoxide. Cystathionine gamma-lyase (CSE)-derived H2S is implicated in the regulation of insulin resistance and glucose metabolism, but the involvement of CSE/H2S system in energy homeostasis and fat mass has not been extensively explored. In this study, a potential functional role of the CSE/H2S system in in vitro adipocyte differentiation and in vivo adipogenesis and the underlying mechanism was investigated. CSE expression and H2S production were increased during adipocyte differentiation, and that the pattern of CSE mRNA expression was similar to that of CCAAT/enhancer-binding protein (C/EBP) β and δ, two key regulators for adipogenesis. C/EBPβ and γ bind to the CCAAT box in CSE promoter and stimulate CSE gene transcription. H2S induced PPARγ transactivation activity by S-sulfhydrating all the cysteine residues in the DNA binding domain and stimulated adipogenesis. High fat diet-induced fat mass was lost in CSE deficient mice, and exogenously applied H2S promoted fat mass accumulation in fruit flies. In conclusion, CSE/H2S system is essential for adipogenesis and fat mass accumulation through enhancement of PPARγ function in adipocytes. This study suggests that the CSE/H2S system is involved in the pathogenesis of obesity in mice.

Aims: Endothelium-dependent vasorelaxation is mediated by endothelium-derived relaxing factor and endothelium-derived hyperpolarizing factor (EDHF). However, the molecular entity of EDHF remains unclear. The present study examined whether hydrogen sulfide (H2S) acts as EDHF and how H2S mediates EDHF pathways from endothelial origination to downstream target of smooth muscle cells (SMCs). Results: We found that knocking-out the expression of cystathionine γ-lyase (CSE) in mice (CSE-knockout [KO]) elevated resting-membrane-potential of SMCs and eliminated methacholine-induced endothelium-dependent relaxation of mesenteric arteries, but not that of aorta. Methacholine, a cholinergic-muscarinic agonist, hyperpolarized SMC in endothelium-intact mesenteric arteries from wide-type mice. This effect was inhibited by muscarinic antagonist (atropine) or the co-application of charybdotoxin and apamin, which blocked intermediate- and small-conductance KCa (IKCa and SKCa) channels, or abolished in CSE-KO mice...

Hydrogen sulfide (H2 S) and nitric oxide (NO) are major gasotransmitters produced in endothelial cells (ECs), contributing to the regulation of vascular contractility and structural integrity. Their interaction at different levels would have a profound impact on angiogenesis. Here, we showed that H2 S and NO stimulated the formation of new microvessels. Incubation of human umbilical vein endothelial cells (HUVECs-926) with NaHS (a H2 S donor) stimulated the phosphorylation of endothelial NO synthase (eNOS) and enhanced NO production. H2 S had little effect on eNOS protein expression in ECs. L-cysteine, a precursor of H2 S, stimulated NO production whereas blockage of the activity of H2 S-generating enzyme, cystathionine gamma-lyase (CSE), inhibited this action. CSE knockdown inhibited, but CSE overexpression increased, NO production as well as EC proliferation. LY294002 (Akt/PI3-K inhibitor) or SB203580 (p38 MAPK inhibitor) abolished the effects of H2 S on eNOS phosphorylation, NO production, cell proliferation and tube formation. Blockade of NO production by eNOS-specific siRNA or nitro-L-arginine methyl ester (L-NAME) reversed, but eNOS overexpression potentiated, the proliferative effect of H2 S on ECs. Our results suggest that H2 S stimulates the phosphorylation of eNOS through a p38 MAPK and Akt-dependent pathway, thus increasing NO production in ECs and vascular tissues and contributing to H2 S-induced angiogenesis.

H2S, a third member of gasotransmitter family along with nitric oxide and carbon monoxide, exerts a wide range of cellular and molecular actions in our body. Cystathionine gamma-lyase (CSE) is a major H2S-generating enzyme in our body. Aging at the cellular level, known as cellular senescence, can result from increases in oxidative stress. The aim of this study was to investigate how H2S attenuates oxidative stress and delays cellular senescence.Here we showed that mouse embryonic fibroblasts isolated from CSE knockout mice (CSE KO-MEFs) display increased oxidative stress and accelerated cellular senescence in comparison with MEFs from wild-type mice (WT-MEFs). The protein expression of p53 and p21 was significantly increased in KO-MEFs, and knockdown of p53 or p21 reversed CSE deficiency-induced senescence. Incubation of the cells with NaHS (a H2S donor) significantly increased the glutathione (GSH) level and rescued KO-MEFs from senescence. Nrf2 is a master regulator of the antioxidant response, and Keap1 acts as a negative regulator of Nrf2. NaHS S-sulfhydrated Keap1 at cysteine-151, induced Nrf2 dissociation from Keap1, enhanced Nrf2 nuclear translocation, and stimulated mRNA expression of Nrf2-targeted downstream genes, such as glutamate-cysteine ligase and GSH reductase.These results provide a mechanistic insight into how H2S signaling mediates cellular senescence induced by oxidative stress.H2S protects against cellular aging via S-sulfhydration of Keap1 and Nrf2 activation in association with oxidative stress.

BACKGROUND AND PURPOSE The accumulation of hypoxia-inducible factor-1α (HIF-1α) is under the influence of hydrogen sulfide (H2S), which regulates hypoxia responses. The regulation of HIF-1α accumulation by H2S has been shown, but the mechanisms for this effect are largely elusive and controversial. This study aimed at addressing the controversial mechanisms for and the functional importance of the interaction of H2S and HIF-1α protein. EXPERIMENTAL APPROACH HIF-1α protein levels and HIF-1α transcriptional activity were detected by Western blotting and luciferase assay. The mechanisms for H2S-regulated HIF-1α protein levels were determined using short interfering RNA transfection, co-immunoprecipitation and 7-methyl-GTP sepharose 4B pull-down assay. Angiogenic activity was evaluated using tube formation assay in EA.hy926 cells. KEY RESULTS The accumulation of HIF-1α protein under hypoxia (1% O2) or hypoxia-mimetic conditions was reversed by sodium hydrosulfide (NaHS). This effect of NaHS was not altered after blocking the ubiquitin-proteasomal pathway for HIF-1α degradation; however, blockade of protein translation with cycloheximide abolished the effect of NaHS on the half-life of HIF-1α protein. Knockdown of eukaryotic translation initiation factor 2α (eIF2α) suppressed the effect of NaHS on HIF-1α protein accumulation under hypoxia. NaHS inhibited the expression of VEGF under hypoxia. It also decreased in vitro capillary tube formation and cell proliferation of EA.hy926 cells under hypoxia, but stimulated the tube formation under normoxia. CONCLUSIONS AND IMPLICATIONS H2S suppresses HIF-1α translation by enhancing eIF2α phosphorylation under hypoxia. The interaction of H2S and HIF-1α inhibits the angiogenic activity of vascular endothelial cells under hypoxia through the down-regulation of VEGF.

Hydrogen sulfide (H(2)S) has emerged as an important gasotransmitter, offering protection against ischemia-reperfusion damage to the heart. Cystathionine gamma-lyase (CSE) is believed to be the major H(2)S-generating enzyme in the heart. Quite contrary to the general contemplation, CSE protein in cardiac tissues has not been convincingly detected and it has become an issue of controversy. In the present study, we isolated cardiac tissues from wild type (WT) and CSE knockout mice or the rat. CSE expression at transcriptional and translational levels were assayed by RT-PCR and Western Blotting with five different antibodies (four commercial products and one homemade), respectively. Cardiac H(2)S production rate was also examined. Our data validated the expression of CSE mRNA in the heart of WT mice or rats, not in CSE KO mice. Using all 5 different anti-CSE antibodies, we could not detect CSE proteins in mouse or rat cardiac tissues or in cultured rat cardiomyocytes. On the other hand, CSE protein was detectable in liver tissues from WT mice with the expected molecular mass of 43.6 kDa. H(2)S production rate of heart tissues in CSE KO mice was significantly decreased compared with that in WT mice. In the presence of an CSE inhibitor, D,L-propargylglycine, H(2)S production rate of heart tissues from WT mice was inhibited by approximately 80%. It appears that CSE mediates mostly endogenous H(2)S production in heart tissues. However, the available anti-CSE antibodies could not detect CSE proteins in rat and mouse heart tissues or rat cardiomyocytes.

Cystathionine gamma-lyase (CSE) is the major H(2)S-generating enzyme in vascular smooth muscle cells (SMCs). CSE/H(2)S system contributes to the maintenance of SMC phenotype, and transcript factor specificity protein-1 (SP1) is a critical regulator of CSE expression during SMC differentiation. The involvements of microRNA-21 (miR-21) in cardiovascular pathophysiology have been known, however miR-21 regulation of CSE and SP1 as well as SMC phenotype are uncertain. Using quantitative real-time PCR, we demonstrated that the expression of miR-21 was upregulated in dedifferentiated human aorta SMCs (HASMCs) and injured mouse carotid arteries. To determine the potential roles of miR-21 in SP1-mediated CSE gene expression and SMC phenotypic change, we showed that miR-21 expression was upregulated by miR-21 precursor. Interestingly, miR-21 overexpression significantly repressed the protein expressions of both CSE and SP1, inhibited H(2)S production, stimulated SMC proliferation, and reduced SMC differentiation marker gene expression, respectively. The mRNA expression of CSE but not SP1 was inhibited by miR-21 precursor. Blockage of SP1 binding by mithramycin or inhibition of CSE activity by DL-propargylglycine did not change miR-21 expression. We further demonstrated that miR-21 repressed SP1 protein expression by directly targeting at SP1 3' untranslational regions, which in turn downregulated CSE mRNA expression and stimulated SMC proliferation. Take together, these results suggest that miR-21 participates in CSE/H(2)S-mediated-SMC differentiation by targeting SP1.

The physiological and biomedical importance of H 2 S in cardiovascular system has been extensively studied, and H 2 S is involved the regulation of vascular tone, angiogenesis, cell growth and differentiation, inflammation, and energy generation, etc. In blood vessels, cystathionine gamma-lyase (CSE) seems to be the major H 2 S-producing gene expressed in both smooth muscle and endothelium. Abnormal metabolism and functions of the CSE/H 2 S pathway have been linked to various cardiovascular diseases, including atherosclerosis and hypertension. It is known now that H 2 S posttranslationally modification of proteins by S -sulfhydration contributes to the major bioactivities of H 2 S. Cardiovascular disorders and epigenetics are closely associated, and epigenetics has become a promising field in cardiovascular research. In this research highlight, I discuss the latest published findings on H 2 S signalling in epigenetic regulation of vascular cell functions via modification of DNA methylation and DNA damage repair.

Hydrogen sulfide (H(2)S) has been found to play an important role as a signal molecule in regulating cell survival. It appears paradoxical that, on one side, H(2)S acts as a physiological intercellular messenger to stimulate cell growth, and on the other side, it may display cytotoxic activity. This article summarizes the current body of evidence demonstrating the cytoprotective versus cytotoxic effects of H(2)S in mammalian cells and describes the janus-faced properties of this important gasotransmitter. This article will also provide a brief description of the current signaling mechanisms that have been demonstrated to be responsible for these different actions. The pharmacologic regulation of H(2)S production and the potential clinical significance of H(2)S are highlighted.

Butyrate is a short-chain fatty acid that arrests growth of various types of cells. H(2)S can be endogenously produced by cystathionine gamma-lyase (CSE) or cystathionine beta-synthase (CBS) or both in colonic tissues. In this study, we observed endogenous H(2)S production in a colon cancer cell line (WiDr) and colonic tissues through the activity of both CSE and CBS. After 24 h of incubation of WiDr cells, butyrate increased cell production of H(2)S and upregulated CBS and CSE expressions. Both butyrate and NaHS (a H(2)S donor) decreased cell viability in a dose-dependent manner. Blockade of CBS, but not CSE, decreased butyrate-stimulated H(2)S production and reversed butyrate-inhibited cell viability. In addition, NaHS treatment stimulated the phosphorylation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK), but not c-Jun N-terminal kinase (JNK). Inhibition of the phosphorylation of either p38 MAPK or ERK did not abolish NaHS-induced cell death. Butyrate treatment increased the phosphorylation of ERK, not p38 MAPK and JNK, but inhibition of ERK and p38 MAPK phosphorylation did not inhibit butyrate-reduced cell viability. In conclusion, butyrate regulates endogenous H(2)S production by stimulating CBS expression in colon cancer cells, but butyrate and H(2)S inhibit cancer cell growth through different mechanisms.

Hydrogen sulfide (H(2)S) has been traditionally known for its toxic effects on living organisms. The role of H(2)S in the homeostatic regulation of pancreatic insulin metabolism has been unclear. The present study is aimed at elucidating the effect of endogenously produced H(2)S on pancreatic insulin release and its role in diabetes development. Diabetes development in Zucker diabetic fatty (ZDF) rats was evaluated in comparison with Zucker fatty (ZF) and Zucker lean (ZL) rats. Pancreatic H(2)S production and insulin release were also assayed. It was found that H(2)S was generated in rat pancreas islets, catalyzed predominantly by cystathionine gamma-lyase (CSE). Pancreatic CSE expression and H(2)S production were greater in ZDF rats than in ZF or ZL rats. ZDF rats exhibited reduced serum insulin level, hyperglycemia, and insulin resistance. Inhibition of pancreatic H(2)S production in ZDF rats by intraperitoneal injection of DL-propargylglycine (PPG) for 4 weeks increased serum insulin level, lowered hyperglycemia, and reduced hemoglobin A1c level (P

Cystathionine gamma-lyase (CSE) is a key enzyme in the trans-sulfuration pathway, which uses L-cysteine to produce hydrogen sulfide (H2S). Functional changes of pancreatic beta cells induced by endogenous H2S have been reported, but the effect of the CSE/H2S system on pancreatic beta cell survival has not been known. In this study, we demonstrate that H2Sat physiologically relevant concentrations induced apoptosis of INS-1E cells, an insulin-secreting beta cell line. Transfection of INS-1E cells with a recombinant defective adenovirus containing the CSE gene (Ad-CSE) resulted in a significant increase in CSE expression and H2S production. Ad-CSE transfection also stimulated apoptosis. The other two end products of CSE-catalyzed enzymatic reaction, ammonium and pyruvate, had no effects on INS-1E cell apoptosis, indicating that overexpression of CSE may stimulate INS-1E cell apoptosis via increased endogenous production of H2S. Both exogenous H2S (100 microM) and Ad-CSE transfection inhibited ERK1/2 but activated p38 MAPK. Interestingly, BiP and CHOP, two indicators of endoplasmic reticulum (ER) stress, were up-regulated in H2S-and CSE-mediated apoptosis in INS-1E cells. After suppressing CHOP mRNA expression, H2S-induced apoptosis of INS-1E cells was significantly decreased. Inhibition of p38 MAPK, but not of ERK1/2, inhibited the expression of BiP and CHOP and decreased H2S-stimulated apoptosis, suggesting that p38 MAPK activation functions upstream of ER stress to initiate H2S-induced apoptosis. It is concluded that H2S induces apoptosis of insulin-secreting beta cells by enhancing ER stress via p38 MAPK activation. Our findings may help unmask a novel role of CSE/H2S system in regulating pancreatic functions under physiological condition and in diabetes.

Aims: Hydrogen sulfide (H2S) exerts a wide range of actions in the body, especially in the modulation of mitochondrial functions. The normal replication of mitochondrial DNA (mtDNA) is critical for cellular energy metabolism and mitochondrial biogenesis. The aim of this study was to investigate whether H2S affects mtDNA replication and the underlying mechanisms. We hypothesize that H2S maintains mtDNA copy number via inhibition of Dnmt3a transcription and TFAM promoter methylation. Results: Here, we demonstrated that deficiency of cystathionine gamma-lyase (CSE), a major H2S-producing enzyme, reduces mtDNA copy number and mitochondrial contents, and it inhibits the expressions of mitochondrial transcription factor A (TFAM) and mitochondrial marker genes in both smooth muscle cells and aorta tissues from mice. Supply of exogenous H2S stimulated mtDNA copy number and strengthened the expressions of TFAM and mitochondrial marker genes. TFAM knockdown diminished H2S-enhanced mtDNA copy number. In addition, CSE deficiency induced the expression of DNA methyltransferase 3a (Dnmt3a) and TFAM promoter DNA methylation, and H2S repressed Dnmt3a expression, resulting in TFAM promoter demethylation. We further found that H2S S-sulfhydrates transcription repressor interferon regulatory factor 1 (IRF-1) and enhances the binding of IRF-1 with Dnmt3a promoter after reduced Dnmt3a transcription. H2S had little effects on the expression of Dnmt1 and Dnmt3b as well as on ten-eleven translocation methylcytosine dioxygenase 1, 2, and 3. Innovation: A sufficient level of H2S is able to inhibit TFAM promoter methylation and maintain mtDNA copy number. Conclusion: CSE/H2S system contributes to mtDNA replication and cellular bioenergetics and provides a novel therapeutic avenue for cardiovascular diseases. Antioxid. Redox Signal. 23, 630–642.

The physiological and biomedical importance of hydrogen sulfide (H2S) has been fully recognized in the cardiovascular system as well as in the rest of the body. In blood vessels, cystathionine γ-lyase (CSE) is a major H2S-producing enzyme expressed in both smooth muscle and endothelium as well as periadventitial adipose tissues. Regulation of H2S production from CSE is controlled by a complex integration of transcriptional, posttranscriptional, and posttranslational mechanisms in blood vessels. In smooth muscle cells, H2S regulates cell apoptosis, phenotypic switch, relaxation and contraction, and calcification. In endothelial cells, H2S controls cell proliferation, cellular senescence, oxidative stress, inflammation, etc. H2S interacts with nitric oxide and acts as an endothelium-derived relaxing factor and an endothelium-derived hyperpolarizing factor. H2S generated from periadventitial adipose tissues acts as an adipocyte-derived relaxing factor and modulates the vascular tone. Extensive evidence has demonstrated the beneficial roles of the CSE/H2S system in various blood vessel diseases, such as hypertension, atherosclerosis, and aortic aneurysm. The important roles signaling in the cardiovascular system merit further intensive and extensive investigation. H2S-releasing agents and CSE activators will find their great applications in the prevention and treatment of blood vessel-related disorders.

Just in the last decade, the knowledge of the physiologic functions of hydrogen sulfide (H2S) in medicine and biology has been tremendously accumulated [1–3]. H2S has emerged to be one important member of the family of gasotransmitters, together with nitric oxide (NO) and carbon monoxide (CO) [1, 2]. The ubiquitous distribution of H2S-producing enzymes and the potent chemical reactivities of H2S in biological system make this molecule unique in regulating cellular and organ functions in both the mammalian and plants [1–3]. Pathophysiological abnormalities related to altered H2S metabolism and function have been demonstrated in many cases [2, 3]. H2S possesses a great therapeutic potential in age-associated diseases by modulation of oxidative stress [4, 5]. This special issue contains review paper and research articles that focus on the topics of H2S signaling in oxidative stress and aging development, including discussions on the potency and efficiency of H2S in dealing with various diseases. A number of contributions have addressed the protective role of H2S in cardiovascular diseases and diabetes. In an original research article, H. Yu et al. demonstrate that H2S decreases NADPH oxidase activity and reactive oxidative species (ROS) production, which lead to reduced mean arterial pressure and heart rate in spontaneously hypertensive rats. H2S, as an antioxidant, may be a potential target for cardiovascular diseases. A research article by S. Jin and colleagues compares H2S generation in ageing diabetic mouse hearts, and they find that H2S levels are reduced in the diabetic heart due to the alterations in H2S-producing enzymes, which might be related with the pathogenesis of diabetic cardiomyopathy. Y. Zong and colleagues explore the possible effects of endogenous H2S on endothelial apoptosis under high-salt stimulation, and their data validate that supplementation of H2S donor markedly inhibits vascular endothelial cell oxidative stress and mitochondria-related apoptosis induced by high salt. Q. Wang and colleagues report that H2S antagonizes advanced glycation end-products induced-epithelial sodium channel activity by targeting the ROS/PI3K/PTEN pathway in A6 cells. The authors conclude that H2S may provide protection against hypertension in diabetic patients. In a review paper by Y. Shen and colleagues, the underlying mechanisms for the cardioprotective effects of H2S against myocardial infarction, arrhythmia, hypertrophy, heart failure, and so forth are discussed. Some mechanisms, including antioxidative action, preservation of mitochondrial function, reduction of apoptosis, anti-inflammatory responses, angiogenic actions, regulation of ion channel, and interaction with NO, are mostly responsible for the cardioprotective effect of H2S. Some papers in this special issue describe new insights into the therapeutic potential in fibrosis. In a review paper, S. Zhang and colleagues summarize studies that supplement with exogenous H2S mitigates the severity of fibrosis in various experimental animal models. The protective role of H2S in the development of fibrosis is primarily attributed to its antioxidation, antiapoptosis, anti-inflammation, proangiogenesis, and inhibition of fibroblasts activities. K. Song and colleagues continue on with this topic that H2S protects fibrosis diseases that relate to heart, liver, kidney, and other organs. In a research article, G. Meng and colleagues provide new evidence on the protective role of GYY4137, a slow-releasing H2S donor, in myocardial fibrosis by inhibiting oxidative stress, blocking TGF-β1/Smad2 signaling pathway, and decreasing in expression of α-SMA. Further clinical studies are needed to translate this potential to clinical use. D. Wu and colleagues highlight the recent findings regarding the role of H2S in ischemia-reperfusion (I/R) injury. In their paper, the authors proposed that treatment with H2S or its donors in proper dose range and time frame will exhibit more potent therapeutic effects against I/R injury in further preclinical research and clinical application. A review article by W. Zhang and her colleagues addresses the reciprocal interaction between H2S and calcium ion channels and transporters through different mechanisms, all of which are essential for the maintenance of intracellular calcium homeostasis by H2S. In an original research article, L. Zhang and colleagues explore the role of H2S in human gastric neoplasias. Their data point that H2S level is lower in noncancerous gastric samples in comparison with human gastric carcinoma mucosa, and the authors further prove that H2S induces apoptosis and inhibits cell migration and invasion of gastric cancer cells by regulating apoptosis related proteins. The therapeutic application of H2S donors against gastric cancer development can be realized. In a review article, B. Wu and colleagues discuss the latest research on the interaction of H2S with oxygen sensing under hypoxia condition. Emerging evidence has elucidated an important protective role of H2S in hypoxia-mediated damage in many mammalian systems. By regulating the functions of hypoxia-inducible factors and the activation of carotid bodies, H2S acts as important oxygen/hypoxia sensor. Not only has it acted as a signalling molecule in mammalian system, but also overwhelming evidence has demonstrated that H2S plays important roles in diverse physiological processes in plants. J. Zhu and Y. Pei discuss in a review article the physiological implications of H2S in plants. H2S modulates various defence responses in plants, including growth and development, abiotic stress, heavy metal toxicity, drought and osmotic stress, hypoxia, senescence, and maturation by interacting with plant hormones, hydrogen peroxide, NO, CO, and other molecules. The same research group also provides evidence that H2S alleviates cadmium-induced cell death in Chinese cabbage roots, and they further verify that, by upregulating antioxidant enzyme activities, H2S removes excessive ROS and reduces cell oxidative damage induced by cadmium. In one original research article, Y. Zhang and colleagues demonstrate that H2S acts as an antioxidant in delaying cell apoptosis and enhancing α-amylase secretion regardless of the presence of gibberellic acid in barley aleurone layers. In addition, D.-B. Zhu and colleagues investigate the effects of SO2 pretreatment on H2S and ROS accumulation in germinating wheat seeds, and their data suggest that SO2 could increase endogenous H2S accumulation and the antioxidant capability and decrease endogenous aluminum content in wheat grain to alleviate aluminum stress. SO2 may be reduced to H2S by sulfite reductase, thus contributing to H2S production. The articles presented in this special issue highlight the current advances in the research field of H2S in medicine and biology. These articles not only enrich our understanding of how H2S regulation of oxidative stress in various disorders occurs but also provide evidence on the therapeutic potential of H2S against aging development and other disorders. We hope that readers will find these contributions interesting and informative.

K(ATP) channels are composed of pore-forming subunits Kir6.x and auxiliary subunits SURx. These channels play important roles in modulating the contractility of vascular smooth muscle cells (SMCs) by altering membrane potentials. The molecular basis of K(ATP) channels in vascular SMCs is unclear and the expression of different K(ATP) channel subunits at protein level in various tissues still undetermined. In this study, using an anti-Kir6.1 antibody, we detected the expression of Kir6.1 proteins in rat vascular tissues including mesenteric artery, pulmonary artery, aorta, and tail artery. Kir6.1 proteins were also identified in heart and other non-vascular tissues including spleen and brain, but they were undetectable in liver and kidney. Immunocytochemical study revealed the expression of Kir6.1 proteins in cultured rat thoracic aortic SMCs. Using the whole-cell patch-clamp technique, it was found that the intracellularly applied anti-Kir6.1 antibody significantly inhibited K(ATP) channel currents in HEK-293 cells that were stably transfected with Kir6.1 cDNA. A better understanding of differential expression of Kir6.1 proteins in various vascular and non-vascular tissues may help discern different molecular basis and functions of K(ATP) channel complexes in these tissues.

1Department of Chemistry and Biochemistry, Laurentian University, Sudbury, ON, Canada 2Department of Surgery and Department of Microbiology and Immunology, Western University, London, ON, Canada 3Department of Pathophysiology, Nanjing Medical University, Nanjing, China 4School of Life Science, Shanxi University, Taiyuan, China 5Department of Chemistry and Biochemistry, University of Oregon, Eugene, OR, USA

The gasotransmitter hydrogen sulfide (H(2)S), which is generated by cystathionine γ-lyase (CSE), signals by modifying proteins through S-sulfhydration and potentially other mechanisms. A target protein for H(2)S is endothelial nitric oxide synthase (eNOS), an enzyme that generates nitric oxide (NO), which causes vasodilation. We investigated whether H(2)S-induced S-sulfhydration affected the S-nitrosylation and phosphorylation of eNOS and the functional effects of changes in these posttranslational modifications on eNOS activity. In vitro, different NO donors induced the S-nitrosylation of eNOS without affecting its S-sulfhydration, whereas the H(2)S donor sodium hydrosulfide (NaHS) decreased the S-nitrosylation of eNOS. Cys(443) was the primary S-sulfhydration site in eNOS and was one site that could be S-nitrosylated. Phosphorylation increases eNOS activity. Although exposure of eNOS-expressing HEK-293 cells to NaHS or vascular endothelial growth factor (VEGF) triggered the phosphorylation of wild-type and C443G-eNOS, VEGF did not affect the S-sulfhydration of eNOS and a mutant of eNOS that could not be phosphorylated was still S-sulfhydrated. eNOS can be present in cells in monomeric or dimeric form, but only eNOS dimers produce NO. In wild-type mice, eNOS proteins were predominantly dimerized, whereas eNOS from CSE-knockout (KO) mice, S-nitrosylated eNOS, and heterologously expressed C443G-eNOS was mostly monomeric. Accordingly, basal production of NO was lower in CSE-KO endothelial cells than in wild-type endothelial cells. Our data suggest that H(2)S increases eNOS activity by inducing the S-sulfhydration of eNOS, promoting its phosphorylation, inhibiting its S-nitrosylation, and increasing eNOS dimerization, whereas NO decreases eNOS activity by promoting the formation of eNOS monomers.

A growing amount of evidence has suggested that hydrogen sulfide (H2S), as a gasotransmitter, is involved in intensive physiological and pathological processes. More and more research groups have found that H2S mediates diverse cellular biological functions related to regulating intracellular calcium concentration. These groups have demonstrated the reciprocal interaction between H2S and calcium ion channels and transporters, such as L-type calcium channels (LTCC), T-type calcium channels (TTCC), sodium/calcium exchangers (NCX), transient receptor potential (TRP) channels,β-adrenergic receptors, and N-methyl-D-aspartate receptors (NMDAR) in different cells. However, the understanding of the molecular targets and mechanisms is incomplete. Recently, some research groups demonstrated that H2S modulates the activity of calcium ion channels through protein S-sulfhydration and polysulfide reactions. In this review, we elucidate that H2S controls intracellular calcium homeostasis and the underlying mechanisms.

Androgen receptor (AR) signaling is indispensable for the development of prostate cancer from the initial androgen-dependent state to a later aggressive androgen-resistant state. This study examined the role of hydrogen sulfide (H(2)S), a novel gasotransmitter, in the regulation of AR signaling as well as its mediation in androgen-independent cell growth in prostate cancer cells. Here we found that H(2)S inhibits cell proliferation of both androgen-dependent (LNCaP) and antiandrogen-resistant prostate cancer cells (LNCaP-B), with more significance on the latter, which was established by long term treatment of parental LNCaP cells with bicalutamide. The expression of cystathionine γ-lyase (CSE), a major H(2)S producing enzyme in prostate tissue, was reduced in both human prostate cancer tissues and LNCaP-B cells. LNCaP-B cells were resistant to bicalutamide-induced cell growth inhibition, and CSE overexpression could rebuild the sensitivity of LNCaP-B cells to bicalutamide. H(2)S significantly repressed the expression of prostate-specific antigen (PSA) and TMPRSS2, two AR-targeted genes. In addition, H(2)S inhibited AR binding with PSA promoter and androgen-responsive element (ARE) luciferase activity. We further found that AR is post-translationally modified by H(2)S through S-sulfhydration. Mutation of cysteine 611 and cysteine 614 in the second zinc finger module of AR-DNA binding domain diminished the effects of H(2)S on AR S-sulfhydration and AR dimerization. These data suggest that reduced CSE/H2S signaling contributes to antiandrogen-resistant status, and sufficient level of H(2)S is able to inhibit AR transactivation and treat castration-resistant prostate cancer.

Here we explored the impact of hydrogen sulfide (H2S) on biophysical properties of the primary human airway smooth muscle (ASM)–the end effector of acute airway narrowing in asthma. Using Magnetic Twisting Cytometry (MTC), we measured dynamic changes in the stiffness of isolated ASM, at the single-cell level, in response to varying doses of GYY4137 (1–10 mM). GYY4137 slowly released appreciable levels of H2S in the range of 10–275 µM, and H2S released was long lived. In isolated human ASM cells, GYY4137 acutely decreased stiffness (i.e. an indicator of the single-cell relaxation) in a dose-dependent fashion, and stiffness decreases were sustained in culture for 24h. Human ASM cells showed protein expressions of cystathionine-γ-lyase (CSE; a H2S synthesizing enzyme) and ATP-sensitive potassium (KATP) channels. The KATP channel opener pinacidil effectively relaxed isolated ASM cells. In addition, pinacidil-induced ASM relaxation was completely inhibited by the treatment of cells with the KATP channel blocker glibenclamide. Glibenclamide also markedly attenuated GYY4137-mediated relaxation of isolated human ASM cells. Taken together, our findings demonstrate that H2S causes the relaxation of human ASM and implicate as well the role for sarcolemmal KATP channels. Finally, given that ASM cells express intrinsic enzymatic machinery of generating H2S, we suggest thereby this class of gasotransmitter can be further exploited for potential therapy against obstructive lung disease.

Phenotype modulation of pulmonary artery smooth muscle cells (PASMCs) plays an important role during hypoxia-induced vascular remodeling and pulmonary hypertension (PAH). We had previously shown that calcium-sensing receptor (CaSR) is expressed in rat PASMCs. However, little is known about the role of CaSR in phenotypic modulation of PASMCs in hypoxia-induced PAH as well as the underlying mechanisms. In this study, we investigated whether CaSR induces the proliferation of PASMCs in small pulmonary arteries from both rats and human with PAH. PAH was induced by exposing rats to hypoxia for 7–21 days. The mean pulmonary arterial pressure (mPAP), right ventricular hypertrophy index (RVI), the percentage of medial wall thickness to the external diameter (WT %), and cross-sectional total vessel wall area to the total area (WA %) of small pulmonary arteries were determined by hematoxylin and eosin (HE), masson trichrome and Weigert’s staining. The protein expressions of matrix metalloproteinase (MMP)-2 and MMP-9, the tissue inhibitors of metalloproteinase (TIMP)-3, CaSR, proliferating cell nuclear antigen (PCNA), phosphorylated extracellular signal-regulated kinase (p-ERK), and smooth muscle cell (SMC) phenotype marker proteins in rat small pulmonary arteries, including calponin, SMα-actin (SMAα), and osteopontin (OPN), were analyzed by immunohistochemistry and Western blotting, respectively. In addition, immunohistochemistry was applied to paraffin-embedded human tissues from lungs of normal human and PAH patients with chronic heart failure (PAH/CHF). Compared with the control group, mPAP, RVI, WT % and WA % in PAH rats were gradually increased with the prolonged hypoxia. At the same time, the expressions of CaSR, MMP-2, MMP-9, TIMP-3, PCNA, OPN, and p-ERK were markedly increased, while the expressions of SMAα and calponin were significantly reduced in lung tissues or small pulmonary arteries of PAH rats. Neomycin (an agonist of CaSR) enhanced but NPS2390 (an antagonist of CaSR) weakened these hypoxic effects. We further found that the expression change of CaSR, PCNA, and SMC phenotypic marker proteins in PAH/CHF lungs was similar to those in PAH rats. Our data suggest that CaSR is involved in the pulmonary vascular remodeling and PAH by promoting phenotypic modulation of small pulmonary arteries.

The physiological and pathological roles of dopamine D2 receptors (DR2) in the regulation of cardiovacular functions have been recognized. DR2 activation protects hypoxia/reoxygenation (H/R)-induced cardiomyocyte injury and apoptosis, and ischemic post-conditioning (PC) plays a critical role in cardioprotection as well; however the involvement of the DR2 activation in the PC-induced cardioprotection is unknown. In the present study, we found that the H/R increased the expressions of DR2 mRNA and protein in cardiomyocytes, which were significantly enhanced by PC. Bromocriptine (Bro, a DR2 agonist) further increased DR2 expression, but Haloperidol (Hal, a DR2 antagonist) reversed the Bro-induced DR2 expressions. PC protected against H/R-induced apoptosis, the rise of [Ca(2+)]i, the expressions of cleaved caspase-3 and -9, release of cytochrome c, and mPTP opening. In addition, PC counteracted the reduction of cell viability caused by H/R, increased the phosphorylation of ERK1/2, PI3K, Akt, GSK-3β and mitochondrial membrane potential. PC further increased Bcl-2 expression, promoted PKC-e translocation to cell membrane, and activated the mitochondrial ATP-sensitive K channels (mKATP). Bro further enhanced the cardioprotective roles of PC, but Hal reversed these effects of Bro. Meanwhile, we found that DR2 was expressed in cell membrane and interacted with PKC-e in PC. In conclusion, these results suggest that PC attenuates cardiomyocyte apoptosis via inhibition of mPTP opening by DR2-mediated activation of ERK1/2, PI3K-Akt-GSK-3β and PKC-e-mKATP. These findings provide a novel target for the treatment of ischemic cardiomyopathy.

Dopamine D2 receptors (DR2) are important regulators in many organs, including cardiac system. Protein kinase C (PKC) activation and translocation is associated with cardioprotection against ischemic post-conditioning (PC); however, the regulatory role of DR2 during this process has been unknown. This study hypothesized that the prevention of cardiomyocyte damage by DR2 activation is associated with PKC translocation to the cell membrane. In the present study, we found that the ischemia/reperfusion (I/R) increased the expressions of DR2 mRNA and protein, which were further enhanced by PC. Bromocriptine (DR2 agonist) up-regulated the PC-induced DR2 expressions, and Haloperidol (DR2 antagonist) reversed the increase of DR2 expressions by Bromocriptine. PC reduced I/R-induced cardiomyocytes damage, apoptosis and myocardial infarct size, and improved cardiac function. Compared with PC, Bromocriptine further enhanced the cardioprotective roles of PC, but Haloperidol canceled the protection effect of Bromocriptine. PC up-regulated PKC-e translocation in the particulate fraction, which was further strengthened by Bromocriptine but canceled by Haloperidol. In the cytosolic fraction, the changes of the PKC-e translocation were opposite to the particulate fraction. These findings suggest that DR2 activation provides cardioprotection via promoting PC-induced translocation of PKC-e.

The calcium-sensing receptor (CaSR), a G protein coupled receptor, is involved in a number of physiological and pathological processes. Embryonic stem cells (ESCs) have a potential role to differentiate into all types of cells. Whether CaSR is functionally expressed in ESCs is unclear. In this study, the expression and distribution of CaSR in 129 mouse ES-D3 cell lines were detected by Western blotting and immunofluorescence; and the intracellular calcium concentration ([Ca(2+)]i) was measured using Laser Confocal Scanning Microscopy. Mouse embryonic stem cells (mESCs) were cultured to embryoid bodies (EBs) and the differentiation of EBs into cardiomyocytes was induced by icariin (ICA). The cardiac specific proteins, a-Actinin and cardiac troponin-I (cTnI), were analyzed by immunofluorescence, and the differentiation rate was analyzed by flow cytometry. The expression of cardiac-specific transcription factors, Nkx2.5 and GATA-4, was detected by Western blotting. We found that the CaSR protein exists in both mESCs and mESC-derived cardiomyocytes (mESC-CMs). Increasing extracellular calcium or neomycin (an agonist of CaSR) increased [Ca(2+)]i and the differentiation rate. These effects were abolished by inhibition of CaSR, phospholipase C, IP3 receptor and Ca(2+) ATPase, or by depletion of the sarcoplasmic reticulum Ca(2+) store, respectively. Activation of CaSR up-regulated protein expression of Nkx2.5 and GATA4 in EBs at an early stage of ICA-induced differentiation. In conclusion, CaSR is functionally expressed in mESCs, and activation of CaSR is involved in the differentiation of mESCs into cardiomyocytes by facilitating the expression of NKx2.5 and GATA-4.

In the study, we investigated how exogenous H(2)S (hydrogen sulfide) influenced streptozotocin (STZ)-induced diabetic myocardial injury through cardiac mitochondrial protection and nitric oxide (NO) synthesis in intact rat hearts and primary neonatal rat cardiomyocytes. Diabetes was induced by STZ (50 mg/kg) and the daily administration of 100 μM NaHS (sodium hydrosulfide, an H(2)S donor) in the diabetes + NaHS treatment group. At the end of 4, 8, and 12 weeks, the morphological alterations and functions of the hearts were observed using transmission electron microscopy and echocardiography system. The percentage of apoptotic cardiomyocytes, the mitochondrial membrane potential, the production of reactive oxygen species (ROS) and the level of NO were measured. The expressions of cystathionine-γ-lyase (CSE), caspase-3 and -9, the mitochondrial NOX4 and cytochrome c were analyzed by western blotting. The results showed the cardiac function injured, morphological changes and the apoptotic rate increased in the diabetic rat hearts. In the primary neonatal rat cardiomyocytes of high glucose group, ROS production was increased markedly, whereas the expression of CSE and the level of NO was decreased. However, treatment with NaHS significantly reversed the diabetic rat hearts function, the morphological changes and decreased the levels of ROS and NO in the primary neonatal rat cardiomyocytes administrated with high glucose group. Furthermore, NaHS down-regulated the expression of mitochondrial NOX4 and caspase-3 and -9 and inhibited the release of cytochrome c from mitochondria in the primary neonatal rat cardiomyocytes. In conclusion, H(2)S is involved in the attenuation of diabetic myocardial injury through the protection of cardiac mitochondria.