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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 (H

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.

Gasotransmitters are small molecules of gases, including nitric oxide (NO), hydrogen sulfide (H2S), and carbon monoxide (CO). These three gasotransmitters can be endogenously produced and regulate ...

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.

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 (H(2)S) 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 H(2)S signaling in biology and medicine, this review proposed H(2)S as a potential defense against COVID-19. It is suggested that H(2)S 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 H(2)S donor(s) or the activators of endogenous H(2)S-generating enzymes should be considered as a preventative treatment or therapy for COVID-19.

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 (HBoth in vitro cell model and in vivo animal model of lipid accumulation were used in this study. Western blotting and real-time PCR were used for analysis of protein and mRNA expression. Acetyl-CoA was analyzed by a coupled enzyme assay, and lipid accumulation was observed with Oil Red O staining.Incubation of human liver carcinoma (HepG2) cells with a mixture of free fatty acids (FFAs) or high glucose reduced CSE expression and HCSE/H

With the development of the precision guidance technology, the earth-penetrating weapon (EPW) is a huge threat to infrastructures. The objective of this research is to investigate the damage characteristics of mass concrete targets under the combined action of penetration and explosion. The validity of the penetration model is discussed by reproducing a previous experimental test reported in the literature. Meanwhile, a field test about the internal explosion in a concrete cube is conducted to verify the validity of the internal explosion model. Subsequently, damage characteristics of a mass concrete target from the upper part of a concrete gravity dam are discussed when subjected to the combined action of the penetration and explosion. In order to improve the structural performance of the mass concrete target to penetration and explosion loadings, high strength concrete material is selected as a preventive measure. Penetration processes and damage patterns of the mass target with normal and high strength concrete material are compared. Then, three internal explosion models are presented to investigate the influence of the initial penetration damage on the failure characteristics of the mass concrete target. The results show that the resistance of the mass target to the combined action of the penetration and explosion can be enhanced significantly by using the high strength concrete material. The initial penetration damage has a significant influence on the damage processes of the mass concrete target subjected to internal blast loading.

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.

Recently, external terrorist activities have become one of the most influential events on structural safety because of the absence of proper mechanisms to detect these events. In this study, the effects of surface explosions on the dynamic response and blast resistance of a submarine tunnel are investigated by using a coupled Lagrange and Euler (CLE) method. The feasibility and accuracy of the numerical method and material models are verified against the experimental data. After that, the numerical model is utilized to investigate the dynamic behavior and damage evolution of the submarine tunnel subjected to surface explosions. The dynamic behavior of the tunnel under various detonation scenarios in terms of the explosive weight and water depth is explored. Both the localized damage mechanism and the global structural response of the tunnel are examined. Empirical formulas are proposed to predict the failure modes of tunnel. Besides, studies of tunnel protection against potential attacks by using carbon fibre reinforced polymer (CFRP) and ultra-high performance concrete (UHPC) are also discussed. Numerical results in this study provide tunnel owners and engineers with thorough and important information on the structural performance of submarine tunnels subjected to blast loads, helping them in choosing effective protection strategies for potential explosion events.

The damage prediction of high dams under the attacks of earth-penetrating weapons has gained significant importance in recent years. For this purpose, a SPH-Lagrangian-Eulerian coupled approach is proposed to describe the damage processes of concrete gravity dams subjected to the combined action of the penetration and explosion. The SPH method is used to model the concrete material with the large deformation near the penetration and explosion regions. The Lagrangian algorithm is adopted to simulate the high-velocity projectile and dam body with the small distortion. And the Eulerian algorithm is employed to describe the dynamic behavior of the water and air media. The validity of the penetration model is calibrated against a previous penetration test. Meanwhile, the SPH-Lagrangian-Eulerian coupled method is verified by implementing an underwater explosion test in a concrete cube. The computed distribution of cracking damage is consistent with the result of the experimental test, which validates the validity of the proposed SPH-Lagrangian-Eulerian coupling method. Subsequently, the penetration processes of a concrete gravity dam under the high-velocity projectile are presented. After the rapid penetration, the explosives are detonated in the dam with the initial penetration damage. The shock wave propagation characteristics in the dam and reservoir water are discussed. The failure processes and dynamic responses of the dam subjected to the combined action of the penetration and explosion are investigated. The influence of the initial penetration damage and the reservoir water on the failure processes of the dam subjected to the internal blast loading is also discussed. The results show that the penetration of the high-velocity projectile only causes a local damage to the concrete gravity dam. However, the combined effects of the penetration and explosion cause significantly more damage to the upper region of the dam.

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 ubiquitous and versatile signal-transduction role of hydrogen sulfide (H2S) has been revealed in mammals and plants in recent Years. It is very important to grasp the physiological concentrations of H2 S; in vivo because of its toxic characteristics at high concentrations. However, the reported data fail to reach an agreement owing to the use of different methods and technique limitations. In fact, this is a common problem for gasotransmitters. What is the exact boundary between the physiological and toxicological concentration? What is the accurate endogenous content? These problems have been plaguing us even though there are a great number of research papers that explore H2S signals.

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.

To increase the tolerance of Chinese cabbage (Brassica campestris L. ssp. pekinensis) to Pieris rapae, we introduced a modified cowpea trypsin inhibitor (CpTI) gene, SCK, into various cultivars. SCK was derived from CpTI, an insect-resistance gene. The protein accumulating capacity of CpTI can be improved by adding a signal peptide sequence at the 5′ end and an endoplasmic reticulum-detained signal sequence at the 3′ end. Using an optimized Agrobacterium tumefaciens-mediated transformation system in Chinese cabbage, we obtained a maximum transformation efficiency of ~6.83%. Insect resistance tests and CpTI enzymatic assays showed that most of the transgenic plants had significant resistance to cabbage worm (Pieris rapae) larvae and that the plants with the highest levels of insect resistance had the greatest CpTI-related capacity, indicating a high correlation between SCK expression and insect resistance. An evaluation of segregation patterns in the independent transgenic line with the highest insect resistance, ‘ZB-08-04’, showed that kanamycin resistant versus sensitive plants segregated in a 3:1 Mendelian fashion. This study provides a potential germplasm resource for Chinese cabbage breeding in the future.

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.

Cysteine is the first organic molecule generated during the assimilation of sulfate. As such, cysteine and its derivatives are always essential signal molecules and thus have important roles in the regulation of many plant processes. O-acetylserine (thiol) lyase (OASTL) catalyzes the last step of the biosynthesis of cysteine. At present, detailed and comprehensive work about these enzymes has only been reported from the plant Arabidopsis thaliana, though sporadic studies on OASTL have been conducted on other dicots, such as spinach and soybean. However, few reports on the functions of OASTLs in monocots have been found in the literature. Here in this study, we obtained four SiOASTL genes (SiOASTL7, SiOASTL8, SiOASTL9 and SiOASTL10) from foxtail millet and analyzed their potential functions. Phylogenetically, the four SiOASTL genes did not belong to any published subfamily of the OASTL genes; instead they constituted a new subfamily specific to the OASTL genes from monocots. In sequencing, we found that with the exception of the pseudogene SiOASTL8, proteins encoded by the other three genes exhibited high similarity with OASTL proteins from Arabidopsis, though the critical PLP-binding sites of both SiOASTL7 and SiOASTL10 were missing. The enzymatic activity assays demonstrated that SiOASTL9 has the ability to catalyze the biosynthesis of both cysteine and S-sulfocysteine, while SiOASTL7 and SiOASTL10 did not possess any previously reported catalyzing abilities. In addition, the gene expression pattern analysis showed that all four genes were widely expressed in various tissues of foxtail millet, and all had a preference in the leaves. Under abiotic stresses, the expression of these genes could be induced by salt and drought stress. Our finding that cadmium could only up-regulate the transcription of SlOASTL8 and SlOASTL9, further indicates the diversified responses of SiOASTLs to abiotic stresses.

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.

Hydrogen sulfide (H2S) has been reported as a significant gasotransmitter, which involved in a range of physiological processes in plant, including growth, development and abiotic stresses resistence. Enzymatic activity assays showed that L-cysteine desulfhydrase (L-CD) was primarily responsible for the generation of endogenous H2S in plant. In this work, we identified two activated L-CDs from cucumber 9930 varieties. Firstly, three L-CD genes, CsaLCD, CsaDES1 and CsaDES2 were acquired from tBLASTn algorithm-based search of NCBI and a cucumber genomic database ( http://www.icugi.org ), using the Arabidopsis LCD and DES1 protein sequences as the query. The CDS of CsaLCD (Csa2P034800.1) and CsaDES2 (Csa1P574810.1) were amplified successfully by PCR, while the CsaDES1 (Csa1P574800.1) could not be obtained in the case of cDNA as the template. The recombinant proteins His-CsaLCD and His-CsaDES2, which were purified from E.coli BL21 strain, exhibited evident L-CD activity. The mRNA level of CsaLCD was induced by 37 °C, low light or PEG8000 treatments, and CsaDES2 was induced by 4 °C or 37 °C treatments, while CsaDES1 itself has no expression. The CDS of CsaLCD or CsaDES2 with 35S promoter were transformated into lcd/des mutant of Arabidopsis, and both their heterologous expression restored the H2S production rate, as well as enhancing the resistance to osmosis stress of the transgenetic seedlings. Therefore, CsaLCD and CsaDES2 are the main enzymes for endogenous H2S production in cucumber, and play a pivotal role in H2S generation during stresses responses.

The importance of H2S in biology and medicine has been widely recognized in recent years, and protein S-sulfhydration is proposed to mediate the direct actions of H2S bioactivity in the body. Thioredoxin 1 (Trx1) is an important reducing enzyme that cleaves disulfides in proteins and acts as an S-denitrosylase. The regulation of Trx1 on protein S-sulfhydration is unclear. Here we showed that Trx1 facilitates protein S-desulfhydration. Overexpression of Trx1 attenuated the basal level and H2S-induced protein S-sulfhydration by direct interaction with S-sulfhydrated proteins, i.e., glyceraldehyde 3-phosphate dehydrogenase and pyruvate carboxylase. In contrast, knockdown of Trx1 mRNA expression by short interfering RNA or blockage of Trx1 redox activity with PX12 or 2,4-dinitrochlorobenzene enhanced protein S-sulfhydration. Mutation of cysteine-32 but not cysteine-35 in the Trp–Cys32–Gly–Pro–Cys35 motif eliminated the binding of Trx1 with S-sulfhydrated proteins and abolished the S-desulfhydrating effect of Trx1. All these data suggest that Trx1 acts as an S-desulfhydrase.

Background Cystathionine gamma-lyase (CSE)-derived hydrogen sulfide (H 2 S) possesses diverse roles in the liver, affecting lipoprotein synthesis, insulin sensitivity, and mitochondrial biogenesis. H 2 S S -sulfhydration is now proposed as a major mechanism for H 2 S-mediated signaling. Pyruvate carboxylase (PC) is an important enzyme for gluconeogenesis. S -sulfhydration regulation of PC by H 2 S and its implication in gluconeogenesis in the liver have been unknown. Methods Gene expressions were analyzed by real-time PCR and western blotting, and protein S -sulfhydration was assessed by both modified biotin switch assay and tag switch assay. Glucose production and PC activity was measured with coupled enzyme assays, respectively. Results Exogenously applied H 2 S stimulates PC activity and gluconeogenesis in both HepG2 cells and mouse primary liver cells. CSE overexpression enhanced but CSE knockout reduced PC activity and gluconeogenesis in liver cells, and blockage of PC activity abolished H 2 S-induced gluconeogenesis. H 2 S had no effect on the expressions of PC mRNA and protein, while H 2 S S -sulfhydrated PC in a dithiothreitol-sensitive way. PC S -sulfhydration was significantly strengthened by CSE overexpression but attenuated by CSE knockout, suggesting that H 2 S enhances glucose production through S -sulfhydrating PC. Mutation of cysteine 265 in human PC diminished H 2 S-induced PC S -sulfhydration and activity. In addition, high-fat diet feeding of mice decreased both CSE expression and PC S -sulfhydration in the liver, while glucose deprivation of HepG2 cells stimulated CSE expression. Conclusions CSE/H 2 S pathway plays an important role in the regulation of glucose production through S -sulfhydrating PC in the liver. General significance Tissue-specific regulation of CSE/H 2 S pathway might be a promising therapeutic target of diabetes and other metabolic syndromes.

Aims Hydrogen sulfide (H2S) as a novel gasotransmitter can be endogenously produced in liver by cystathionine gamma-lyase (CSE). The dysfunctions of CSE/H2S system have been linked to various liver diseases. Acetyl-CoA is the key intermediate from the metabolism of lipid. This study examined the roles of H2S in hepatic acetyl-CoA and lipid metabolism. Materials and methods Both in vitro cell model and in vivo animal model of lipid accumulation were used in this study. Western blotting and real-time PCR were used for analysis of protein and mRNA expression. Acetyl-CoA was analyzed by a coupled enzyme assay, and lipid accumulation was observed with Oil Red O staining. Key findings Incubation of human liver carcinoma (HepG2) cells with a mixture of free fatty acids (FFAs) or high glucose reduced CSE expression and H2S production, promoted intracellular accumulation of acetyl-CoA and lipid. Supply of exogenous NaHS or cysteine reduced acetyl-CoA contents and lipid accumulation, while blockage of CSE activity promoted intracellular lipid accumulation. Furthermore, H2S blocked FFAs-induced transcriptions of de novo lipogenesis, inflammation, and fibrosis-related genes. In vivo, knockout of CSE gene stimulated more hepatic acetyl-CoA and lipid accumulation in mice induced by high-fat choline-deficient diet. The expressions of lipogenesis, inflammation, and fibrosis-related genes were significantly higher in liver tissues from CSE knockout mice when compared with wild-type mice. Significance CSE/H2S system is indispensable for maintaining the homeostasis of acetyl-CoA and lipid accumulation and protecting from the development of inflammation and fibrosis in liver under excessive caloric ingestion.

Diabetic cardiomyopathy (DCM) is a severe complication of type 1 diabetic (T1D) patients, manifested as combined diastolic and systolic dysfunction. DCM is associated with impaired calcium homeostasis secondary to decreased calcium-sensitive receptor (CaSR) expression. Spermine, a direct agonist of CaSR, was found deficient in cardiomyocytes of T1D rats. However, the role of spermine in DCM was unclear. Here, we examined the cardioprotective effect of exogenous spermine on DCM in streptozotocin (STZ) induced-T1D rats and high-glucose (HG)-incubated neonatal rat cardiomyocytes. Exogenous spermine significantly attenuated cardiac dysfunction in T1D rats, characterized by improved echocardiography, less fibrosis, reduced myocardial endoplasmic reticulum (ER) stress and oxidative stress, and increased expression of myocardial membrane CaSR. In cultured neonatal rat cardiomyocytes, exogenous spermine attenuated myocardial injury induced by HG treatment, demonstrated by restored cellular glucose uptake capacity, reduced expression of apoptotic markers, lowered level of oxidative stress, ER stress and unfolded protein response, and upregulated cell membrane CaSR. Mechanistically, the cardioprotective effect of spermine appeared dependent upon effective elimination of reactive oxygen species (ROS) and up-regulation of CaSR expression by suppressing the Nrf2-ROS-p53-MuRF1 axis. Taken together, these results suggest that exogenous spermine protects against DCM in vivo and in vitro, partially via suppressing ROS and p53-mediated downregulation of cell membrane CaSR.

The damage prediction and protective measures of tunnels subjected to blast loads have gained importance in recent years due to increasing accidental events and terrorist attacks. This paper performs numerical modeling to assess the damage characteristics of an underwater tunnel subjected to blast loads and explore the potential mitigation measures. Firstly, a three-dimensional numerical model of an underwater tunnel based on coupled Lagrange and Euler (CLE) method is developed. The accuracy of the CLE method and material models is verified with blast testing data. Subsequently, the dynamic response and damage evolution of the tunnel subjected to underwater explosions are investigated. Potential failure patterns due to various underwater blast loads are explored. Intensive numerical simulations are then carried out to investigate the damage levels of the tunnel. Based on numerical data, empirical relations are suggested to predict the tunnel damage levels based on charge weight and standoff distances. CFRP (carbon fiber reinforced polymer) cloth is used to improve the resistance of the tunnel to underwater explosions. Mitigation effects of different strengthening schemes are investigated, and the optimum strengthening thickness of the CFRP cloth is recommended. The results show that the rigidity and load carrying ability of the tunnel are significantly improved by bonding the CFRP cloth. The recommended thickness of the CFRP cloth is 0.5–0.835 mm.

Hydrogen sulfide (HHuman primary hepatocellular carcinoma cell line (HepG2) and doxorubicin (Dox)-resistant cells were used in this study. Cell survival was analyzed by MTT, Annexin V-FITC/propidium iodide staining and clonogenic assay. Western blotting was used for analysis of protein expression, and immunoprecipitation was used to determine interactions of LXR/RXR.The expression of HOur study provides a novel solution for reversing drug resistance in cancer cells by targeting H

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. H

Aims: Hydrogen sulfide (H2S) inhibits the proliferation of vascular smooth muscle cells (VSMCs). However, how cystathionine-gamma-lyase (CSE), a major enzyme that produces H2S, is regulated remains unknown. Whether calcium-sensing receptor (CaSR) inhibits the proliferation of VSMCs by regulating the endogenous CSE/H2S pathway in diabetic rat has not been previously investigated. Methods and Results: The morphological and ultrastructure alterations were tested by transmission electron microscopy, changes in the H2S concentration and the relaxation of the mesenteric secondary artery loop of diabetic rats were determined by Multiskan spectrum microplate spectrophotometer and isometric force transducer. Additionally, the expression levels of CaSR, CSE and Cyclin D1 in the mesenteric arteries of rats were examined by western blotting. The intracellular calcium concentration, the expression of p-CaMK II (phospho-calmodulin kinases II), CSE activity, the concentration of endogenous H2S and the proliferation of cultured VSMCs from rat thoracic aortas were measured by using confocal microscope, western blotting, microplate spectrophotometer, MTT and BrdU, respectively. The VSMC layer thickened, the H2S concentration dropped, the relaxation of the mesenteric secondary artery rings weakened, and the expression of CaSR and CSE decreased whereas the expression of Cyclin D1 increased in diabetic rats compared with the control group. The [Ca2+]i of VSMCs increased upon treatment with CaSR agonists (10 µM Calindol and 2.5 mM CaCl2), while it decreased upon administration of calhex231, U73122 and 2-APB. The expression of p-CaMK II and CSE increased upon treatment with CaSR agonists in VSMCs. CSE activity and the endogenous H2S concentration decreased in response to high glucose, while it increased with treatment of CaSR agonists. The proliferation rate increased in response to high glucose, and CaSR agonists or NaHS significantly reversed the proliferation of VSMCs caused by high glucose. Conclusions: Our results demonstrated that CaSR regulated the endogenous CSE/H2S pathway to inhibit the proliferation of VSMCs in both diabetic and high glucose models.

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.

Abnormal hepatic lipid metabolism is a key component of fatty liver development with excess fat deposition in the liver through steatosis. Cystathionine gamma-lyase (CSE) is one of the enzymes that catalyze hydrogen sulfide (H2S) production in the liver. The aim of the present study was to investigate the role of CSE/H2S in hepatic regulation of cholesterol and fatty acid metabolism. Wild-type (WT) and CSE knockout (CSE-KO) mice fed with high-fat diet (HFD) were analyzed for liver morphological and biochemical changes. HFD feeding of CSE-KO mice, not WT mice, markedly increased cholesterol levels in plasma and livers, and the sizes of the liver and gall bladder. Typical histological and biochemical changes of fatty liver disease were found in CSE-KO mice with damaged liver functions. The levels of plasma and liver triglyceride were significantly lower in HFD-fed CSE-KO mice than in HFD-fed WT mice. Moreover, the expression of nuclear receptors transcriptional factors, especially LXRα, in the liver was decreased in both control diet- or HFD-fed CSE-KO mice. Decreased expression of CYP7A1, an LXRα targeted gene, halted catabolism of cholesterol into bile and subsequently led to cholesterol accumulation in the liver and gall bladder. Since deficiency in CSE/H2S pathway results in high susceptibility to HFD-induced fatty liver, targeting at CSE/H2S pathway in the liver may represent a novel strategy against the development of fatty liver damage.

Protein S-sulfhydration is a newly discovered post-translational modification of specific cysteine residue(s) in target proteins, which is involved in a broad range of cellular functions and metabolic pathways. By changing local conformation and the final activity of target proteins, S-sulfhydration is believed to mediate most cellular responses initiated by H2S, a novel gasotransmitter. In comparison to protein S-sulfhydration, nitric oxide-mediated protein S-nitrosylation has been extensively investigated, including its formation, regulation, transfer and metabolism. Although the investigation on the regulatory mechanisms associated with protein S-sulfhydration is still in its infancy, accumulated evidence suggested that protein S-sulfhydration may share similar chemical features with protein S-nitrosylation. Glutathione persulfide acts as a major donor for protein S-sulfhydration. Here, we review the present knowledge on protein S-sulfhydration, and also predict its formation and regulation mechanisms based on the knowledge from protein S-nitrosylation.

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.

Hydrogen sulfide (H

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...

Background— Cystathionine γ-lyase (CSE) produces hydrogen sulfide (H 2 S) in the cardiovascular system. The deficiency of CSE in mice leads to a decreased endogenous H 2 S level, an age-dependent increase in blood pressure, and impaired endothelium-dependent vasorelaxation. To date, there is no direct evidence for a causative role of altered metabolism of endogenous H 2 S in atherosclerosis development. Methods and Results— Six-week-old CSE gene knockout and wild-type mice were fed with either a control chow or atherogenic paigen-type diet for 12 weeks. Plasma lipid profile and homocysteine levels, blood pressure, oxidative stress, atherosclerotic lesion size in the aortic roots, cell proliferation, and adhesion molecule expression were then analyzed. CSE-knockout mice fed with atherogenic diet developed early fatty streak lesions in the aortic root, elevated plasma levels of cholesterol and low-density lipoprotein cholesterol, hyperhomocysteinemia, increased lesional oxidative stress and adhesion molecule expression, and enhanced aortic intimal proliferation. Treatment of CSE-knockout mice with NaHS, but not N -acetylcysteine or ezetimibe, inhibited the accelerated atherosclerosis development. Double knockout of CSE and apolipoprotein E gene expression in mice exacerbated atherosclerosis development more than that in the mice with only apolipoprotein E or CSE knockout. Conclusions— Endogenously synthesized H 2 S protects vascular tissues from atherogenic damage by reducing vessel intimal proliferation and inhibiting adhesion molecule expression. Decreased endogenous H 2 S production predisposes the animals to vascular remodeling and early development of atherosclerosis. The CSE/H 2 S pathway is an important therapeutic target for protection against atherosclerosis.

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.

L-type voltage-dependent Ca(2+) channels (VDCCs) in pancreatic beta cells play a critical role in regulating insulin secretion. The gasotransmitter H(2)S is mostly generated from L-cysteine in pancreatic beta cells by cystathionine γ-lyase (CSE) and has been reported to inhibit insulin release by opening ATP-sensitive K(+) channels. However, whether and how H(2)S affects VDCCs in beta cells is unknown.The whole-cell patch-clamp technique was used to record VDCCs in beta cells from Cse (also known as Cth)-knockout (KO) and wild-type (WT) mice. Insulin secretion from pancreatic islets and endogenous H(2)S production in pancreas were measured.The H(2)S donor NaHS reversibly decreased L-type VDCC current density in a concentration-dependent fashion in WT pancreatic beta cells, and the current density was further inhibited by nifedipine. Furthermore, NaHS inhibited the channel recovery from depolarisation-induced inactivation, but did not shift the current-voltage (I-V) relationship. ACS67, another H(2)S donor, also inhibited L-type VDCCs in beta cells. Inhibiting CSE activity with DL-propargylglycine increased the basal L-channel activity of beta cells from WT mice, but not that of beta cells from Cse-KO mice. Beta cells from Cse-KO mice displayed higher L-type VDCC density than those from WT mice. Insulin secretion from pancreatic islets was elevated in Cse-KO mice compared with WT mice. NaHS dose-dependently inhibited glucose-stimulated insulin secretion, which was further inhibited by nifedipine. Bay K-8644 increased glucose-stimulated insulin secretion, but this was counteracted by NaHS and nifedipine.Exogenous and endogenous H(2)S inhibit L-type VDCC activity and pancreatic insulin secretion, constituting a novel mechanism for the regulation of insulin secretion by the CSE/H(2)S system.

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.

Hydrogen sulfide (H 2 S), once regarded as a toxic gas with a very bad smell of rotten eggs, now is recognized as an important gasotransmitter and regulates numerous physiological and pathophysiological processes in our body. Recent studies have demonstrated that H 2 S is able to directly alter protein activity by modifying the free thiol groups in the target cysteine residue(s) to form persulfides, a process termed as protein S -sulfhydration, which mediates the major bioactivities of H 2 S in cellular functions. Compared with all other tissues, liver generates huge amount of H 2 S mainly from the enzyme cystathionine gamma-lyase. H 2 S participates in the regulation of liver functions and attenuates fatty liver development. In this research highlight, I discuss the latest published findings on H 2 S regulation of liver glucose generation and metabolism via posttranslational modification of gluconeogenic enzymes.

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 pathological roles of hydrogen sulfide (H(2)S) in the regulation of cardiovacular functions have been recognized. Vascular smooth muscle cells (SMCs) express cystathionine gamma-lyase (CSE) and produce significant amount of H(2)S. Although growing evidence demonstated the anti-atherosclerotic effect of H(2)S, less is known about the contribution of the endogenous CSE/H(2)S pathway to the development of vascular remodeling. This study investigated the roles of the CSE/H(2)S pathway on SMC migration and neoimtimal formation by using CSE knockout (KO) mice. SMCs and aortic explants isolated from CSE KO mice exhibited more migration and outgrowth compared with that from wild-type (WT) mice, and exogenously applied NaHS (a H(2)S donor) at 100 μM significantly inhibited SMC migration and outgrowth. SMCs became more elongated and spread in the absence of CSE, and fibronectin significantly stimulated adhesion and migration of SMCs from CSE KO mice (KO-SMCs) in comparison with SMCs from WT mice (WT-SMCs). The expressions of α5- and β1-integrins were significantly higher in KO-SMCs, and functional blocking of α5β1-integrin effectively abrogated KO-SMC migration. CSE deficiency also enhanced matrix metalloproteinase-2 (MMP-2) expression, and the selective blocking of MMP-2 decreased KO-SMC migration. NaHS treatment decreased both the expressions of α5- and β1-integrins and MMP-2. We further found that the expressions of α5- and β1-integrins as well as MMP-2, were stimulated by fibronectin, and that the blockage of α5β1-integrin reduced but overexpression of α5β1-integrin induced MMP-2 expression in both WT-SMCs and KO-SMCs. We also noticed that CSE deficiency in mice led to increased neointima formation in carotid arteries 4 weeks after ligation, which were attenuated by NaHS administration. In conclusion, inhibition of SMC migration by H(2)S may be a novel target for the treatment of vascular occlusive disorder.