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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 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.

Reinforced concrete (RC) is one of the most commonly used construction materials. However, impact and blast loads could completely destroy RC structures, causing tremendous casualties and property losses. Therefore, analyzing the damage mechanism and dynamic behaviour of RC structures under blast loading is significant. This paper presents a study on the behaviour of RC slabs subjected to air and underwater contact detonation. Two RC slabs were tested under real blast loads. One was tested under air contact explosion of 210 g TNT, the other was tested subjected to underwater contact explosion of 6 g TNT. Damage evolution and damage mechanism of the RC slabs subjected to the air and underwater contact explosions are investigated by using high-fidelity numerical models. The damage patterns obtained from the numerical models match well with those of the tests. The effect of experimental environments on the structural behaviour of the slabs subjected to the underwater explosions is also discussed. The different response of the RC slabs subjected air and underwater explosions with identical charge mass are analyzed contrastively by employing the validated coupled method. The results show that the present model can give a reliable prediction of damage characteristics for RC structures under various blast scenarios. The experimental environments have important influence on the structural response and bubble development. The damage of the RC slabs induced by underwater contact explosion is significantly larger than that caused by air contact blast with equal charge mass.

Columns are the key load-bearing elements in infrastructures. Common cross-section shapes of columns are square and circle. Those columns may be subjected to various blasting scenarios. This paper investigates the effects of cross-section shape on the anti-knock performance of RC columns subjected to air and underwater blast loads. The reliability of the fully coupled Lagrangian-Eulerian numerical model is verified by comparing with the results of the field blast test. The damage characteristics and dynamic response of the RC column with different cross-section shapes subjected to contact and close-in air explosions are investigated. The anti-knock performance of the RC column with different cross-section shapes subjected to contact and close-in underwater explosion is also studied. Nonlinear dynamic response behavior and damage mechanism of the RC column subjected to contact and close-in explosion are compared. The improvement effects of the reinforcement ratio and ultra-high performance concrete on the blast resistance of the underwater RC column are investigated. The results show that the cross-section shape has an important influence on the anti-knock performance of the column, whether subjected to air or underwater explosions. Employing circular cross-section can effectively improve the blast resistance of the column.

This paper is aim to investigate the damage characteristics of a shallow-buried water diversion box culvert subjected to a surface explosion. For this purpose, the soil model is verified by comparing with the analytical formulas of the Technical Manual (TM5-855-1), and the accuracy of the reinforced concrete model is validated with a previous explosion test. Subsequently, a three-dimensional (3D) numerical model of the water diversion culvert is established. A fully coupled Eulerian and Lagrangian (CEL) approach is presented to model the soil-structure-water interaction. The shock wave propagation characteristics in the soil, culvert and water medium are captured. The dynamic responses and damage processes of the shallow-buried box culvert in design water diversion condition (i.e. unconfined full flow) subjected to surface explosion are investigated. Other three water diversion conditions (i.e. pressure flow, free flow and no water) are employed to evaluate the influence of the water diversion condition on the antiknock performance of the culvert. The effects of the burial depth and charge weight on the dynamic behavior of the culvert to surface explosion are also discussed. The results show that the surface blast loads will cause a great threat to the whole security of the shallow-buried water diversion box culvert. The water diversion condition has a significant influence on the dynamic behavior of the culvert when subjected to a surface explosion.

Aims Hydrogen sulfide (H2S), an important gasotransmitter, is involved in a variety of cellular functions and pathophysiologic processes. Drug resistance due to alterations in drug trafficking and metabolism severely limits the effectiveness of cancer therapy. This study examined the role of H2S in drug resistance in liver cancer cells. Materials and methods Human 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. Key findings The expression of H2S-generating enzyme cystathionine gamma-lyase (CSE) was inhibited by doxorubicin treatment in HepG2 cells, and H2S sensitized Dox-inhibited cell survival and colony formation. In addition, H2S promoted cellular retention of Dox by suppressing the expressions of ABCA1 and ABCG8. H2S significantly blocked Dox-induced heterodimer formation between LXRα and RXRβ and attenuated the binding of LXRα/RXRβ to the promoters of ABCA1 and ABCG8 genes. RXRβ but not LXRα was S-sulfhydrated by H2S, and blockage of RXRβ S-sulfhydration abolished the inhibitory role of H2S on LXRα/RXRβ heterodimer formation. CSE expression was reduced in Dox-resistant cells in comparison with their parental cells, while H2S could reverse drug resistance in Dox-resistant cells. Significance Our study provides a novel solution for reversing drug resistance in cancer cells by targeting H2S signalling.

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 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.

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.

Disulfiram (DSF), a sulfur-containing compound, has been used to treat chronic alcoholism and cancer for decades by inactivating aldehyde dehydrogenase (ALDH). Hydrogen sulfide (H2S) is a new gasotransmitter and regulates various cellular functions by S-sulfhydrating cysteine in the target proteins. H2S exhibits similar properties to DSF in the sensitization of cancer cells. The interaction of DSF and H2S on ALDH activity and liver cancer cell survival are not clear. Here it was demonstrated that DSF facilitated H2S release from thiol-containing compounds, and DSF and H2S were both capable of regulating ALDH through inhibition of gene expression and enzymatic activity. The supplement of H2S sensitized human liver cancer cells (HepG2) to DSF-inhibited cell viability. The expression of cystathionine gamma-lyase (a major H2S-generating enzyme) was lower but ALDH was higher in mouse liver cancer stem cells (Dt81Hepa1-6) in comparison with their parental cells (Hepa1-6), and H2S was able to inhibit liver cancer stem cell adhesion. In conclusion, these data point to the potential of combining DSF and H2S for inhibition of cancer cell growth and tumor development by targeting ALDH.

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.

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.

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.

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.

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.

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.

Hydrogen sulfide (H(2)S) is a novel gasotransmitter that regulates cell proliferation and other cellular functions. Sulforaphane (SFN) is a sulfur-containing compound that exhibits anticancer properties, and young sprouts of broccoli are particularly rich in SFN. There is consistent epidemiological evidence that the consumption of sulfur-containing vegetables, such as garlic and cruciferous vegetables, may help reduce the occurrence of prostate cancer. Here we found that a large amount of H(2)S is released when SFN is added into cell culture medium or mixed with mouse liver homogenates, respectively. Both SFN and NaHS (a H(2)S donor) decreased the viability of PC-3 cells (a human prostate cancer cell line) in a dose-dependent manner, and supplement of methemoglobin or oxidized glutathione (two H(2)S scavengers) reversed SFN-reduced cell viability. We further found both cystathionine gamma-lyase (CSE) and cystathionine beta-synthase are expressed in PC-3 cells and mouse prostate tissues. H(2)S production in prostate tissues from CSE knockout mice was only 20% of that from wild-type mice, suggesting CSE is a major H(2)S-producing enzyme in prostate. CSE overexpression enhanced H(2)S production and inhibited cell viability in PC-3 cells. In addition, both SFN and NaHS activated p38 mitogen-activated protein kinases (MAPK) and c-Jun N-terminal kinase (JNK). Pre-treatment of PC-3 cells with methemoglobin decreased SFN-stimulated MAPK activities. Suppression of both p38 MAPK and JNK reversed H(2)S- or SFN-reduced viability of PC-3 cells. Our results demonstrated that H(2)S mediates the inhibitory effect of SFN on the proliferation of PC-3 cells, which suggests that H(2)S-releasing diet or drug might be beneficial in the treatment of prostate cancer.

Reduced β-cell mass and increased activities of ATP-sensitive K(+) channels in pancreatic β cells are associated with the pathogenesis of diabetes. Cystathionine γ-lyase (CSE) is a major hydrogen sulfide (H(2)S)-producing enzyme in pancreatic β cells. Herein, we examine the effects of genetic and pharmacologic ablation of CSE on β-cell functions and their correlation with streptozotocin (STZ)-induced diabetes. Compared with wild-type mice, CSE knockout (CSE KO) mice that received STZ injections exhibited a delayed onset of diabetic status. The application of dl-propargylglycine (PPG) to inhibit CSE activity protected wild-type mice from STZ-induced hyperglycemia and hypoinsulinemia. STZ significantly increased pancreatic H(2)S production in wild-type mice but not in CSE KO mice. STZ induced more apoptotic β-cell death in wild-type mice than in CSE KO mice. STZ exposure decreased the viability of cultured INS-1E cells, which was partly reversed by PPG co-treatment. STZ also significantly stimulated H(2)S production in cultured INS-1E cells. In addition, STZ stimulated ATP-sensitive K(+) currents in pancreatic β cells from wild-type mice but not in the presence of PPG or in β cells from CSE KO mice. Sodium hydrosulfide injection instantly increased blood glucose, decreased plasma insulin, and deteriorated glucose tolerance in mice. Take together, these results provide evidence that the CSE/H(2)S system plays a critical role in regulating β-cell functions.

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.

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.

Objective Both estrogen and hydrogen sulfide (H 2 S) are involved in the regulation of atherosclerosis development. We previously reported that male mice with cystathionine gamma-lyase (CSE) gene knockout (CSE-KO) exhibited reduced vascular H 2 S production and increased adhesion molecule expression, which led to early development of atherosclerosis. Estrogen offers significant protection against proliferative cardiovascular diseases in females. The objective of this study is to investigate the impact of estrogen on the anti-atherogenic effect of H 2 S. Methods and results Plasma estrogen (17 β -estradiol) levels were significantly decreased in female CSE-KO mice in comparison with female wild-type (WT) mice. Age-matched female CSE-KO and WT mice were fed with control or atherogenic diet for 12-weeks. Plasma lipid levels, except triglyceride levels, were significantly increased in atherogenic diet-fed WT and CSE-KO mice when compared with control diet-fed groups. However, the extent of lipid change was greater in CSE-KO mice than in WT mice. Atherogenic diet fed female CSE-KO mice, but not WT mice, developed early atherosclerotic lesion. Atherogenic diet induced more atherosclerotic lesion, oxidative stress and intra-cellular adhesion molecule-1 (ICAM-1) expression and nuclear factor kappa B (NF κ B) pathway in female CSE-KO mice when compared with female WT mice. To explore the role of estrogen in early development of atherosclerosis, female WT and CSE-KO mice fed with atherogenic diet were administered subcutaneously with 17 β -estradiol at 80 μg/kg/day for 12 weeks. This treatment suppressed atherogenic factors such as hypercholesterolemia, oxidative stress, ICAM-1 expression and NF κ B pathway in female WT mice, but not in female CSE-KO mice. Conclusions Deficiency of CSE/H 2 S pathway lowered plasma estrogen levels in female mice and nullified the effects of estrogen on hypercholesterolemia, oxidative stress, ICAM-1 expression and atherosclerosis development. Thus, H 2 S is irreplaceable for these vascular effects of estrogen. The impact of this H 2 S dependency of estrogen on atherosclerosis development and the underlying mechanisms remain to be clarified.

Hydrogen sulfide (H2S), a novel 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 mammalian. The maintenance of genome integrity is essential for the proper function and survival of all organisms, and DNA damage may result in blockages of transcription and replication, mutagenesis, cellular senescence, and carcinogenesis. In this study, we demonstrated that more DNA damages occur in mouse embryonic fibroblasts (MEFs) from CSE knockout mice in comparison with that from wild-type mice, and H2S at physiologically relevant concentration attenuates DNA damage and cellular senescence in human umbilical vein endothelial cells (HUVECs). Treatment of HUVECs with 10 μM NaHS stimulated poly (ADP-ribosyl) ation (PAR) activation, while knockdown of CSE expression by RNA interference or inhibition of CSE activity by 5 mM DL-propargyglycine attenuated PAR activation. We also observed that H2S stimulates more PAR, XRCC-1 and DNA ligase III complex in HUVECs, while H2S had little effect on the release of apoptosis inducing factor from mitochondria. We further found that PAR levels are significantly higher in liver, kidney and MEFs from wild-type mice when compared with those from CSE knockout mice. H2S induced the interaction of PAR and phosphorylated ERK1/2, and U0126 (an inhibitor of MEK1/2) reversed H2S-induced PAR activation. MEK1, but not MEK2 and PARP-1 were S-sulfhydrated by H2S, following increased ERK1 phosphorylation and PAR activation. These results provide mechanistic insight into how H2S signalling mediates DNA damage and cellular senescence (supported by a grant-in-aid from Heart and Stroke Foundation of Canada to G.Y.).