Cystathionine Gamma-lyase Protein Research Articles

THE FUNCTION OF THE CYSTATHIONINE-GAMMA-LYASE/HYDROGEN SULFIDE AXIS IN THE PATHOGENESIS OF ULCERATIVE COLITIS

Abstract INTRODUCTION Current combination therapies in ulcerative colitis (UC) target different inflammatory cascades, including tumor necrosis factor-alpha (TNF-alpha) signaling. However, most patients do not achieve long-term remission, suggesting that the molecular pathophysiology of UC is still poorly understood. Endogenously produced hydrogen sulfide (H2S) has emerged as a critical physiological mediator of gastrointestinal homeostasis, limiting mucosal inflammation and promoting tissue healing in response to epithelial injury. Inhibition of cystathionine-gamma-lyase (CSE)-dependent H2S production in animal models of UC has been shown to exacerbate colitis and delay tissue repair. Our research group has strong evidence revealing that the CSE/H2S axis regulates TNF-alpha signaling in colonic epithelial cells. Therefore, we hypothesize that decreased CSE/H2S pathway activity in colonic mucosa cells leads to dysregulation of TNF-alpha signaling and uncontrolled inflammatory/immune responses with aberrant wound repair mechanisms contributing to the pathogenesis of UC. RESULTS We found a significant decrease in CSE mRNA and protein expression in colonic biopsies from patients with UC. Using a preclinical animal model of epithelium injury-induced chronic colitis (DSS), we confirmed a sustained downregulation of CSE, as well as upregulation of TNF-alpha and tumor necrosis receptor 2 expressions in colonic mucosa samples. Treatment with the slow-releasing H2S-donor GYY4137 significantly reduced the severity of DSS-induced inflammation and improved mucosal integrity and restitution responses. In epithelial cell and colonoid cultures, the inhibition of CSE-dependent H2S synthesis decreased the activation of extracellular-signal-regulated-kinase-1/2 (ERK1/2), increased IL-6 and TNF-alpha mRNA levels and suppressed basic and TNF-alpha–mediated colonocyte migration. CONCLUSION These studies demonstrate that CSE regulates mucosal TNF-alpha signaling, pro- and anti-inflammatory responses as well as colonic epithelial repair. Adding the H2S donor GYY4137 reduces inflammation, suggesting the restoration of normal TNF-alpha signaling function. Acknowledgments: KM is supported by the DoD Career Development Award (W81XWH2010641) and ACS Research Scholar Grant (RSG–21–027–01–CSM).

Hepatocellular cystathionine γ lyase/hydrogen sulfide attenuates nonalcoholic fatty liver disease by activating farnesoid X receptor.

Hydrogen sulfide (H2 S) plays a protective role in NAFLD. However, whether cystathionine γ lyase (CSE), a dominant H2 S generating enzyme in hepatocytes, has a role in the pathogenesis of NAFLD is currently unclear. We showed that CSE protein expression is dramatically downregulated, especially in fibrotic areas, in livers from patients with NAFLD. In high-fat diet (HFD)-induced NAFLD mice or an oleic acid-induced hepatocyte model, the CSE/H2 S pathway is also downregulated. To illustrate a regulatory role for CSE in NAFLD, we generated a hepatocyte-specific CSE knockout mouse (CSELKO ). Feeding an HFD to CSELKO mice, they showed more hepatic lipid deposition with increased activity of the fatty acid de novo synthesis pathway, increased hepatic insulin resistance, and higher hepatic gluconeogenic ability compared to CSELoxp control mice. By contrast, H2 S donor treatment attenuated these phenotypes. Furthermore, the protection conferred by H2 S was blocked by farnesoid X receptor (FXR) knockdown. Consistently, serum deoxycholic acid and lithocholic acid (FXR antagonists) were increased, and tauro-β-muricholic acid (FXR activation elevated) was reduced in CSELKO . CSE/H2 S promoted a post-translation modification (sulfhydration) of FXR at Cys138/141 sites, thereby enhancing its activity to modulate expression of target genes related to lipid and glucose metabolism, inflammation, and fibrosis. Sulfhydration proteomics in patients' livers supported the CSE/H2 S modulation noted in the CSELKO mice. FXR sulfhydration is a post-translational modification affected by hepatic endogenous CSE/H2 S that may promote FXR activity and attenuate NAFLD. Hepatic CSE deficiency promotes development of nonalcoholic steatohepatitis. The interaction between H2 S and FXR may be amenable to therapeutic drug treatment in NAFLD.

Effect of hydrogen sulfide on the intimal hyperplasia of vein grafts in rabbits

Objective To observe the effect of exogenous hydrogen sulfide (H2S) on the intimal hyperplasia of vein grafts in rabbits and and its possible molecular mechanism. Methods Forty New Zealand rabbits were randomly divided into 5 group, including blank control group (Ctr group); two weeks of simple transplantation; four weeks of simple transplantation; transplantation + NaHS 2 weeks group (NaHS: 30 μmol/kg. 3 days, 2 weeks of continuous administration), transplantation + NaHS 4 weeks group (NaHS: 30 μmol/kg. 3 days, 4 weeks of continuous administration). Rabbit jugular vein transplantation animal model was established. Administration by intraperitoneal injection once 3 days. Serum H2S level was detected by enzyme linked immunosorbent assay (ELISA). The structure and hyperplasia of vascular tissue were observed by hematoxylin-eosin staining (HE) staining. real-time quantitative polymerase chain reaction (Real-time PCR) was used to determine the expression levels of cystathionine-gamma-lyase (CSE) mRNA. CSE, Calmodulin (p-CaM/ CaM)and Cyclin D1 protein levels were measured by Western blotting. Results After NaHS treatment, the intimal hyperplasia of vein grafts was relieved. Compared with the corresponding group, the serum H2S level increased in the transplantation + NaHS 2 weeks group and transplantation + NaHS 4weeks group (33.65±4.10 vs. 23.56±4.82, P<0.05; 36.50±4.62 vs. 23.72±3.95, P<0.05). And the expression of CSE mRNA and protein increased significantly (mRNA: 0.54±0.04 vs. 0.15±0.05, P<0.05; 1.08±0.13 vs. 0.11±0.02, P<0.05; Protein: 0.56±0.06 vs. 0.47±0.03, P<0.05; 1.14±0.07 vs. 0.38±0.04, P<0.05). The expression of p-CaM/CaM and Cyclin D1 protein decreased significantly (p-CaM: 0.25±0.05 vs. 0.52±0.06, P<0.05; 1.21±0.09 vs. 2.39±0.15, P<0.05; CaM: 0.42±0.04 vs. 0.73±0.07, P<0.05, 0.47±0.05 vs. 1.17±0.06 P<0.05; Cyclin D1: 0.28±0.06 vs. 0.95±0.11, P<0.05; 0.29±0.04 vs. 1.02±0.10, P<0.05). Conclusion Exogenous hydrogen sulfide could inhibit the intima proliferation of vein grafts which related to the reduction of the expression of p-CaM/CaM and Cyclin D1. Key words: Hydrogen sulfide; Vein grafts; Transplantation; Intima hyperplasia

H2S protects lipopolysaccharide-induced inflammation by blocking NFκB transactivation in endothelial cells

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.

Augmented H2S production via cystathionine-beta-synthase upregulation plays a role in pregnancy-associated uterine vasodilation.

Endogenous hydrogen sulfide (H2S) synthesized via metabolizing L-cysteine by cystathionine-beta-synthase (CBS) and cystathionine-gamma-lyase (CSE) is a potent vasodilator and angiogenic factor. The objectives of this study were to determine if human uterine artery (UA) H2S production increases with augmented expression and/or activity of CBS and/or CSE during the menstrual cycle and pregnancy and whether exogenous H2S dilates UA. Uterine arteries from nonpregnant (NP) premenopausal proliferative (pPRM) and secretory (sPRM) phases of the menstrual cycle and pregnant (P) women were studied. H2S production was measured by the methylene blue assay. CBS and CSE mRNAs were assessed by quantitative real-time PCR, and proteins were assessed by immunoblotting and semiquantitative immunofluorescence microscopy. Effects of H2S on rat UA relaxation were determined by wire myography ex vivo. H2S production was greater in NP pPRM and P than NP sPRM UAs and inhibited by the specific CBS but not CSE inhibitor. CBS but not CSE mRNA and protein were greater in NP pPRM and P than NP sPRM UAs. CBS protein was localized to endothelium and smooth muscle and its levels were in a quantitative order of P >NP UAs of pPRM>sPRM. CSE protein was localized in UA endothelium and smooth muscle with no difference among groups. A H2S donor relaxed P > NP UAs but not mesentery artery. Thus, human UA H2S production is augmented with endothelium and smooth muscle CBS upregulation, contributing to UA vasodilation in the estrogen-dominant physiological states in the proliferative phase of the menstrual cycle and pregnancy.

18 Hydrogen sulfide stimulates human myometrial angiogenesis: Influence of endogenous estrogens

Introduction Angiogenesis regulates myometrial spiral artery expansion and remodeling that are key mechanisms for upregulating uterine blood flow during pregnancy. Hydrogen sulfide (H 2 S), mainly synthesized from l -cysteine by cystathionine-beta synthase (CBS) and cystathionine-gamma lyase (CSE), is a potent vasodilator and proangiogenic factor. However, it is unknown if pregnancy regulates H 2 S biosynthesis and if H 2 S plays a role in myometrial angiogenesis. Hypothesis Correlating with endogenous estrogens during the menstrual cycle and pregnancy, H 2 S biosynthesis is augmented to stimulate myometrial angiogenesis in vivo and in vitro. Methods Myometrial tissues were obtained from hysterectomies from women with different endogenous estrogens ( n = 5–7/group), including late pregnant (very high estrogen), premenopausal proliferative (high estrogen) and secretory (low estrogen) of the menstrual cycle, and postmenopausal (very low estrogen). Tissue levels of CBS and CSE mRNA and protein were determined by real-time qPCR and immunoblotting, respectively. Tissue sections were analyzed by immunofluorescence microscopy with antibodies of CBS or CSE with an endothelial marker protein CD31. Images were analyzed for quantifying cellular CBS and CSE protein expression and angiogenesis index. Primary myocytes and microvascular endothelial cells (hMMEC) were enzymatically isolated for in vitro angiogenesis assays. Results Myometrial H 2 S levels and angiogenesis index were greatest in association with significant upregulation of CBS but not CSE mRNA and protein and elevated estrogens in pregnant vs. non-pregnant women. CBS protein was localized in myocytes and microvessels. A slow releasing H 2 S donor significantly stimulated hMMEC proliferation, migration and tube formation in vitro (in vivo angiogenesis). Co-culture with myocytes also stimulated hMMEC migration in vitro, which was blocked by a specific inhibitor of CBS. Conclusion Pregnancy augmented H 2 S biosynthesis due to enhanced CBS expression in myocytes and microvessels stimulates myometrial angiogenesis in the presence of elevated estrogens during the menstrual cycle and pregnancy (Funded by NIH RO1 HL70562 and R21 HL98746).

Caffeic Acid Phenethyl Ester inhibit Hepatic Fibrosis by Nitric Oxide Synthase and Cystathionine Gamma-Lyase in Rats

BackgroundOur aim was to study the effect of caffeic acid phenethyl ester (CAPE) on iNOS and cystathionine gamma-lyase (CSE) of hepatic fibrosis rat, and discuss the anti-hepatic fibrosis mechanism of caffeic acid phenethyl ester.Material/MethodsWe observed changes of NO and H2S in serum of hepatic fibrosis rats. Enzyme-linked immunosorbent assay was used to test OD value of iNOS and CSE in serum of each. The expressions of iNOS and CSE protein in the liver were also detected by immunohistochemistry.ResultsCompared with the model group, the expression of NO and iNOS was decreased obviously and the level of H2S and CSE was increased in the CAPE group.ConclusionsCAPE has the effect of anti-hepatic fibrosis, which can be realized through adjusting the expression level of iNOS and CSE.

P2 Cystathionine gamma-lyase is expressed in human atherosclerotic plaque microvessels: A possible role for hydrogen sulfide in plaque vulnerability

Atherosclerotic plaque vulnerability is associated with intraplaque angiogenesis ( i . e . increased microvessel density). Vulnerable plaques are prone to rupture with severe risk of infarction. Hydrogen sulfide (H 2 S) is a gasotransmitter with angiogenic properties and is endogenously produced by endothelial cystathionine gamma-lyase (CSE). We hypothesize that endogenous intraplaque H 2 S production promotes angiogenesis. We here studied CSE expression in human carotid endarterectomy plaques and the effects of CSE inhibition on microvessel angiogenesis in vitro. Plaques ( n = 6) and microvascular endothelial cells (HMEC-1) were studied for CSE expression using immunostaining and PCR analysis. HMEC-1 tube formation assays were performed in the presence of the selective irreversible CSE inhibitor DL-propargylglycine (PAG). CSE mRNA expression was abundantly expressed in atherosclerotic plaques. At the protein level, CSE co-localized with vWF-positive microvessel endothelial cells. In vitro , HMEC-1 expressed both CSE and vWF protein. CSE inhibition using PAG dose-dependently reduced tube-formation up to 24% with 10 mM PAG ( p < 0.01). High levels of CSE in atherosclerotic plaques may be a potential risk for plaque vulnerability through intraplaque angiogenesis. On the other hand, CSE and H 2 S may also have protective effects in atherosclerosis, through protection against inflammation and scavenging of reactive oxygen species.

P70 Involvement of endogenous hydrogen sulfide and T-type calcium channels in cyclic AMP-induced neuronal differentiation in ng108-15 cells

Hydrogen sulfide (H 2 S) is a gasotransmitter synthesized by multiple enzymes including cystathionine-gamma-lyase (CSE) in the mammalian body. H 2 S donors cause neuronal differentiation characterized by neuritogenesis via activation of Ca v 3.2 T-type Ca 2+ channels (T-channels) in NG108-15 cells. Recently, we have shown that the cAMP/PKA pathway induces prompt phosphorylation and sensitization of Ca v 3.2 in NG108-15 cells. Thus, we investigated roles of the endogenous H 2 S/Ca v 3.2 pathway in the cAMP-dependent neuritogenesis in NG108-15 cells. Neuritogenesis was produced by stimulation with dibutyryl cAMP (db-cAMP) or Na 2 S, an H 2 S donor, for 16 h. The db-cAMP-induced neuritogenesis was suppressed by inhibitors of PKA, CSE, and T-channels, while the Na 2 S-induced neuritogenesis was reduced by inhibitors of T-channels, but not PKA. Stimulation with db-cAMP for 6 h caused functional and protein upregulation of Ca v 3.2, but did not alter CSE protein expression. A CSE inhibitor suppressed the persistently enhanced T-currents by 6-h db-cAMP stimulation, although it did not affect the rapid facilitation of T-currents by 10-min db-cAMP stimulation. Our data suggest that the cAMP/PKA pathway does not only cause rapid phosphorylation-mediated Ca v 3.2 sensitization, but also long-lasting functional facilitation of Ca v 3.2 through protein upregulation and of the CSE/H 2 S pathway that further enhances Ca v 3.2 function, resulting in neuritogenesis and possibly neuronal differentiation in NG108-15 cells.

Decrease in cystathionine‐gamma‐lyase (CSE) activity mediates the impaired H2S homeostasis in U937 monocytes, livers of type 1 diabetic rats and peripheral blood mononuclear cells of type 1 diabetic patients: role of hyperglycemia and hyperketo (767.5)

H2S is a novel signaling molecule. This study investigated the molecular mechanism that causes impaired H2S levels in diabetes using U937 monocytes, livers from streptozotocin‐treated type 1 (T1D) diabetic rats, and PBMC from T1D patients. T1D is associated with both hyperketonemia (acetoacetate, AA and β‐hydroxybutyrate, BHB) and hyperglycemia. Treatment of monocytes with high glucose (HG, 25 mM) and/or AA (4 mM) increased ROS (44%), and decreased CSE (a major enzyme that catalyzes H2S formation) protein expression (53%) and activity (39%), and reduced H2S levels (45%); however, BHB treatment had no effect (n=4). Signal silencing studies with CSE siRNA (71% knock down) showed a decrease in H2S levels (83%) in monocytes compared to controls. Liver plays a major role in the H2S homeostasis. Livers from T1D rats showed a significantly higher ROS production, lower CSE protein expression (48%) and activity (0.268±0.032 vs 0.175±0.014 nmol/min/mg protein), and lower H2S levels (46%) compared to controls (n=6). Further studies with T1D patients (n=17) also demonstrated a decrease in CSE protein expression (44%) and activity (0.099±0.023 vs 0.044±0.005 nmol/min/mg protein) in PBMC compared to those of age‐matched normals (n=18). This study demonstrates for the first time that both hyperglycemia and hyperketonemia mediate a reduction in CSE activity which can contribute to the impaired H2S signaling in diabetes.Grant Funding Source: Supported by NIH RO1 AT007442 and Malcolm Feist Chair in Diabetes

P40 The up-regulation role of H2S in 3T3L1 on adipogenesis

The endogenous gas, hydrogen sulfide (H2S), plays an important role in regulating cardiovascular function and inflammation. It also contributes to the pathogenesis of insulin resistance and glucose metabolism [1] , [2] , [3] . Recently, it has been reported that large amounts of H2S are formed from L-cysteine by cystathionine gamma lyase (CSE) in fat tissues and that H2S reduces glucose uptake in rat adipose tissue [3] . However, the biological roles of H2S in adipocytes and its contribution to adipogenesis are not yet clear. Here, we investigate whether H2S-synthesizing enzymes; cystathionine beta-synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (3-MST), are endogenously expressed in adipocytes and play a role in adipogenesis. In the present experiments, we stimulated the mouse fibroblast-like cell line, 3T3L1, to differentiate into adipocytes for up to one week as a model of adipogenesis in vitro. Over 7 days differentiation, whole cell lysate at day 0, 1, 3, 5 and 7 were collected for Western blotting of CBS, CSE and 3-MST. Endogenous CBS and 3-MST in adipocytes peaked at day 5 of differentiation whereas CSE protein expression was up-regulated in a time-dependent manner and maximally expressed at day 7 (n = 3, P [4] , [5] , [6] , but that these enzymes are also present in large amounts in adipose tissue and that their up-regulation during adipogenesis promotes adipocyte maturation and prevent lipid degradation. Thus, it is possible that modulation of the H2S system may be useful for obesity treatment.

Cystathionine Gamma-Lyase Expression Is Regulated by Exogenous Hydrogen Peroxide in the Mammalian Cells

Hydrogen sulfide (H2S), as an endogenous signaling molecule in mammals, shows a variety of biological effects. Cystathionine gamma-lyase (CSE)/H2S pathway has been implicated in scavenging reactive oxygen species (ROS) in the mammalian cells. Therefore, we first investigated the regulatory effects of exogenously applied hydrogen peroxide (H2O2) on CSE expression in the mammalian cells. African green monkey kidney fibroblastlike cells (COS-7 cells) or human embryonic kidney 293 cells (HEK 293 cells) were transfected with CSE promoter-luciferase reporter constructs and treated with H2O2 of 1, 5, and 10 microM for 0.5 and 1.5 h at 37 degrees C. The transfected cells were assayed for firefly luciferase activities normalized by Renilla luciferase activity. Human lung adenocarcinoma cells (A549 cells) or human liver cancer cells (SMMC-7721 cells) were treated with H2O2 of 1, 5, and 10 microM for 0.5 and 1.5 h at 37 degrees C, and were then harvested and analyzed by Western blotting and quantitative RT-PCR. Our results showed that the treatment of a medium concentration (5 microM) of H2O2 at a longer time (1.5 h) upregulated CSE expression in the mammalian cells at the levels of the promoter, message RNA, and protein. Collectively, exogenously applied H2O2 can not only markedly affect CSE mRNA and protein expression, but also can affect the CSE promoter activity in the mammalian cells. Our observations indicate that that exogenous H2O2 can upregulate the expression of the CSE gene in the mammalian cells, which will provide the possibility of the scavenging effect of the CSE gene indirectly on ROS in the mammalian cells. However, the regulatory mechanism involved in the effects of exogenously applied H2O2 on CSE expression in the mammalian cells need be further studied.

Cystathionine Gamma‐Lyase Deficiency Impairs H2S Biosynthesis And Vessel Reactivity In Type‐2 Diabetes

Together with the precursor L‐cysteine, cystathionine gamma‐lyase (CSE) is a key enzyme involved in the biosynthesis of hydrogen sulphide (H2S), a potent gaseous vasodilator. Since impaired vasodilation is a hallmark of diabetes the purpose of the current study was to assess the role of H2S in diabetes‐induced vascular dysfunction. Mesenteric arterioles from type‐2 diabetic (db/db) mice were found to express ~30% less CSE protein and 5 fold less CSE mRNA compared to nondiabetic (db/m) controls. In pressure myography experiments, addition of L‐cysteine (1 – 50 μM), to arterioles pre‐contracted with phenylephrine, dose‐dependently induced vasorelaxation in both db/m and db/db vessels, however, the efficacy of L‐cysteine was significantly lower in db/db. The CSE inhibitors D,L‐propargylglycine (PAG) and amino‐oxyacetic acid (AOAA) completely ablated L‐cysteine‐induced relaxation confirming that L‐cysteine operated through the H2S pathway. Incubating db/db vessels overnight with sulforaphane (500 nM), a phytochemical found in cruciferous vegetables, doubled its mRNA level. Taken together, these data suggest that reduced CSE expression and impaired H2S production may contribute to altered vascular reactivity in type‐2 diabetes and hint at the potential for sulforaphane to therapeutically correct H2S deficiency.

Is cystathionine gamma-lyase protein expressed in the heart?

Hydrogen sulfide (H2S) 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 H2S-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 H2S 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 43.6kDa. H2S 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, H2S production rate of heart tissues from WT mice was inhibited by approximately 80%. It appears that CSE mediates mostly endogenous H2S production in heart tissues. However, the available anti-CSE antibodies could not detect CSE proteins in rat and mouse heart tissues or rat cardiomyocytes.

P56 MiRNA-30 regulates the production of endogenous H2S by Cystathionine gamma-lyase (CSE) in primary cardiac myocytes

Cystathionine gamma-lyase (CSE) is the major H2S-generating enzyme in myocardial cell. H2S has been reported to be cardioprotective during myocardial infarction (MI) injury. MiRNAs are identified as crucial gene regulators in response to myocardial infarction. However, there is little evidence indicating a functional role for miRNA in regulating the generation of H2S. The MI rat model was induced by ligating the left anterior descending coronary artery. Real-time PCR and western bolt results show that the expression of CSE mRNA and protein were increased in the infarct region and reduced in non-infarct and border zone regions 48 h after MI induction. The concentration of H2S in plasma was reduced significantly in MI model. Correspondingly, using real time PCR, we demonstrated that the miR-30 family, which includes 5 closely related miRNAs (miR-30a, miR-30b-5p, miR-30, miR-30d, miR-30e) were significantly down regulated in the infarct region and highly expressed in the non-infarct and border zone regions. To determine the potential roles of miR-30 family members in CSE gene expression, we transfected miRNA mimics into neonatal rat cardiomyocytes to upregulate the expression of miR-30 family numbers. Interestingly, overexpression of each number of the miR-30 family significantly repressed the protein expression of CSE tested by western blot and immunofluorescence, and reduced the H2S production in culture media. The mRNA expression of CSE was also inhibited by each number of the miR-30 family, respectively. In contrast, downregulation of all miR-30 family numbers by LNA modified miR-30 family inhibitor significantly increased the level of CSE mRNA and protein and the H2S concentration in culture media. We further demonstrated that miR-30 family members repressed CSE protein expression by directly targeting at CSE untranslational regions by luciferase activity assay. This result was consistent with computational prediction that CSE is a target of miR-30 family members. Take together, these results suggest that miR-30 family participate in regulating the production of endogenous H2S by targeting CSE.

P15 The vascular effect of zofenopril, a sulfhydrylated ACE inhibitor, involves l-cys/H2S pathway

Background Hydrogen sulphide (H2S) is a novel gaseous mediator enzymatically produced by cystathionine-beta-synthase (CBS) and cystathionine-gamma-lyase (CSE). It is involved in physiological homeostatic processes and several pathological conditions; however, more importantly, H2S has a prominent role in cardiovascular system, where CSE deletion results in hypertension [1] .ACE inhibitors are widely used in controlling blood pressure in hypertensive patients and they represent first line treatment in different cardiovascular diseases, since they also show additional beneficial effects unrelated to ACE inhibition [2] , [3] .In particular, therapeutic use of zofenopril, a sulfhydrylated ACE inhibitor, has raised hypothesis over a potential role of thiol group in such beneficial effects, other than antioxidant activity.Here, we aimed to investigate on vascular effect of zofenoprilat, water-soluble metabolite of zofenopril, with respect to H2S pathway activation. Methods In order to pursue our goal, we performed isolated organ bath experiments by using aorta and carotid arteries harvested from Wistar Kyoto rats, where we tested vascular effect of zofenoprilat, compared to other ACE inhibitors, in presence of CSE inhibitor PAG or after endothelium removal.In addition, we also performed in vivo experiment, where we tested vascular response after two weeks treatment with zofenopril compared to enalapril in spontaneously hypertensive rats.We also determined vascular response to l -cys, precursor for H2S production, and quantified H2S levels in plasma and tissues and CSE and CBS protein levels upon different treatments. Results In vitro data showed that zofenoprilat, but not enalaprilat, was able to relax both aorta and carotid arteries in a concentration dependent fashion (100 nM–1 mM), reaching ∼60% vasodilation in both cases.Endothelium removal significantly reduced zofenoprilat induced vasorelaxation in aorta (EC50:8.5 μM vs 381 μM), but this effect was not observed in carotid.Next, CSE inhibition by PAG nearly abrogated zofenoprilat vasodilation in both aorta and carotid, indicating a role for H2S in this effect.In vivo data, first highlighted a significantly stronger effect of zofenopril vs enalapril treatment in lowering blood pressure (p l -cys was significantly improved in zofenopril vs enalapril treated animals (p Conclusion In conclusion, we show that sulfhydrylated ACE inhibitor zofenopril triggers H2S pathway and regulates different signal transduction cascades, other than inhibition of ACE. We suggest that this action could be, at least partly, explicative of the additional effects reported in the clinical literature about sulfhydrylated ACE inhibitors. Our data also imply the chance to understand, by using a drug already adopted in clinic, the role played by the l -Cys/H2S pathway in human hypertension.

Interaction of Methylglyoxal and Hydrogen Sulfide in Rat Vascular Smooth Muscle Cells

Hydrogen sulfide (H(2)S) is a gasotransmitter with multifaceted physiological functions, including the regulation of glucose metabolism. Methylglyoxal (MG) is an intermediate of glucose metabolism and plays an important role in the pathogenesis of insulin resistance syndromes. In the present study, we investigated the effect of MG on H(2)S synthesis and the interaction between these two endogenous substances. In cultured vascular smooth muscle cells (VSMCs), MG (10, 30, and 50 microM) significantly decreased cellular H(2)S levels in a concentration-dependent manner, while H(2)S donor, NaHS (30, 60, and 90 microM), significantly decreased cellular MG levels. The expression level and activity of H(2)S-producing enzyme, cystathionine gamma-lyase (CSE), were significantly decreased by MG treatment. NaHS (30-90 microM) significantly inhibited MG (10 or 30 microM)-induced ROS production. Cellular levels of GSH, cysteine, and homocysteine were also increased by MG or NaHS treatment. Furthermore, direct reaction of H(2)S with MG in both concentration- and time-dependent manners were observed in in vitro incubations. In conclusion, MG regulates H(2)S level in VSMCs by downregulating CSE protein expression and directly reacting with H(2)S molecule. Interaction of MG with H(2)S may be one of future directions for the studies on glucose metabolism and the development of insulin resistance syndromes.

Contractile and Vasorelaxant Effects of Hydrogen Sulfide and Its Biosynthesis in the Human Internal Mammary Artery

This study aimed to test these hypotheses: cystathionine gamma-lyase (CSE) is expressed in a human artery, it generates hydrogen sulfide (H(2)S), and H(2)S relaxes a human artery. H(2)S is produced endogenously in rat arteries from cysteine by CSE. Endogenously produced H(2)S dilates rat resistance arteries. Although CSE is expressed in rat arteries, its presence in human blood vessels has not been described. In this study, we showed that both CSE mRNA, determined by reverse transcription-polymerase chain reaction, and CSE protein, determined by Western blotting, apparently occur in the human internal mammary artery (internal thoracic artery). Artery homogenates converted cysteine to H(2)S, and the H(2)S production was inhibited by dl-propargylglycine, an inhibitor of CSE. We also showed that H(2)S relaxes phenylephrine-precontracted human internal mammary artery at higher concentrations but produces contraction at low concentrations. The latter contractions are stronger in acetylcholine-prerelaxed arteries, suggesting inhibition of nitric oxide action. The relaxation is partially blocked by glibenclamide, an inhibitor of K(ATP) channels. The present results indicate that CSE protein is expressed in human arteries, that human arteries synthesize H(2)S, and that higher concentrations of H(2)S relax human arteries, in part by opening K(ATP) channels. Low concentrations of H(2)S contract the human internal mammary artery, possibly by reacting with nitric oxide to form an inactive nitrosothiol. The possibility that CSE, and the H(2)S it generates, together play a physiological role in regulating the diameter of arteries in humans, as has been demonstrated in rats, should be considered.

Pro‐apoptotic effect of endogenous H2S on human aorta smooth muscle cells

Cystathionine gamma-lyase (CSE) is a key enzyme in the trans-sulfuration pathway, which uses L-cysteine to produce hydrogen sulfide (H2S). The CSE/H2S system has been shown to play an important role in regulating cellular functions in different systems. In the present study, we overexpressed CSE in human aorta smooth muscle cells (HASMCs) using a recombinant defective adenovirus containing CSE gene (Ad-CSE). Infection of HASMCs with Ad-CSE resulted in a significant increase in the expression of CSE protein and H2S production. Ad-CSE transfection inhibited cell growth and stimulated apoptosis, as evidenced by cell viability assay, Hoechst 33258 staining, TUNEL, and caspase 3 activation. CSE-mediated apoptosis was associated with an increased ERK and p38 MAPK activation, up-regulation of p21(Cip/WAK-1), and down-regulation of cyclin D1 expression. After inhibiting endogenous background CSE gene expression, direct administration of H2S at 100 microM induced apoptosis of HASMCs. The other two endproducts of CSE-catalyzed enzymatic reaction, ammonium and pyruvate, failed to do so. These results demonstrate that overexpression of CSE stimulates SMC apoptosis due to an increased endogenous production of H2S. Adenovirus-mediated transfer of CSE gene may provide a novel therapeutic approach in treating vascular diseases linked to abnormal cellular proliferation and vascular remodeling.