Cystathionine Γ-lyase Protein Research Articles

The cystathionine γ-lyase/hydrogen sulfide pathway mediates the trimetazidine-induced protection of H9c2 cells against hypoxia/reoxygenation-induced apoptosis and oxidative stress.

Objective:Trimetazidine is a piperazine-derived metabolic agent. It exerts cardioprotective effects against myocardial ischemia/reperfusion (I/R) injury. In addition, studies confirm that the cystathionine γ-lyase (CSE)/hydrogen sulfide (H2S) pathway serves a beneficent role in attenuating myocardial I/R injury. However, the underlying role of the CSE/H2S pathway in the trimetazidine-induced protection against myocardial I/R injury remains elusive. Therefore, this study investigated whether trimetazidine ameliorates hypoxia/reoxygenation (H/R)-induced H9c2 cardiomyocyte injuries in an in vitro cell model of myocardial I/R injury, by enhancing the CSE/H2S pathway.Methods:The H9c2 cell viability was determined with a cell counting Kit-8.Results:Trimetazidine significantly increased the cell viability and decreased lactate dehydrogenase (LDH) release in H/R-treated H9c2 cells. Additionally, trimetazidine increased the H2S levels and the CSE mRNA and protein levels, promoting the CSE/H2S pathway under H/R conditions. The inhibition of the CSE/H2S pathway, induced by transfection with specific siRNA against human CSE (si-CSE), eliminated the trimetazidine-induced upregulation of cell viability, downregulation of LDH release, increase of caspase-3 activity and apoptosis regulator BAX expression, and the decrease of apoptosis regulator Bcl-2 expression, which suggests involvement of the CSE/H2S pathway in trimetazidine-induced cardioprotection. Furthermore, trimetazidine mitigated the H/R-induced increase in reactive oxygen species production and NADPH oxidase 2 expression, and decrease in superoxide dismutase activity and glutathione level, in H9c2 cells. These effects were also reversed by si-CSE.Conclusion:This study revealed that the CSE/H2S pathway mediates the trimetazidine-induced protection of H9c2 cardiomyocytes against H/R-induced damage by inhibiting apoptosis and oxidative stress.

Reduced cystathionine-γ-lyase (CSE) expression is involved in high glucose induced MMP14 expression in adipocytes and adipose tissues.

In the present study, we investigate the effect of reduced cystathionine-γ-lyase (CSE) expression in high glucose induced metalloproteinases14 (MMP14) expression in adipocytes and visceral adipose tissues. Diabetic mice were prepared by injections of STZ and the expression of CSE, MMP14 in visceral adipose tissues were determined. Adipocytes were differentiated from 3T3-L1 cells and treated with high glucose (HG), H2S slow-releasing compound GYY4137 or transfected with CSE siRNA. Then the expression of CSE, MMP14 were determined by western blotting. CSE knockout mice were generated by crossing CSE+/- heterozygous mice and given intraperitoneally (i.p.) injections of GYY4137, and then the expression of CSE and MMP14 in visceral adipose tissues were determined by quantitative real-time PCR and western blotting. The following results were obtained from the study. In adipose tissues of diabetic mice, the mRNA and protein expression of MMP14 increased while the mRNA and protein expression of CSE decreased. In 3T3-L1 adipocytes, both HG DMEM and CSE siRNA transfection increased the mRNA and protein of MMP14. The addition of GYY4137 inhibited HG-induced upregulation of MMP14 expression. In CSE knockout mice, the mRNA and protein expression of MMP14 in adipose tissues increased, which could be inhibited by i.p. injections of GYY4137. In conclusion, high glucose increased the expression of MMP14 in adipocytes and visceral adipose tissues through inhibiting the expression of CSE.

6 weeks consumption of pure fresh coconut milk caused up-regulation of eNOS and CSE protein expression in middle-aged male rats

Coconut milk (CCM) has been an important cooking ingredient in the Asia-Pacific region since ancient time. Due to its high content of saturated fatty acids, it has been considered atherogenic. We have tested if chronic consumption of fresh coconut milk by middle-aged male rat affects vascular function, plasma glucose and lipid profiles. Compared to control, CCM caused lower maximal contraction to phenylephrine of thoracic aortic rings and increased relaxation to acetylcholine that was abolished by N G-nitro-L-arginine (L-NA) or disruption of the endothelium. DL-propargylglycine caused slight increase in baseline tension of L-NA treated aortic rings of CCM-treated rats and produced higher contractile response of the aortic rings to low concentrations of phenylephrine. The aortic eNOS- and cystathionine-γ-lyase(CSE) proteins expression of the CCM-treated rats were also higher than in controls. Except for lower fasting plasma glucose there were no changes in blood chemistry for the CCM treated rats. CCM consumption caused up-regulation of eNOS and CSE protein expression which resulted in increased production of NO and H2S from the blood vessels with attenuation of vasocontraction to phenylephrine and increased relaxation to acetylcholine. These novel benefits may be expected to reduce the development of cardiovascular risk factors in the aging rat.

Estradiol-17β stimulates H2 S biosynthesis by ER-dependent CBS and CSE transcription in uterine artery smooth muscle cells in vitro.

Endogenous hydrogen sulfide (H2 S), synthesized by cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), is a potent vasodilator that can be stimulated by estradiol-17β (E 2 β) in uterine artery (UA) smooth muscle (UASMC) in vivo; however, the underlying mechanisms are unknown. This study tested a hypothesis that E 2 β stimulates H 2 S biosynthesis by upregulating CBS expression via specific estrogen receptor (ER). Treatment with E 2 β stimulated time- and concentration- dependent CBS and CSE messenger RNA (mRNA) and protein expressions, and H 2 S production in cultured primary UASMC isolated from late pregnant ewes, which were blocked by ICI 182,780. Treatment with specific ERα or ERβ agonist mimicked these E 2 β-stimulated responses, which were blocked by specific ERα or ERβ antagonist. Moreover, E 2 β activated both CBS and CSE promoters and ICI 182,780 blocked the E 2 β-stimulated responses. Thus, E 2 β stimulates H 2 S production by upregulating CBS and CSE expression via specific ER-dependent transcription in UASMC in vitro.

Cystathionine γ-Lyase Modulates Flow-Dependent Vascular Remodeling.

Objective- Flow patterns differentially regulate endothelial cell phenotype, with laminar flow promoting vasodilation and disturbed flow promoting endothelial proinflammatory activation. CSE (cystathionine γ-lyase), a major source of hydrogen sulfide (H2S) in endothelial cells, critically regulates cardiovascular function, by both promoting vasodilation and reducing endothelial activation. Therefore, we sought to investigate the role of CSE in the endothelial response to flow. Approach and Results- Wild-type C57Bl/6J and CSE knockout ( CSE-/-) mice underwent partial carotid ligation to induce disturbed flow in the left carotid. In addition, endothelial cells isolated from wild-type and CSE -/- mice were exposed to either laminar or oscillatory flow, an in vitro model of disturbed flow. Interestingly, laminar flow significantly reduced CSE expression in vitro, and only disturbed flow regions show discernable CSE protein expression in vivo, correlating with enhanced H2S production in wild-type C57BL/6J but not CSE-/- mice. Lack of CSE limited disturbed flow-induced proinflammatory gene expression (ICAM-1[intercellular adhesion molecule 1], VCAM-1 [vascular cell adhesion molecular 1]) and monocyte infiltration and CSE-/- endothelial cells showed reduced NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) activation and proinflammatory gene expression in response to oscillatory flow in vitro. In addition, CSE-/- mice showed reduced inward remodeling after partial carotid ligation. CSE-/- mice showed elevated vascular nitrite levels (measure of nitric oxide [NO]) in the unligated carotids, suggesting an elevation in baseline NO production, and the NO scavenger 2-(4-carboxyphenyl)-4,5-dihydro-4,4,5,5-tetramethyl-1H-imidazolyl-1-oxy-3-oxide normalized the reduced inward remodeling, but not inflammation, of ligated carotids in CSE-/- mice. Conclusions- CSE expression in disturbed flow regions critically regulates both endothelial activation and flow-dependent vascular remodeling, in part through altered NO availability.

Abstract 460: Diabetes Impairs Reparative Property of Bone Marrow-derived Endothelial Progenitor Cells: Role of Mir-499-mediated Hydrogen Sulfide Deficiency

Background: miR-499 has been considered as a potential biomarker for myocardial infarction. Overexpression of miR-499 impaired heart function and induced cardiac hypertrophy in mice. Recent studies suggested miR-499 suppresses tumor growth through the inhibition of vascular endothelial growth factor (VEGF) mediated angiogenesis. Furthermore, emerging evidences indicate that miR-499 is increased in heart of diabetic rodents and patients. Here, we tested the hypotheses that miR-499 impairs angiogenic ability of endothelial cells (ECs) under diabetic conditions. Methods and Results: miR-499 levels were examined in endothelial progenitor cells (EPCs), microvascular endothelial cells (MVECs) and plasma in diabetic db/db mice (8-week, male). Gender- and age-matched non-diabetic db/+ mice served as controls. EPCs were isolated from bone marrow density-gradient centrifugation. MVECs were isolated using collagenase. We found that miR-499 levels were significantly increased in EPCs, MVECs as well as plasma of db/db mice. miR-499 antagomiR rescued high glucose (25 mM D-glucose for 48 h)-impaired tube formation and migration of human cardiac microvascular endothelial cells (HCMVECs). Mechanistically, by luciferase assay, we found that miR-499 directly targets cystathionine γ-lyase (CSE), a major hydrogen sulfide (H 2 S)-synthesizing enzyme in cardiovascular cells. Overexpression of miR-499 by transfection of miR-499 mimics decreased CSE protein levels and intracellular H 2 S production in HCMVECs. Conclusions: Our results suggest that miR-499-induced CSE downregulation/inactivation thus H 2 S reduction may play an important role in diabetes-impaired angiogenic capacity. Silencing miR-499 in diabetic EPCs/ECs may provide a novel tool for the treatment of ischemic tissue injury in patients with diabetes.

Increased H2S and its synthases in urothelial cell carcinoma of the bladder, and enhanced cisplatin-induced apoptosis following H2S inhibition in EJ cells.

H2S, synthesized by cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (MPST), functions as a signalling molecule in mammalian cells. H2S serves complex functions in physiological and pathological processes, including in bladder cancer. In the present study, H2S production, the expression of the associated enzymes and the effect of H2S on human urothelial cell carcinoma of the bladder (UCB) tissue and cell lines were evaluated, and whether decreasing H2S levels influenced cell viability and tumour growth following treatment with cisplatin (CDDP) was assessed in UCB cells in vitro and in vivo. H2S production and the expression of CBS, CSE and MPST in bladder tissue specimens and the UCB cell lines 5637, EJ and UM-UC-3 were analysed using a sulfur-sensitive electrode and western blotting. UCB cells were subjected to different treatments, and viability and protein expression were determined. H2S production was inhibited to examine its influence on EJ cell tumour growth following CDDP treatment in vivo. It was identified that CBS, CSE and MPST protein were up-regulated in UCB tissues and cells. The H2S production and enzyme expression levels were the highest in UCB tissue and EJ cells. The inhibition of endogenous H2S biosynthesis decreased EJ cell viability and tumour growth in response to CDDP treatment. H2S levels and the associated biosynthetic enzymes were increased in human UCB tissue and cells compared with adjacent tissue and normal cells, which may have increased the resistance to CDDP-induced apoptosis in UCB. Therefore, H2S and its production may be an alternative therapeutic target for UCB.

Hyperhomocysteinemia potentiates diabetes-impaired EDHF-induced vascular relaxation: Role of insufficient hydrogen sulfide

Insufficient hydrogen sulfide (H2S) has been implicated in Type 2 diabetic mellitus (T2DM) and hyperhomocysteinemia (HHcy)-related cardiovascular complications. We investigated the role of H2S in T2DM and HHcy-induced endothelial dysfunction in small mesenteric artery (SMA) of db/db mice fed a high methionine (HM) diet. HM diet (8 weeks) induced HHcy in both T2DM db/db mice and non-diabetic db/+ mice (total plasma Hcy: 48.4 and 31.3 µM, respectively), and aggravated the impaired endothelium-derived hyperpolarization factor (EDHF)-induced endothelium-dependent relaxation to acetylcholine (ACh), determined by the presence of eNOS inhibitor N(ω)-nitro-L-arginine methyl ester (L-NAME) and prostacyclin (PGI2) inhibitor indomethacin (INDO), in SMA from db/db mice but not that from db/+ mice. A non-selective Ca2+-active potassium channel (KCa) opener NS309 rescued T2DM/HHcy-impaired EDHF-mediated vascular relaxation to ACh. EDHF-induced relaxation to ACh was inhibited by a non-selective KCa blocker TEA and intermediate-conductance KCa blocker (IKCa) Tram-34, but not by small-conductance KCa (SKCa) blocker Apamin. HHcy potentiated the reduction of free sulfide, H2S and cystathionine γ-lyase protein, which converts L-cysteine to H2S, in SMA of db/db mice. Importantly, a stable H2S donor DATS diminished the enhanced O2- production in SMAs and lung endothelial cells of T2DM/HHcy mice. Antioxidant PEG-SOD and DATS improved T2DM/HHcy impaired relaxation to ACh. Moreover, HHcy increased hyperglycemia-induced IKCa tyrosine nitration in human micro-vascular endothelial cells. EDHF-induced vascular relaxation to L-cysteine was not altered, whereas such relaxation to NaHS was potentiated by HHcy in SMA of db/db mice which was abolished by ATP-sensitive potassium channel blocker Glycolamide but not by KCa blockers. ConclusionsIntermediate HHcy potentiated H2S reduction via CSE-downregulation in microvasculature of T2DM mice. H2S is justified as an EDHF. Insufficient H2S impaired EDHF-induced vascular relaxation via oxidative stress and IKCa inactivation in T2DM/HHcy mice. H2S therapy may be beneficial for prevention and treatment of micro-vascular complications in patients with T2DM and HHcy.

The expression of cystathionine gamma-lyase is regulated by estrogen receptor alpha in human osteoblasts.

Hydrogen sulfide (H2S), generated in the osteoblasts predominantly via cystathionine-γ-lyase (CSE), is bone protective. Previous studies suggested that the onset of bone loss due to estrogen deficiency is associated to decreased levels of H2S and blunted gene expression of CSE. However, there are still a lot of unknowns on how H2S levels influence bone cells function. The present study aims to explore the mechanisms by which estrogen may regulate CSE expression, in particular the role of estrogen receptor alpha (ERα) in human osteoblasts (hOBs). Vertebral lamina derived hOBs were characterized and then assessed for CSE expression by western blot analysis in the presence or absence of ERα overexpression. Bioinformatic analysis, luciferase reporter assay and ChIP assay were performed to investigate ERα recruitment and activity on hCSE gene promoter.Three putative half Estrogen Responsive Elements (EREs) were identified in the hCSE core promoter and were found to participate in the ERα – mediated positive regulation of CSE expression. All osteoblast samples responded to ERα over-expression increasing the levels of CSE protein in a comparable manner. Notably, the ERα recruitment on the regulatory regions of the CSE promoter occurred predominantly in female hOBs than in male hOBs. The obtained results suggest that CSE/H2S system is in relation with estrogen signaling in bone in a gender specific manner.

Reduced adipose tissue H2S in obesity

Hydrogen sulfide (H2S) is an endogenously produced signaling molecule synthesized by cystathionine γ-lyase (CSE), cystathionine β-synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (3-MST). Given that H2S exerts significant effects on bioenergetics and metabolism, the goal of the current study was to determine the expression of H2S-producing enzymes in adipose tissues in models of obesity and metabolic disruption. Mice fed a western diet expressed lower mRNA levels of all three enzymes in epididymal fat (EWAT), while only CSE and 3-MST were reduced in brown adipose tissue (BAT). At the protein level 3-MST was reduced in all fat depots studied. Using db/db mice, a genetic model of obesity, we found that CSE, CBS and 3-MST mRNA were reduced in white fat, while only CSE was reduced in BAT. CBS and CSE protein levels were suppressed in all three fat depots. In a model of age-related weight gain, no reduction in the mRNA of any of the enzymes was noted. Smaller amounts of 3-MST protein were found in EWAT, while both CSE and 3-MST were reduced in BAT. Tissue levels of H2S were lower in WAT in HFD mice; both WAT and BAT contained lower H2S amounts in db/db animals. Taken together, our data suggest that obesity is associated with a decreased expression of H2S-synthesizing enzymes and reduced H2S levels in adipose tissues of mice. We propose that the reduction in H2S may contribute to the metabolic response associated with obesity. Further work is needed to determine whether restoring H2S levels may have a beneficial effect on obesity-associated metabolic alterations.

Changes in hydrogen sulfide in rats with hepatic cirrhosis in different stages.

This study aimed to observe changes in the hydrogen sulfide (H2S) system in the blood and liver tissue of rats with hepatic cirrhosis at different stages by studying the effect of H2S on the course of hyperdynamic circulation in rats with hepatic cirrhosis. H2S concentration in the blood from the portal vein and inferior vena cava of hepatic cirrhosis rat model induced with carbon tetrachloride was detected on the 15th, 30th, and 52nd day. The expression of cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) protein, and CBS and CSE mRNA in the liver was detected by immunohistochemistry and reverse transcriptase polymerase chain reaction (RT-PCR), respectively. The results indicated that H2S concentration in the blood from the portal vein and inferior vena cava of rats with hepatic cirrhosis was significantly lower than that in the control group. H2S was gradually decreased with the development of the disease and significantly lower in the blood from portal vein than in the blood of inferior vena cava at the mid-stage and the late stage groups. The expression levels of CBS and CSE protein, and CBS and CSE mRNA in the livers with hepatic cirrhosis at different stages were all higher than those in the control group, and the expression gradually increased with the development of the disease. The expression of CBS was lower than CSE in the same stages. The results indicated that the CSE mRNA was expressed predominantly in the cirrhosis groups as compared with CBS mRNA. Among experimental rats, the H2S system has an important effect on the occurrence and development of hyperdynamic circulation in rats with hepatic cirrhosis. This finding adds to the literature by demonstrating that H2S protects vascular remodelling in the liver, and that CSE is indispensable in this process.

3-Mercaptopyruvate Sulfurtransferase, Not Cystathionine β-Synthase Nor Cystathionine γ-Lyase, Mediates Hypoxia-Induced Migration of Vascular Endothelial Cells.

Hypoxia-induced angiogenesis is a common phenomenon in many physiological and patho-physiological processes. However, the potential differential roles of three hydrogen sulfide producing systems cystathionine γ-lyase (CSE)/H2S, cystathionine β-synthase (CBS)/H2S, and 3-mercaptopyruvate sulfurtransferase (MPST)/H2S in hypoxia-induced angiogenesis are still unknown. We found that minor hypoxia (10% oxygen) significantly increased the migration of vascular endothelial cells while hypoxia (8% oxygen) significantly inhibited cell migration. The present study was performed using cells cultured in 10% oxygen. RNA interference was used to block the endogenous generation of hydrogen sulfide by CSE, CBS, or MPST in a vascular endothelial cell migration model in both normoxia and hypoxia. The results showed that CBS had a promoting effect on the migration of vascular endothelial cells cultured in both normoxic and hypoxic conditions. In contrast, CSE had an inhibitory effect on cell migration. MPST had a promoting effect on the migration of vascular endothelial cells cultured in hypoxia; however, it had no effect on the cells cultured in normoxia. Importantly, it was found that the hypoxia-induced increase in vascular endothelial cell migration was mediated by MPST, but not CSE or CBS. The western blot analyses showed that hypoxia significantly increased MPST protein levels, decreased CSE protein levels and did not change CBS levels, suggesting that these three hydrogen sulfide-producing systems respond differently to hypoxic conditions. Interestingly, MPST protein levels were elevated by hypoxia in a bi-phasic manner and MPST mRNA levels increased later than the first stage elevation of the protein levels, implying that the expression of MPST induced by hypoxia was also regulated at a post-transcriptional level. RNA pull-down assay showed that some candidate RNA binding proteins, such as nucleolin and Annexin A2, were dissociated from the 3′-UTR of MPST mRNA in hypoxia which implied their involvement in MPST mRNA regulation.

The expressions of CSE/H2S and RvE1 in the patients with ulcerative colitis

Objective To investigate the expressions of endogenous cystathionine-γ-lyase (CSE)/hydrogen sulfide (H2S) and resolvin E1 (RvE1) in patients with ulcerative colitis (UC), and its effect on the pathogenesis of ulcerative colitis. Methods The distribution and expression of CSE proteins in the rectum mucosa in 60 cases of UC and 30 cases of normal control group were detected by Strept Avidin-Biotin Complex (SABC) immunohistochemistry. The average optical density value of CSE was analyzed with an Image Analyzing systems. The expression of CSE mRNA in the rectum mucosa was detected by real-time polymerase chain reaction (RT-PCR). The levels of H2S and RvE1 in sera were detected by spectrophotometry. Results The expressions of CSE proteins in three groups were detected in the rectum mucosa membrane epithelia. The average optical density value of CSE and the expression of CSE mRNA in patients with active UC were higher than that in normal group and remission of UC. The levels of H2S and RvE1 in patients with active UC were significantly higher than that in normal group and remission of UC. Conclusions The abnormal expressions of CSE/H2S and RvE1 in activity of the UC might play an important role in the pathogenesis of UC. Key words: Cystathionine gamma-lyase/ME; Hydrogen sulfide/ME; Fatty acids, unsaturated/ME; Colitis, ulcerative/ME

Endogenous hydrogen sulfide contributes to uterine quiescence during pregnancy.

Recent evidence suggests that uterine activation for labor is associated with inflammation within uterine tissues. Hydrogen sulfide (H2S) plays a critical role in inflammatory responses in various tissues. Our previous study has shown that human myometrium produces H2S via its generating enzymes cystathionine-γ-lyase (CSE) and cystathionine-β-synthetase (CBS) during pregnancy. We therefore explored whether H2S plays a role in the maintenance of uterine quiescence during pregnancy. Human myometrial biopsies were obtained from pregnant women at term. Uterine smooth muscle cells (UMSCs) isolated from myometrial tissues were treated with various reagents including H2S. The protein expression of CSE, CBS and contraction-associated proteins (CAPs) including connexin 43, oxytocin receptor and prostaglandin F2α receptor determined by Western blot. The levels of cytokines were measured by ELISA. The results showed that CSE and CBS expression inversely correlated to the levels of CAPs and activated NF-κB in pregnant myometrial tissues. H2S inhibited the expression of CAPs, NF-κB activation and the production of interleukin (IL)-1β, IL-6 and tumor necrosis factor α (TNFα) in cultured USMCs. IL-1β treatment reversed H2S inhibition of CAPs. Knockdown of CSE and CBS prevented H2S suppression of inflammation. H2S modulation of inflammation is through KATP channels and phosphoinositide 3-kinase (PI3K) and extracellular signal-regulated kinase (ERK) signaling pathways. H2S activation of PI3K and ERK signaling is dependent on KATP channels. Our data suggest that H2S suppresses the expression of CAPs via inhibition of inflammation in myometrium. Endogenous H2S is one of the key factors in maintenance of uterine quiescence during pregnancy.

NFAT regulation of cystathionine γ-lyase expression in endothelial cells is impaired in rats exposed to intermittent hypoxia.

Sleep apnea is a risk factor for cardiovascular disease, and intermittent hypoxia (IH, 20 episodes/h of 5% O2-5% CO2 for 7 h/day) to mimic sleep apnea increases blood pressure and impairs hydrogen sulfide (H2S)-induced vasodilation in rats. The enzyme that produces H2S, cystathionine γ-lyase (CSE), is decreased in rat mesenteric artery endothelial cells (EC) following in vivo IH exposure. In silico analysis identified putative nuclear factor of activated T cell (NFAT) binding sites in the CSE promoter. Therefore, we hypothesized that IH exposure reduces Ca2+ concentration ([Ca2+]) activation of calcineurin/NFAT to lower CSE expression and impair vasodilation. In cultured rat aortic EC, inhibiting calcineurin with cyclosporine A reduced CSE mRNA, CSE protein, and luciferase activity driven by a full-length but not a truncated CSE promoter. In male rats exposed to sham or IH conditions for 2 wk, [Ca2+] in EC in small mesenteric arteries from IH rats was lower than in EC from sham rat arteries (Δfura 2 ratio of fluorescence at 340 to 380 nm from Ca2+ free: IH = 0.05 ± 0.02, sham = 0.17 ± 0.03, P < 0.05), and fewer EC were NFATc3 nuclear positive in IH rat arteries than in sham rat arteries (IH = 13 ± 3, sham = 59 ± 11%, P < 0.05). H2S production was also lower in mesenteric tissue from IH rats vs. sham rats. Endothelium-dependent vasodilation to acetylcholine (ACh) was lower in mesenteric arteries from IH rats than in arteries from sham rats, and inhibiting CSE with β-cyanoalanine diminished ACh-induced vasodilation in arteries from sham but not IH rats but did not affect dilation to the H2S donor NaHS. Thus, IH lowers EC [Ca2+], NFAT activity, CSE expression and activity, and H2S production while inhibiting NFAT activation lowers CSE expression. The observations that IH exposure decreases NFATc3 activation and CSE-dependent vasodilation support a role for NFAT in regulating endothelial H2S production.NEW & NOTEWORTHY This study identifies the calcium-regulated transcription factor nuclear factor of activated T cells as a novel regulator of cystathionine γ-lyase (CSE). This pathway is basally active in mesenteric artery endothelial cells, but, after exposure to intermittent hypoxia to mimic sleep apnea, nuclear factor of activated T cells c3 nuclear translocation and CSE expression are decreased, concomitant with decreased CSE-dependent vasodilation.

MicroRNA-186 promotes macrophage lipid accumulation and secretion of pro-inflammatory cytokines by targeting cystathionine γ-lyase in THP-1 macrophages

Background and aimsSeveral studies suggest that cardiomyocyte-enriched miR-186 is involved in cardiac injury and myocardial infarction, and also plays an important role in atherosclerotic diseases, but the underlying mechanism is unknown. Cystathionine-γ-lyase (CSE) is the predominant enzyme to produce H2S in the cardiovascular system. Here, miR-186 was identified to bind to the 3′UTR of CSE. In this study, we aimed at exploring whether miR-186 affects lipid accumulation and secretion of pro-inflammatory cytokines by targeting CSE and its underlying mechanism in human THP-1 macrophages and peripheral blood monocyte-derived macrophages (PBMDM). PBMDM just as a control group for the comparison with the THP-1 macrophages. MethodsMiR-186 target genes, CSE 3′UTR sequence and free energy were predicted and analyzed by bioinformatics analyses and dual-luciferase reporter assays. The expression of CSE mRNA and protein were measured by real-time quantitative PCR and western blot analyses. The lipid accumulation in THP-1 macrophages was detected by high performance liquid chromatography (HPLC). The effects of miR-186 on secretion of IL-6, IL-1β and TNF-α were examined by ELISA. Endogenous H2S was detected by spectrophotometry. Using small interfering RNA (siRNA) approach to decrease the expression of CSE protein and mRNA. ResultsWe found that miR-186 directly inhibited CSE protein and mRNA expression through targeting CSE 3′UTR by bioinformatics analyses and dual-luciferase reporter assays. HPLC assays showed that miR-186 increased the lipid accumulation in human THP-1 macrophages. We also showed that miR-186 enhanced secretion of pro-inflammatory cytokines in human THP-1 macrophages. Using siRNA approach, we found that CSE siRNA could inhibit the miR-186 inhibitor-induced decrease in the expression of LPL protein and mRNA in human THP-1 macrophages, which was accompanied a decrease in the level of H2S. ConclusionsMicroRNA-186 promotes macrophage lipid accumulation and pro-inflammatory cytokine secretion by targeting cystathionine γ-lyase in THP-1 macrophages.

Abstract 570: Cystathionine γ-Lyase Modulates Flow-dependent Vascular Remodeling

Disturbed flow causes endothelial dysfunction and development of atherosclerotic lesions. The gaseous signaling molecule H 2 S and cystathionine γ-lyase (CSE), its major enzymatic source in the vasculature, protect against cardiovascular diseases including atherosclerosis, peripheral artery disease, and cardiac ischemia in a nitric oxide (NO) dependent manner. Therefore, we sought to investigate the role of CSE in the endothelial response to disturbed flow. Wild-type C57Bl/6 (WT) and CSE knockout (CSE-/-) mice underwent partial carotid ligation to induce disturbed flow in the left carotid with the right carotid serving as an internal control. Additionally, endothelial cells isolated from WT and CSE-/- mice were exposed to oscillatory flow, a model of disturbed flow, in vitro. While disturbed flow decreased endothelial CSE mRNA expression, CSE protein expression showed strong induction under disturbed flow conditions both in vitro and in vivo. This induction correlated with enhanced free sulfide and sulfane sulfur production in WT but not in CSE-/- mice. Intimal mRNA isolated 2 days post-ligation showed increased VCAM-1 and ICAM-1 expression in WT mice which was prevented in CSE-/- mice. Similarly, endothelial cells isolated from CSE-/- mice show reduced NF-B activation and proinflammatory gene expression in response to oscillatory flow in vitro. Morphometric analysis of carotid arteries collected 7 days post-ligation revealed reduced macrophage infiltration and medial thickening in the ligated carotid of CSE-/- mice. Interestingly, ligation increased the carotid nitrite level in WT mice but not in CSE-/- mice. However, nitrite level of the non-ligated carotid was significantly higher in the CSE-/- mice compared to WT mice. Shear induced phosphorylation of eNOS Ser1179 in vitro was not different between WT and CSE knockout endothelial cells, suggesting alternative regulatory mechanisms. Collectively, CSE in mouse carotid arteries plays a critical role in flow dependent vascular remodeling, which may be mediated by CSE derived free sulfide and sulfane sulfur. CSE deficiency completely inhibits disturbed flow-induced NF-κB activation and macrophage recruitment, consistent with the role of inflammation in vascular remodeling.

LPS Up-Regulates Cystathionine γ -Lyase Gene Expression in Primary Human Macrophages via NF-κB/ERK Pathway.

Hydrogen sulfide (H2S) is an endogenous inflammatory mediator produced by the activity of cystathionine γ-lyase (CSE) in mammals. Macrophages are a key element of the immune system and play a crucial role in inflammation. To determine the role of H2S and macrophages in inflammation, we investigated the expression of CSE in human primary macrophages. Our results show that H2S is produced by the activity of CSE in these cells. To investigate the role of common signalling pathway in biosynthesis of CSE in human primary macrophages, specific inhibitors were used to block NF-κB, ERK, p38 and JNK. Inhibition of NF-κB, ERK significantly reduced levels of CSE gene and protein expression in these cells but inhibition of JNK and p38 did not have an inhibitory effect on the expression of CSE gene in macrophages. Inhibition of NF-κB and ERK prevented the effect of LPS on H2S synthesizing activity in human primary macrophages. These data showed that H2S acts as an inflammatory mediator via NF-κB/ERK pathway in macrophages.

Transcriptional Regulation of Cystathionine-γ-Lyase in Endothelial Cells by NADPH Oxidase 4-Dependent Signaling

The gasotransmitter, hydrogen sulfide (H2S) is recognized as an important mediator of endothelial cell homeostasis and function that impacts upon vascular tone and blood pressure. Cystathionine-γ-lyase (CSE) is the predominant endothelial generator of H2S, and recent evidence suggests that its transcriptional expression is regulated by the reactive oxygen species, H2O2. However, the cellular source of H2O2 and the redox-dependent molecular signaling pathway that modulates this is not known. We aimed to investigate the role of Nox4, an endothelial generator of H2O2, in the regulation of CSE in endothelial cells. Both gain- and loss-of-function experiments in human endothelial cells in vitro demonstrated Nox4 to be a positive regulator of CSE transcription and protein expression. We demonstrate that this is dependent upon a heme-regulated inhibitor kinase/eIF2α/activating transcription factor 4 (ATF4) signaling module. ATF4 was further demonstrated to bind directly to cis-regulatory sequences within the first intron of CSE to activate transcription. Furthermore, CSE expression was also increased in cardiac microvascular endothelial cells, isolated from endothelial-specific Nox4 transgenic mice, compared with wild-type littermate controls. Using wire myography we demonstrate that endothelial-specific Nox4 transgenic mice exhibit a hypo-contractile phenotype in response to phenylephrine that was abolished when vessels were incubated with a CSE inhibitor, propargylglycine. We, therefore, conclude that Nox4 is a positive transcriptional regulator of CSE in endothelial cells and propose that it may in turn contribute to the regulation of vascular tone via the modulation of H2S production.

H2S, a novel gasotransmitter, involves in gastric accommodation.

H2S is produced mainly by two enzymes:cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE), using L-cysteine (L-Cys) as the substrate. In this study, we investigated the role of H2S in gastric accommodation using CBS+/− mice, immunohistochemistry, immunoblot, methylene blue assay, intragastric pressure (IGP) recording and electrical field stimulation (EFS). Mouse gastric fundus expressed H2S-generating enzymes (CBS and CSE) and generated detectable amounts of H2S. The H2S donor, NaHS or L-Cys, caused a relaxation in either gastric fundus or body. The gastric compliance was significantly increased in the presence of L-Cys (1 mM). On the contrary, AOAA, an inhibitor for CBS, largely inhibited gastric compliance. Consistently, CBS+/− mice shows a lower gastric compliance. However, PAG, a CSE inhibitor, had no effect on gastric compliances. L-Cys enhances the non-adrenergic, non-cholinergic (NANC) relaxation of fundus strips, but AOAA reduces the magnitude of relaxations to EFS. Notably, the expression level of CBS but not CSE protein was elevated after feeding. Consistently, the production of H2S was also increased after feeding in mice gastric fundus. In addition, AOAA largely reduced food intake and body weight in mice. Furthermore, a metabolic aberration of H2S was found in patients with functional dyspepsia (FD). In conclusion, endogenous H2S, a novel gasotransmitter, involves in gastric accommodation.