Cystathionine Γ-lyase Protein Expression Research Articles

Curcumin reduces myocardial ischemia-reperfusion injury, by increasing endogenous H2S levels and further modulating m6A.

Our previous research shows that Curcumin (CUR) attenuates myocardial ischemia-reperfusion injury (MIRI) by reducing intracellular total RNA m6A levels. However, the mechanism remains unknown. For ischemia-reperfusion (IR), H9c2 cells were cultured for 6h in serum-free low-glycemic (1g/L) medium and a gas environment without oxygen, and then cultured for 6h in high-glycemic (4.5g/L) medium supplemented with 10% FBS and a 21% oxygen environment. The effects of different concentrations of CUR (5, 10, and 20 µM) treatments on signaling molecules in conventionally cultured and IR-treated H9c2 cells were examined. CUR treatment significantly up-regulated the H2S levels, and the mRNA and protein expression of cystathionine γ-lyase (CSE), and down-regulated the mRNAs and proteins levels of thiosulfate sulfurtransferase (TST) and ethylmalonic encephalopathy 1 (ETHE1) in H9c2 cells conventionally cultured and subjected to IR. Exogenous H2S supply (NaHS and GYY4137) significantly reduced intracellular total RNA m6A levels, and the expression of RNA m6A "writers" METTL3 and METTL14, and increased the expression of RNA m6A "eraser" FTO in H9c2 cells conventionally cultured and subjected to IR. CSE knockdown counteracted the inhibitory effect of CUR treatment on ROS production, promotion on cell viability, and inhibition on apoptosis of H9c2 cells subjected to IR. CUR attenuates MIRI by regulating the expression of H2S level-regulating enzymes and increasing the endogenous H2S levels. Increased H2S levels could regulate the m6A-related proteins expression and intracellular total RNA m6A levels.

Hydrogen Sulfide Ameliorated High Choline-Induced Cardiac Dysfunction by Inhibiting cGAS-STING-NLRP3 Inflammasome Pathway.

Although it is an essential nutrient, high choline intake directly or indirectly via its metabolite is associated with increased risk of cardiovascular disease, the mechanism of which remains to be elucidated. The present study was performed to investigate whether hydrogen sulfide (H2S) was involved in high choline-induced cardiac dysfunction and explore the potential mechanisms. We found that ejection fraction (EF) and fractional shortening (FS), the indicators of cardiac function measured by echocardiography, were significantly decreased in mice fed a diet containing 1.3% choline for 4 months as compared to the control, while applying 3,3-dimethyl-1-butanol (DMB) to suppress trimethylamine N-oxide (TMAO, a metabolite of choline) generation ameliorated the cardiac function. Subsequently, we found that feeding choline or TMAO significantly increased the protein levels of cyclic GMP-AMP (cGAMP) synthase (cGAS), stimulator of interferon genes (STING), NOD-like receptor protein 3 (NLRP3), caspase-1, and interleukin-1β (IL-1β) as compared to the control, which indicated the activation of cGAS-STING-NLRP3 inflammasome axis. Moreover, the protein expression of cystathionine γ-lyase (CSE), the main enzyme for H2S production in the cardiovascular system, was significantly increased after dietary supplementation with choline, but the plasma H2S levels were significantly decreased. To observe the effect of endogenous H2S, CSE knockout (KO) mice were used, and we found that the EF, FS, and plasma H2S levels in WT mice were significantly decreased after dietary supplementation with choline, while there was no difference between CSE KO + control and CSE KO + choline group. To observe the effect of exogenous H2S, mice were intraperitoneally injected with sodium hydrosulfide (NaHS, a H2S donor) for 4 months, and we found that NaHS improved the cardiac function and reduced the protein levels of cGAS, STING, NLRP3, caspase-1, and IL-1β in mice receiving dietary choline. In conclusion, our studies revealed that high choline diet decreased plasma H2S levels and induced cardiac dysfunction via cGAS-STING-NLRP3 inflammasome axis while H2S treatment could restore the cardiac function by inhibiting cGAS-STING-NLRP3 inflammasome axis.

Exogenous hydrogen sulfide restores CSE and CBS but no 3-MST protein expression in the hypothalamus and brainstem after severe traumatic brain injury.

Hydrogen sulfide (H2S) is a gasotransmitter endogenously synthesized by cystathionine-γ-lyase (CSE), cystathionine-β-synthase (CBS), and 3-mercaptopiruvate sulfurtransferase (3-MST) enzymes. H2S exogenous administration prevents the development of hemodynamic impairments after traumatic brain injury (TBI). Since the hypothalamus and the brainstem highly regulate the cardiovascular system, this study aimed to evaluate the effect of NaHS subchronic treatment on the changes of H2S-sythesizing enzymes in those brain areas after TBI and in physiological conditions. For that purpose, animals were submitted to a lateral fluid percussion injury, and the changes in CBS, CSE, and 3-MST protein expression were measured by western blot at days 1, 2, 3, 7, and 28 in the vehicle group, and 7 and 28days after NaHS treatment. After severe TBI induction, we found a decrease in CBS and CSE protein expression in the hypothalamus and brainstem; meanwhile, 3-MST protein expression diminished only in the hypothalamus compared to the Sham group. Remarkably, i.p. daily injections of NaHS, an H2S donor, (3.1mg/kg) during seven days: (1) restored CBS and CSE but no 3-MST protein expression in the hypothalamus at day 28 post-TBI; (2) reestablished only CSE in brainstem 7 and 28days after TBI; and (3) did not modify H2S-sythesizing enzymes protein expression in uninjured animals. Mainly, our results show that the NaHS effect on CBS and CSE protein expression is observed in a time- and tissue-dependent manner with no effect on 3-MST expression, which may suggest a potential role of H2S synthesis in hypothalamus and brainstem impairments observed after TBI.

Roles of the RhoA-ROCK Signaling Pathway in the Endothelial H2S Production and Vasodilation in Rat Cerebral Arteries.

Cerebral endothelial H2S protects against cerebral ischemia-reperfusion injury through vasodilation, but its cerebral vasodilation mechanism and regulation of production are poorly understood. The RhoA-ROCK pathway plays important roles in vascular function. In this study, the roles of this pathway in the endothelial H2S production and vasodilation in rat cerebral arteries were investigated. Acetylcholine significantly increased H2S-generating enzyme cystathionine-γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST) protein expressions and H2S production in rat cerebrovascular endothelial cells (ECs), but the increases were markedly decreased by the M receptor blocker atropine or the CSE inhibitor dl-propargylglycine. Pretreatment with dl-propargylglycine or the 3-MST inhibitor l-aspartic acid markedly reduced the acetylcholine-increased H2S; CSE protein expression and H2S levels in the ECs were obviously attenuated by the RhoA agonist U46619 but increased by the RhoA inhibitor C3 transferase. U46619 also reduced 3-MST protein expression; Acetylcholine markedly inhibited RhoA protein expression and activity, but the inhibition was obviously reversed by atropine, dl-propargylglycine, and l-aspartic acid, respectively; Acetylcholine-induced endothelium-dependent vasodilation in rat cerebral basilar artery was significantly attenuated by pretreatment with dl-propargylglycine or l-aspartic acid or RhoA inhibitor CCG-1423 or ROCK inhibitor KD025, and was further decreased by co-pretreatment with dl-propargylglycine (or l-aspartic acid) and CCG-1423 (or KD025); NaHS significantly relaxed rat cerebral basilar artery vascular smooth muscle cells and inhibited ROCK1/2 activities, phosphorylated myosin light chain (MLC) protein expression, and KCl-increased [Ca2+]i, but these relaxation and inhibitions were markedly attenuated by pretreatment with C3 transferase or ROCK inhibitor Y27632. Our results demonstrated that endothelial H2S production is promoted by activation of the M receptor but inhibited by the RhoA-ROCK pathway in rat cerebral arteries; the endothelial H2S induces cerebral vasodilation by inhibiting this pathway to reduce phosphorylation of MLC and [Ca2+]i in vascular smooth muscle cells.

The effect of fluid shear stress in hydrogen sulphide production and cystathionine γ-lyase expression in human early endothelial progenitor cells.

BackgroundPhysiological fluid shear stress has been shown to have a beneficial impact on vascular homeostasis. Endothelial progenitor cells (EPCs) make a significant contribution to maintaining endothelial integrity. Therefore, we hypothesised that shear stress-induced endothelium protection plays a role in hydrogen sulphide (H2S) production and up-regulation of cystathionine γ-lyase (CSE) expression in EPCs.MethodsHuman EPC-derived CSE activity was detected by colorimetric assay, and H2S production was evaluated by membrane adsorption method. Cell proliferation, migration, and adhesion were assessed by MTT, Transwell, and endothelial cell-mediated adhesion assays, respectively. Real-time polymerase chain reaction (RT-PCR) was carried out to analyse gene expression. Protein expression was analysed by western blot.ResultsHuman EPCs were treated with shear stress levels of 5–25 dyn/cm2 for up to 3 h, and 25 dyn/cm2 for up to 24 h. H2S production and CSE mRNA expression in the EPCs were increased by shear stress in a dose-dependent manner in vitro. Likewise, time-dependent shear stress also significantly enhanced CSE protein expression. Compared to static condition, shear stress improved EPCs proliferation, migration and adhesion capacity. Knockdown of CSE expression by small interfering RNA substantially eliminated the shear stress-induced above functions of human EPCs in vitro.ConclusionsThis study gives new insight into the regulatory effect of physiological shear stress on the CSE/H2S system in human EPCs. Our findings may contribute to the development of vascular protective research, although the relevant evidence is admittedly indirect.

Estrogen Regulates Local Cysteine Metabolism in Mouse Myometrium.

Sulfur amino acid metabolism influences reproductive physiology, and transsulfuration in particular may be critical for normal cellular function. The sex hormone estrogen (E2) modulates gene expression and redox balance in some tissues by inducing the transsulfuration enzymes cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE). The role of sex hormones in sulfur amino acid metabolism by uterine smooth muscle is not known. Here, we show that CBS and CSE proteins increase in the mouse myometrium during estrus and diestrus, respectively, suggesting that E2 reciprocally regulates myometrial CBS and CSE expression. In ovariectomized mice, exogenous E2 upregulates CBS and downregulates CSE levels. E2 promotes CBS mRNA and protein expression but attenuates CSE protein expression without affecting CSE mRNA. This pattern of E2-stimulated changes in transsulfuration enzyme expression is specific to the uterine smooth muscle. E2 does not change vaginal or cervical expression of CBS or CSE significantly, and E2 decreases expression of CSE in the liver without affecting CBS. E2 also downregulates myometrial cysteinesulfinic acid decarboxylase (CSAD) and decreases myometrial biochemical synthesis of the gaso-transmitter hydrogen sulfide (H2S). These findings suggest that myometrial sulfur amino acid metabolism may regulate uterine redox homeostasis, with implications for the source and metabolism of myometrial cysteine in high E2 states such as estrus and pregnancy.

Effects of hydrogen sulphide donor, sodium hydrosulphide treatment on the erectile dysfunction in L‐NAME‐induced hypertensive rats

Men with hypertension often develop erectile dysfunction (ED). The present study aimed to examine the effects of sodium hydrosulphide (NaHS), a hydrogen (H2 S) donor, treatment on ED in nitric oxide synthase (NOS) inhibitor (L-NAME)-induced hypertensive rats. Forty adult Sprague-Dawley rats were divided into four groups: control, NaHS (0.037mgkgday-1 )-treated control, L-NAME-induced hypertension (40mgkgday-1 ) and NaHS-treated L-NAME-induced hypertension. The ratio of intracavernosal pressure to mean arterial pressure and isometric tension of corpus cavernosum (CC) were measured. The penile expression of endothelial and neuronal NOS (eNOS and nNOS), inflammation markers [nuclear factor kappa B (NF-κB) and inhibitor kappa B alpha (IκBα)], H2 S-producing enzymes[cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE)], the smooth muscle/collagen ratio and H2 S concentrations were determined. The blood pressure was significantly increased in the hypertensive group, but not reversed by NaHS. The erectile response in hypertensive rats was partially prevented by NaHS. The relaxation response to electrical field stimulation was increased in CC from NaHS-treated hypertensive rats. NaHS treatment restored decreased protein expression of eNOS, nNOS and CSE as well as smooth muscle/collagen ratio and H2 S levels and increased NF-κB and IκBα protein expression in the penile tissue of hypertensive rats. NaHS promoted the recovery of erectile responses in hypertensive rats by improvement of neuronal function and downregulation of fibrosis and NF-κB signalling.

Upregulation of cystathionine-γ-lyase/hydrogen sulfide pathway underlies the celecoxib counteraction of cyclosporine-induced hypertension and renal insult in rats

We recently reported that celecoxib, a selective cyclooxygenase-2 (COX2) inhibitor, counteracts the adverse circulatory and renal actions of cyclosporine (CSA). Despite the seemingly advantageous nature of this interaction particularly in clinical settings that necessitate the combined use of the two drugs such as immune-related arthritis, the underlying mechanism remains elusive. This prompted us to test the hypothesis that the facilitation of the cystathionine-γ-lyase (CSE)/hydrogen sulfide (H2S) signaling accounts for such favorable effects of celecoxib on CSA nephrotoxicity. The data showed that the 10-day co-treatment of rats with celecoxib (10 mg/kg/day) ameliorated the hypertensive and biochemical and renal structural damages caused by CSA (20 mg/kg/day). Celecoxib also reversed the CSA-evoked (i) reductions in the tubular and glomerular protein expression of CSE and levels of H2S, prostaglandin E2 (PGE2), and total antioxidant capacity (TAC), and (ii) increases in inflammatory (tumor necrosis factor-α, TNF-α), fibrotic (transforming growth factor-β1, TGF-β1) and apoptotic (caspase-3) cytokines. These celecoxib effects disappeared when rats were treated concomitantly with the CSE inhibitor DL-propargylglycine (DL-PAG), indicating the importance of the CSE-derived H2S in mediating the renoprotective action of celecoxib. This view is bolstered by the observation that the beneficial hemodynamic and renal actions of celecoxib were replicated after supplementation of rats with sodium sulfide (Na2S, H2S donor). Together, the increased abundance of renal CSE and H2S and subsequent dampening down of inflammatory, fibrotic, oxidant, and apoptotic pathways play pivotal roles in the capacity of celecoxib to compromise the troublesome hypertensive and nephrotoxic insults caused by CSA in rats.

Comparative localization of cystathionine beta synthases and cystathionine gamma lyase in canine, non-human primate and human retina

Chronic exposure of the retina to light and high concentrations of polyunsaturated fatty acid in photoreceptor cells make this tissue susceptible to oxidative damage. As retinal degenerative diseases are associated with photoreceptor degeneration, the antioxidant activity of both hydrogen sulfide (H2S) and glutathione (GSH) may play an important role in ameliorating disease progression. H2S production is driven by cystathionine-γ-lyase (CSE) and cystathionine-β–synthase (CBS), the key enzymes that also drive transsulfuration pathway (TSP) necessary for GSH production. As it is currently unclear whether localized production of either H2S or GSH contributes to retinal homeostasis, we undertook a comparative analysis of CBS and CSE expression in canine, non-human primate (NHP) and human retinas to determine if these antioxidants could play a regulatory role in age-related or disease-associated retinal degeneration. Retinas from normal dogs, NHPs and humans were used for the study. Laser capture microdissection (LCM) was performed to isolate individual layers of the canine retina and analyze CBS and CSE gene expression by qRT-PCR. Immunohistochemistry and western blotting were performed for CBS and CSE labeling and protein expression in dog, NHP, and human retina, respectively. Using qRT-PCR, western blot, and immunohistochemistry (IHC), we showed that CBS and CSE are expressed in the canine, NHP, and human retina. IHC results from canine retina demonstrated increased expression levels of CBS but not CSE with post-developmental aging. IHC results also showed non-overlapping localization of both proteins with CBS presenting in rods, amacrine, horizontal, and nerve fiber cell layers while CSE was expressed by RPE, cones and Mϋller cells. Finally, we demonstrated that these enzymes localized to all three layers of canine, NHP and human retina: photoreceptors, outer plexiform layer (OPL) and notably in the ganglion cells layer/nerve fiber layer (GCL/NFL). QRT-PCR performed using RNA extracted from tissues isolated from these cell layers using laser capture microdissection (LCM) confirmed that each of CBS and CSE are expressed equally in these three layers. Together, these findings reveal that CSE and CBS are expressed in the retina, thereby supporting further studies to determine the role of H2S and these proteins in oxidative stress and apoptosis in retinal degenerative diseases.

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.

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.

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.

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.

Hydrogen Sulfide Facilitates Vaginal Lubrication by Activation of Epithelial ATP-Sensitive K(+) Channels and Cystic Fibrosis Transmembrane Conductance Regulator.

Hydrogen sulfide (H2S) plays a large role in female and male sexual responses characterized by a smooth muscle relaxant effect. Moreover, H2S is a novel pro-secretory neuromodulator that modulates epithelial ion transport. However, whether H2S has a role in regulating vaginal epithelial ion transport and fluid secretion has not been extensively studied. To identify the effects of H2S on vaginal epithelial ion transport and lubrication in an exploratory investigation. The mRNA, protein expression, and localization of cystathionine γ-lyase (CSE) and H2S production in vaginal epithelium were examined by reverse transcriptase polymerase chain reaction, Western blot, H2S synthesizing activity assay, and immunohistochemistry, respectively. The effect of H2S on vaginal epithelial ion transport, vaginal fluid secretion, and ionic concentration was investigated using a short-circuit current (ISC), a measurement of vaginal lubrication, and ion chromatography, respectively. The mRNA, protein expression, and localization of CSE, H2S formation, changes of ISC responses, vaginal lubrication, and K(+) and Cl(-) concentrations were studied. CSE mRNA and protein were predominantly expressed in vaginal epithelium. Sodium hydrosulfide hydrate (NaHS) caused concentration-dependent changes in ISC across isolated rat vaginal epithelium, which consisted of an initial decrease phase and then an increase phase. The increase phase in ISC was mainly Cl(-) dependent and abolished by cystic fibrosis transmembrane conductance regulator inhibitor, whereas the decrease phase was sensitive to the adenosine triphosphate-sensitive K(+) (KATP) channel blocker. Furthermore, intravaginal treatment of NaHS significantly enhanced vaginal lubrication invivo, and this effect was prevented by cystic fibrosis transmembrane conductance regulator and KATP channel inhibitors. In addition, the ionic concentrations of K(+) and Cl(-) in rat vaginal fluid were significantly increased by NaHS treatment. The CSE-H2S pathway participates in the regulation of vaginal epithelial K(+) and Cl(-) ion transport to modulate lumen fluid secretion.

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.

Hydrogen sulfide attenuates ferric chloride-induced arterial thrombosis in rats

ABSTRACTHydrogen sulfide (H2S) is a novel gaseous transmitter, regulating a multitude of biological processes in the cardiovascular and other systems. However, it remains unclear whether it exerts any effect on arterial thrombosis. In this study, we examined the effect of H2S on ferric chloride (FeCl3)-induced thrombosis in the rat common carotid artery (CCA). The results revealed a decrease of the H2S-producing enzyme cystathionine γ-lyase (CSE) expression and H2S production that persisted until 48 h after FeCl3 application. Intriguingly, administration with NaHS at appropriate regimen reduced the thrombus formation and enhanced the blood flow, accompanied with the alleviation of CSE and CD31 downregulation, and endothelial cell apoptosis in the rat CCA following FeCl3 application. Moreover, the antithrombotic effect of H2S was also observed in Rose Bengal photochemical model in which the development of thrombosis is contributed by oxidative injury to the endothelium. The in vitro study demonstrated that the mRNA and protein expression of CSE, as well as H2S production, was decreased in hydrogen peroxide (H2O2)-treated endothelial cells. Exogenous supplement of NaHS and CSE overexpression consistently alleviated the increase of cleaved caspase-3 and endothelial cell damage caused by H2O2. Taken together, our findings suggest that endogenous H2S generation in the endothelium may be impaired during arterial thrombosis and that modulation of H2S, either exogenous supplement or boost of endogenous production, may become a potential venue for arterial thrombosis therapy.

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.

Estrogen Replacement Therapy in Ovariectomized Nonpregnant Ewes Stimulates Uterine Artery Hydrogen Sulfide Biosynthesis by Selectively Up-Regulating Cystathionine β-Synthase Expression.

Estrogens dramatically dilate numerous vascular beds with the greatest response in the uterus. Endogenous hydrogen sulfide (H2S) is a potent vasodilator and proangiogenic second messenger, which is synthesized from L-cysteine by cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE). We hypothesized that estrogen replacement therapy (ERT) selectively stimulates H2S biosynthesis in uterine artery (UA) and other systemic arteries. Intact and endothelium-denuded UA, mesenteric artery (MA), and carotid artery (CA) were obtained from ovariectomized nonpregnant ewes (n = 5/group) receiving vehicle or estradiol-17β replacement therapy (ERT). Total RNA and protein were extracted for measuring CBS and CSE, and H2S production was determined by the methylene blue assay. Paraffin-embedded UA rings were used to localize CBS and CSE proteins by immunofluorescence microscopy. ERT significantly stimulated CBS mRNA and protein without altering CSE mRNA or protein in intact and denuded UA. Quantitative immunofluorescence microscopic analyses showed CBS and CSE protein localization in endothelium and smooth muscle and confirmed that ERT stimulated CBS but not CSE protein expression in UA endothelium and smooth muscle. ERT also stimulated CBS, but not CSE, mRNA and protein expression in intact and denuded MA but not CA in ovariectomized ewes. Concomitantly, ERT stimulated UA and MA but not CA H2S production. ERT-stimulated UA H2S production was completely blocked by a specific CBS but not CSE inhibitor. Thus, ERT selectively stimulates UA and MA but not CA H2S biosynthesis by specifically up-regulating CBS expression, implicating a role of H2S in estrogen-induced vasodilation and postmenopausal women's health.