Cystathionine Gamma-lyase Activity Research Articles

Cystathionine Gamma-Lyase Regulates TNF-α-Mediated Injury Response in Human Colonic Epithelial Cells and Colonoids.

Cystathionine gamma-lyase (CSE) and TNF-α are now recognized as key regulators of intestinal homeostasis, inflammation, and wound healing. In colonic epithelial cells, both molecules have been shown to influence a variety of biological processes, but the specific interactions between intracellular signaling pathways regulated by CSE and TNF-α are poorly understood. In the present study, we investigated these interactions in normal colonocytes and an organoid model of the healthy human colon using CSE-specific pharmacological inhibitors and siRNA-mediated transient gene silencing in analytical and functional assays in vitro. We demonstrated that CSE and TNF-α mutually regulated each other's functions in colonic epithelial cells. TNF-α treatment stimulated CSE activity within minutes and upregulated CSE expression after 24 h, increasing endogenous CSE-derived H2S production. In turn, CSE activity promoted TNF-α-induced NF-ĸB and ERK1/2 activation but did not affect the p38 MAPK signaling pathway. Inhibition of CSE activity completely abolished the TNF-α-induced increase in transepithelial permeability and wound healing. Our data suggest that CSE activity may be essential for effective TNF-α-mediated intestinal injury response. Furthermore, CSE regulation of TNF-α-controlled intracellular signaling pathways could provide new therapeutic targets in diseases of the colon associated with impaired epithelial wound healing.

Norswertianolin Promotes Cystathionine γ-Lyase Activity and Attenuates Renal Ischemia/Reperfusion Injury and Hypertension.

Cystathionine gamma-lyase (CSE)/hydrogen sulfide (H2S) plays a protective role in cardiovascular diseases including hypertension and ischemia/reperfusion (I/R) injury. This study was aimed to screen natural small molecule compounds that activate CSE activity and then evaluate its effect(s) on kidney I/R injury and hypertension. Applying computer molecular docking technology, we screened the natural small molecule compound norswertianolin (NW)-specific binding to CSE. Using the microscale thermophoresis technology, we confirmed that the Leu68 site was the essential hydrogen bond site of NW binding to CSE. NW supplementation significantly increased CSE expression and its activity for H2S generation both in vivo and in vitro. In the model of acute and long-term kidney I/R injury, NW pretreatment dramatically attenuated kidney damage, associated with decreasing blood urea nitrogen (BUN), serum creatinine (Cr) level, reactive oxygen species (ROS) production, and cleaved caspase 3 expression. In spontaneously hypertensive rats (SHRs), NW treatment also lowered blood pressure, the media/lumen ratio of the femoral artery, and the mRNA level of inflammatory cytokines. In conclusion, NW acts as a novel small molecular chemical compound CSE agonist, directly binding to CSE, heightening CSE generation–H2S activity, and then alleviating kidney I/R injury and hypertension. NW has a potential therapeutic merit for cardiovascular diseases.

Effects of Sodium Thiosulfate During Resuscitation From Trauma-and-Hemorrhage in Cystathionine Gamma Lyase (CSE) Knockout Mice.

Sodium thiosulfate (Na2S2O3) is a clinically established drug with antioxidant and sulphide-releasing properties. Na2S2O3 mediated neuro- and cardioprotective effects in ischemia/reperfusion models and anti-inflammatory effects in LPS-induced acute lung injury. Moreover, Na2S2O3 improved lung function during resuscitation from hemorrhagic shock in swine with pre-existing atherosclerosis, characterized by decreased expression of cystathionine γ-lyase (CSE), a major source of hydrogen sulfide (H2S) synthesis in the vasculature. Based on these findings, we investigated the effects of Na2S2O3 administration during resuscitation from trauma-and-hemorrhage in mice under conditions of whole body CSE deficit. After blast wave-induced blunt chest trauma and surgical instrumentation, CSE knockout (CSE-/-) mice underwent 1 h of hemorrhagic shock (MAP 35 ± 5 mm Hg). At the beginning of resuscitation comprising retransfusion, norepinephrine support and lung-protective mechanical ventilation, animals received either i.v. Na2S2O3 (0.45 mg g-1, n = 12) or vehicle (saline, n = 13). Hemodynamics, acid-base status, metabolism using stable isotopes, and visceral organ function were assessed. Blood and organs were collected for analysis of cytokines, mitochondrial respiratory capacity, and immunoblotting. Na2S2O3 treatment improved arterial paO2 (P = 0.03) coinciding with higher lung tissue glucocorticoid receptor expression. Norepinephrine requirements were lower in the Na2S2O3 group (P < 0.05), which was associated with lower endogenous glucose production and higher urine output. Na2S2O3 significantly increased renal tissue IκBα and heme oxygenase-1 expression, whereas it lowered kidney IL-6 and MCP-1 levels. Na2S2O3 exerted beneficial effects during resuscitation of murine trauma-and-hemorrhage in CSE-/- mice, confirming and extending the previously described organ-protective and anti-inflammatory properties of Na2S2O3. The findings make Na2S2O3 a potentially promising therapeutic option in the context of impaired CSE activity and/or reduced endogenous H2S availability.

H2S Donors Reverse Age-Related Gastric Malfunction Impaired Due to Fructose-Induced Injury via CBS, CSE, and TST Expression.

ObjectiveExcess of fructose consumption is related to life-treating conditions that affected more than a third of the global population. Therefore, to identify a newer therapeutic strategy for the impact prevention of high fructose injury in age-related malfunctions of the gastric mucosa (GM) in the animal model is important.MethodsAdult and aged male rats were divided into control groups (standard diet, SD) and high fructose diet (HFD) groups; acute water immersion restraint stress (WIRS) was induced for evaluation of GM adaptive response and effects of testing the therapeutic potential of H2S-releasing compounds (H2S donors). Histological examination of gastric damage was done on hematoxylin-eosin stained slides. Cystathionine beta-synthase (CBS), Cystathionine gamma-lyase (CSE), and Thiosulfate-dithiol sulfurtransferase (TST) activities and oxidative index were assessed during exogenous administration of H2S donors: sodium hydrosulfide (NaHS) and the novel hybrid H2S-releasing aspirin (ATB-340). The results showed that HFD increased gastric damage in adult and aged rats. HFD-associated malfunction characterized by low activities of H2S key enzymes, inducing increased oxidation. Pretreatment with NaHS, ATB-340 of aged rats in the models of HFD, and WIRS attenuated gastric damage in contrast to vehicle-treated group (p < 0.05). The effect of ATB-340 was characterized by reverse oxidative index and increased CBS, CSE, and TST activities. In conclusion, H2S donors prevent GM age-related malfunctions by enhancement of CBS, CSE, and TST expression against fructose excess injury though reduction of oxidative damage.

Cystathionine gamma-lyase/H2S system suppresses hepatic acetyl-CoA accumulation and nonalcoholic fatty liver disease in mice

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

Amelioration of metaflammation induced in rats by exogenous hydrogen sulfide: Focus on mesenteric adipocyte oxidative stress

Metaflammation (metabolic inflammation) is low‐grade chronic inflammatory status linking to nutrient excess. Visceral adipocytes in mesenterium (AM) related to high fructose diet (HFD) are sources for metaflammation process, seriously threatening human health. Novel pathogenesis of AM is still unclear and there are no specific effective drugs for treatment of AM. Recently hydrogen sulfide (H2S) demonstrated potent vasodilatation actions which is promised for AM that have reduced vascularization Therefore, the aim of this study was designed to investigate the effects and underlying mechanisms of H2S on the prevention and treatment of experimental rats with AM during HFD.MethodsThe study was done on rats with standard diet (SD, control group) and 28 days HFD, and acute water immersion restraint stress (WRS) was induced for evaluation AM adaptive response and testing therapeutic potential of exogenous H2S donor NaHS (100 μmol/kg/day) and the hybrid H2S‐Aspirin (ATB 340, H2S‐ASA, 17,5 mg/kg/day) vs conventional aspirin (ASA, 10 mg/kg/day) on AM were evaluated by damage index by electronic microscopic study, and glucose level, activity of Cystathionine gamma‐lyase (CSE), Cystathionine beta‐synthase (CBS), Thiosulfate‐dithiol sulfurtransferase (TST) and Carbonyl group (CG) level in blood were examined biochemically (Institutional Ethical Committee Approval: 23/04/2018 № 4).ResultsIn HSD group with NaHS treatment protected the endothelial and sub‐endothelial structures in mesenterium destructions were absent, the nucleus of endothelial cells it was observed an uneven condensation of chromatin. Effect of hybrid H2S‐aspirin characterized as a protective on AM against HFD and WRS‐induced injury, exhibited a downward trend in the inflammatory infiltration, lowering the level CG, which may indicate a reduction in oxidative stress under the influence of hybrid H2S‐aspirin. ATB‐340 showed an increase CSE, CBS and TST activities vs ASA pretreatment (p&lt;0.05). The use of aspirin and NaHS has shown an increase on 67% CSE and CBS activities in HSD rats vs saline group.ConclusionThese findings suggested that exogenous H2S prevented and ameliorated AM metflammation via reduction of oxidative damage, and provided a theoretical basis for effective tool for prevention of HFD‐related AM against long‐term postprandial hyperglycemia.AcknowledgmentAuthors would like to thank Prof. JL Wallace for ATB 340 providing and his expert suggestions.

Diallyl trisulfide, a H2 S donor, inhibits cell growth of human papillary thyroid carcinoma KTC-1 cells through a positive feedback loop between H2 S and cystathionine-gamma-lyase.

Diallyl trisulfide (DATS), derived from garlic, is a well-known hydrogen sulfide (H2 S) donor. H2 S has recently emerged as a novel gasotransmitter involved in the regulation of cancer progression. The present study demonstrated that DATS along with other two H2 S donors, NaHS and GYY4137, significantly inhibited papillary thyroid carcinoma KTC-1 cells growth. DATS treatment triggered a rapid H2 S generation within 5 min in KTC-1 cells. Iodoacetamide, a potent thiol blocker reagent, partially rescued the cell membrane damage and ultimate cell death induced by DATS, indicating H2 S contributed to the apoptosis-inducing efficacy of DATS on thyroid cancer cells. Specifically, DATS treatment significantly upregulated the expression and enzymatic activity of cystathionine gamma-lyase (CTH), one of H2 S-producing enzymes, which was responsible for endogenous H2 S generation. After DATS treatment, H2 S quickly permeated cell membranes and activated NF-κΒ/p65 signaling pathway in KTC-1 cells. Nuclear translocated NF-κB bound to the promoter of CTH to enhance its transcription. These evidences proved that exogenous H2 S elevated CTH expression. CTH, in turn, catalytically generated a much higher level of endogenous H2 S. This positive feedback sustained excess H2 S production, which resulted in PTC cells growth inhibition. These findings may shed light on the development of novel H2 S-based antitumor agents.

Hydrogen sulfide modulates epithelial-mesenchymal transition and angiogenesis in non-small cell lung cancer via HIF-1α activation

Hydrogen sulfide (H2S) has been frequently implicated in tumor progression. However, the exact regulation mechanism of H2S in human non-small cell lung cancer (NSCLC) has not been fully elucidated. Here, analysis of NSCLC biopsies and adjacent non-tumor tissues revealed selectively high levels of endogenous H2S-producing enzymes, cystathionine-beta-synthase (CBS), cystathionine-gamma-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (MPST). Similarly, quantitative real-time PCR (qRT-PCR) and western blot results showed that NSCLC cell lines (A549, 95D) expressed higher levels of CBS, CSE and MPST in mRNA and enzyme proteins, respectively. Moreover, NSCLC cell lines produced more H2S than did the normal lung epithelial cell line BEAS-2B. H2S was further detected to induce NSCLC migration and invasion, as well as the epithelial mesenchymal transition (EMT) process. Small interfering RNA (siRNA) silencing of CBS or CSE activity reduced proliferation and metastasis potential of tumor cells. In addition, H2S modulated hypoxia-inducible factor-1α (HIF-1α) to stimulate vascular endothelial growth factor (VEGF) expression, which contributes to tumor angiogenesis. Treatment of nude mice with pharmacological inhibition of CBS or CSE activity decreased xenograft growth and suppressed angiogenesis. Collectively, these results indicate H2S plays an important part in NSCLC growth and angiogenesis by HIF-1α activation, which potentially provide new insight in therapeutic strategies.

Sodium thiosulfate post-conditioning protects rat hearts against ischemia reperfusion injury via reduction of apoptosis and oxidative stress

Pharmacological agents given at the time of reperfusion can protect the heart from ischemia reperfusion injury (IR). Being a calcium chelator, antioxidant and mitochondrial potassium channel modulator, sodium thiosulfate (STS) was chosen to treat myocardial IR injury. Isolated rat heart model was used to induce IR injury and the hemodynamic changes were monitored using PowerLab (AD Instruments, Australia). STS at a dose of 1 mM given at the early stage of reperfusion significantly reduced the infarct size and recovered the failing heart from reperfusion injury. Its action was based on reduction of apoptosis as evidenced from decreased activity of caspase-3 in the myocardium, lowered expression of casp-3 and PARP, which was supported by absence of significant DNA fragmentation and histological derangement of fibers compared to the injury control. An evaluation of the inter-dependency of H2S and STS biosynthesis in the STS treated groups showed no significant changes in the level of STS, H2S and rhodanese, except the cystathionine gamma lyase activity that improved upon treatment. The mechanism underlying the antiapoptotic, mitochondrial preservation and antioxidant effects of STS were related to the biosynthesis of H2S. The fact that inhibition of cystathionine gamma lyase limited the STS mediated cardio protection supports this observation.

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.

Abstract 1857: Dysregulation of transsulfuration enzymes contribute to malignant transformation in a murine model of colitis-associated carcinogenesis

Abstract Introduction: Ulcerative colitis (UC) is a highly morbid, chronic inflammatory disease characterized by mucosal ulceration of the colonic mucosa and is associated with the higher risk of colitis-associated cancer (CAC). The exact mechanism(s) causing UC progression to CAC is currently unknown. The balanced activity of transsulfuration pathway enzymes, cystathionine-gamma-lyase (CSE) and cystathionine-beta-synthase (CBS), and their production of endogenous hydrogen sulfide gas, are critical to maintenance of the colonic homeostasis. CSE activity is suggested to be important for wound healing and mucosal protection. Consistent with this, we have previously showed a decrease CSE expression in human colonic mucosa obtained from patients with chronic UC, compared to normal colonic mucosa by immunocytochemistry. By contrast, increased CBS expression is implicated in the progression of sporadic colorectal carcinoma. However, the role of these enzymes in CAC is unknown. We hypothesize that the dysregulation in CSE/CBS expression and activity is important to the progression from UC to CAC. Methods: CSE null mice and wild type Sv129/B6 (control) mice were used in azoxymethane-dextran sodium sulfate (AOM-DSS) colon cancer model which mimics human CAC. The disease development was followed up to day 80. Confocal microscopy and Western blot was used to assess the gene expression during cancer development. Size, number, and time interval to tumor formation, as well inflammation were assessed. Results: Abrogation of CSE expression using CSE null animals in AOM-DSS model of CAC accelerated the time to tumor development and resulted in an increase in both tumor size (p&amp;lt;0.001) and number (p&amp;lt;0.001) compared to wild-type controls. CBS protein expression was increased within the colonic tumor when compared to the normal margin in AOM-DSS treated animals by Western blot analysis and tissue immunostaining. Interestingly, CAC liver metastases, an exceedingly rare finding in this mouse model, were identified. Conclusion: Taken together, our human and murine data suggest that dysregulation in the transsulfuration pathway enzymes CSE and CBS expression/activity may be critical contributor to the CAC development in UC and may serve as a potential biomarker for disease progression in the future. Citation Format: Paul Johnson, Ches’Que M. Phillips, Carl Grim, John R. Zatarain, Aakash H. Gajjar, Suimin Qiu, Rui Wang, Celia Chao, Iryna V. Pinchuk, Mark R. Hellmich. Dysregulation of transsulfuration enzymes contribute to malignant transformation in a murine model of colitis-associated carcinogenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1857.

Hydrogen sulfide accelerates the recovery of kidney tubules after renal ischemia/reperfusion injury.

Progression of acute kidney injury to chronic kidney disease (CKD) is associated with inadequate recovery of damaged kidney. Hydrogen sulfide (H2S) regulates a variety of cellular signals involved in cell death, differentiation and proliferation. This study aimed to identify the role of H2S and its producing enzymes in the recovery of kidney following ischemia/reperfusion (I/R) injury. Mice were subjected to 30 min of bilateral renal ischemia. Some mice were administered daily NaHS, an H2S donor, and propargylglycine (PAG), an inhibitor of the H2S-producing enzyme cystathionine gamma-lyase (CSE), during the recovery phase. Cell proliferation was assessed via 5'-bromo-2'-deoxyuridine (BrdU) incorporation assay. Ischemia resulted in decreases in CSE and cystathionine beta-synthase (CBS) expression and activity, and H2S level in the kidney. These decreases did not return to sham level until 8 days after ischemia when kidney had fibrotic lesions. NaHS administration to I/R-injured mice accelerated the recovery of renal function and tubule morphology, whereas PAG delayed that. Furthermore, PAG increased mortality after ischemia. NaHS administration to I/R-injured mice accelerated tubular cell proliferation, whereas it inhibited interstitial cell proliferation. In addition, NaHS treatment reduced post-I/R superoxide formation, lipid peroxidation, level of GSSG/GSH and Nox4 expression, whereas it increased catalase and MnSOD expression. Our findings demonstrate that H2S accelerates the recovery of I/R-induced kidney damage, suggesting that the H2S-producing transsulfuration pathway plays an important role in kidney repair after acute injury.

Calcium Sensing Receptor Regulating Smooth Muscle Cells Proliferation Through Initiating Cystathionine-Gamma-Lyase/Hydrogen Sulfide Pathway in Diabetic Rat

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

P1 Gender dimorphism of hydrogen sulfide production and physiological effects in cardiovascular system

H 2 S is signalling molecule that regulates vascular tone and myocardial contractility. The role of gender and sex hormones in H 2 S production and physiological effects on rats cardiovascular system (CVS) remains unclear what became research objective. Sex hormones level in Wister rats was modulated by castration and hormone replacement therapy. H 2 S content, cystathionine gamma-lyase (CSE) activity in heart and aorta, sex hormones level in blood, rat aortic ring sensitivity to H 2 S were estimated. CSE activity, H 2 S content in CVS in females was higher by 19–23% ( p < 0.05) than in males. Aortic ring sensitivity to H 2 S was higher in females than in males (EC 50 in females – 70.5 ± 5.84 mM vs – 95.2 ± 4.57 mM in males, p < 0.05). Castration resulted in oppositely directed changes of H 2 S production in CVS: ovariectomy was accompanied by decrease in CSE activity and H 2 S content (by 19–26%, p < 0.05), whereas orhiectomy caused opposite changes. Statistic decrease in H 2 S-induced vasodilatation of aortic ring in females and its increase in males were noted. In castrated animals that underwent hormone replacement therapy CSE activity, H 2 S content in CVS and aorta ring sensitivity to H 2 S did not differ ( p > 0.05) from control. H 2 S level, CSE activity in CVS, EC 50 (H 2 S) in aorta positively correlated with serum estradiol ( r = 0.40–0.56, p < 0.05) but negatively correlated with serum testosterone ( r = −0.30–0.47, p < 0.05). Thus, sex hormones are involved in regulation of H 2 S production and its physiological effects in rats’ CVS.

P65 Inhibition of cystathionine gamma-lyase increases endothelium-dependent relaxation of aortic smooth muscles in old rats

Purpose One of the reasons of increasing the risk of cardiovascular diseases during aging is a dysfunction of endothelium. At the same time we can see more and more publications about vascular protection of hydrogen sulfide (H2S). Cystathionine gamma-lyase (CSE) is the main enzyme for H2S synthesis in blood vessels. Methods The segments of aorta from adult and old rats were used. Activation of isolated samples was carried out by phenylephrine (Phe). Acetylcholine (Ach)-mediated vasorelaxation in the aortic vessel were investigated. The activity of CSE was inhibited with propargylglycine (PPG) (incubation 30 min). H2S was determined by N,N-dimethyl-p-phenylenediamine method, NO de novo synthesis – by NOs activity and nitrite determination. Results The experiments on isolated segments of aorta have shown that the effect of Ach endothelium-dependent vasodilator results in less profound relaxation of aortic smooth muscles (SM) in old animals (12.4%) compared to adult ones (66.3%). PPG addition resulted in growing aortic SM relaxation amplitude from 12.4% to 49.5% (p NO 2 - pools. Also increased the activity of cNOS and decreased the activity of iNOS (p Conclusions Therefore, we can state that use of PPG has led to growing production of two powerful vasodilators – H2S and NO and to the restoration of endothelium-dependent aortic SM vasorelaxation in old animals.

P9 Disturbances in hydrogen sulfide synthesis and degradation in rats’ myocardium in aging

Hydrogen sulfide (H2S) is signaling molecule, vasodilator, cytoprotector, antioxidant and it may be involved in pathogenesis of age-associated heart diseases. The question remains open about H2S synthesis and utilization in myocardium within aging what became our study objective. 60 male Wistar rats of three age groups (1–2 months, 6–8 months, 24–26 months) were randomized into subgroups: control (normal saline), NaHS (3 mg/kg i.p.), DL-propargylglycine (50 mg/kg i.p). Modulators of H2S metabolism were administered for 14 days. It was determined the level of free and acid-labile H2S (Wilinski B., 2011), cystathionine gamma-lyase (CSE), cysteine aminotransferase + 3-mercaptopyruvate sulfurtransferase (CAT/3MST), sulfite oxidase, thioredoxin reductase activity in myocardium. H2S level, CSE, CAT/3MST, sulfite oxidase, thioredoxin reductase activity in myocardium of 24–26-month-old rats were 30–50% lower than in myocardium of 6–8 and 1–2 month-old rats. Propargylglycine administration caused decrease in H2S level and CSE activity on 33.3 and 78.3% in 24–26 month-old rats, 22 and 61.8 % in 6–8 month-old rats, 14 and 50.3% in 1–2 month-old rats (p

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

Regulation of cystathionine gamma-lyase/H₂S system and its pathological implication.

Hydrogen sulfide (H2S) is a highly diffusible gasotransmitter, that influence cellular and organ functions by a number of different mechanisms. Cystathionine gamma-lyase (CSE) is one major H2S-producing enzyme with L-cysteine as the main substrate in mammalian cells. Since the discovery of endogenously-produced H2S as a biological mediator, there has been an explosion of interest in CSE expression and regulation. CSE expression and activity and ultimately the amount of H2S synthesis is controlled by a complex integration of transcriptional, post-transcriptional and post-translational mechanisms. Considering the key role that CSE/H 2 S system plays in both health and diseases, a better understanding of the regulation of CSE/H 2 S system will help us to develop novel and more effective strategies to target CSE and alter H2S production inside cells. In this review, we summarize the altered expression and activity of CSE and abnormal H2S production in various pathophysiological conditions. The current knowledge on the signaling and regulatory pathways for CSE expression and H2S production are also elucidated. As such, our understanding of the pathogenesis of human diseases will be better achieved and the corresponding new therapy can be devised.

PL02 Modulation of H2S levels in cells and tissues using pharmacological agents: Advances and pitfalls

The biological roles of endogenous H 2 S are multiple and rapidly expanding. Hydrogen sulfide exerts its effects in most organ systems including the central and peripheral nervous system, the cardiovascular, gastrointestinal and respiratory systems; H 2 S also participates in the regulation of cellular metabolism and immunological/inflammatory responses. Much of the enzymatically generated H 2 S is derived from two pyridoxal-5′-phosphate (PLP)-dependent enzymes responsible for the metabolism of l -cysteine: cystathionine beta synthase (CBS) and cystathionine gamma lyase (CSE); a third pathway that catalyzes the production of H 2 S from l -cysteine via the combined action of 3-mercaptopyruvate sulphurtransferase and cysteine aminotransferase (3MST/CAT) has also been described. CSE and CBS catalyze several H 2 S-generating reactions using cysteine and/or homocysteine as substrates. CBS is the predominant H 2 S-generating enzyme in the brain and nervous system and is highly expressed in liver and kidney, while CSE is mainly expressed in the liver and in vascular and non-vascular smooth muscle. To study the role of this novel gasotransmitter, investigators have relied on genetic models, knock down of H 2 S-producing enzymes by siRNA, and on pharmacological inhibitors. As genetic and siRNA approaches require additional expertise and access to specialized facilities, most researchers prefer to use pharmacological inhibitors. The most commonly used agents to inhibit H 2 S biosynthesis include propargylglycine (PAG), β -cyanoalanine (BCA), aminooxyacetic acid (AOAA), trifluoroalanine and hydroxylamine. Using recombinant human H 2 S-producing enzymes we have found that PAG exhibits selectivity towards CSE vs. CBS, but its potency is lower than that of l -aminoethoxyvinylglycine (AVG). BCA, although selective for CSE, it also inhibits CBS when used at high concentrations. In contrast, none of the compounds tested exhibited significant selectivity towards CBS. In addition, the above-mentioned compounds did not inhibit 3MST. It should also be stressed that even the inhibitors that were found to be selective for CSE have been shown to inhibit other PLP-dependent enzymes. Inhibiting CSE or CBS activity will affect the levels of several metabolites and intermediates in the transulfuration pathway. For example, CBS inhibition leads to increased homocysteine levels. To avoid this unwanted side effect when long term inhibition of CBS or CSE is desired (such as in conditions where H 2 S production is up regulated contributing to disease progression), scavenging of H 2 S would be the preferred approach. Our efforts to develop H 2 S scavengers will be presented during the meeting. We conclude that design and development of new inhibitors for each of the H 2 S-synthesizing enzymes, as well as H 2 S scavengers is an unmet need for the field, so that the role of H 2 S-regulated pathways in the development and treatment of disease can be evaluated.

OP019 H2S attenuates DNA damage by targeting at MEK1 and PAR

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