Metabolic control analysis of the transsulfuration pathway and the compensatory role of the cysteine transport in Trypanosoma cruzi.

Trypanosoma cruzi is the causal agent of American Trypanosomiasis or Chagas Disease in humans. The current drugs for its treatment benznidazole and nifurtimox have inconveniences of toxicity and efficacy; therefore, the search for new therapies continues. Validation through genetic strategies of new drug targets against the parasite metabolism have identified numerous essential genes. Target validation can be further narrowed by applying Metabolic Control Analysis (MCA) to determine the flux control coefficients of the pathway enzymes. That coefficient is a quantitative value that represents the degree in which an enzyme/transporter determines the flux of a metabolic pathway; those with the highest coefficients can be promising drug targets. Previous studies have demonstrated that cysteine (Cys) is a key precursor for the synthesis of trypanothione, the main antioxidant metabolite in the parasite. In this research, MCA was applied in an ex vivo system to the enzymes of the reverse transsulfuration pathway (RTP) for Cys synthesis composed by cystathionine beta synthase (CBS) and cystathionine gamma lyase (CGL). The results indicated that CGL has 90% of the control of the pathway flux. Inhibition of CGL with propargylglycine (PAG) decreased the levels of Cys and trypanothione and depleted those of glutathione in epimastigotes (proliferative stage in the insect vector); these metabolite changes were prevented by supplementing with Cys, suggesting a compensatory role of the Cys transport (CysT). Indeed, Cys supplementation (but not PAG treatment) increased the activity of the CysT in epimastigotes whereas in trypomastigotes (infective stage in mammals) CysT was increased when they were incubated with PAG. Our results suggested that CGL could be a potential drug target given its high control on the RTP flux and its effects on the parasite antioxidant defense. However, the redundant Cys supply pathways in the parasite may require inhibition of the CysT as well. Our findings also suggest differential responses of the Cys supply pathways in different parasite stages.

Endogenous Hydrogen Sulfide Persulfidates Caspase-3 at Cysteine 163 to Inhibit Doxorubicin-Induced Cardiomyocyte Apoptosis.

Doxorubicin (DOX) is an efficient antitumor anthracycline drug, but its cardiotoxicity adversely affects the prognosis of the patients. In this study, we explored whether endogenous gasotransmitter hydrogen sulfide (H2S) could protect against DOX-induced cardiomyocyte apoptosis and its mechanisms. The results indicated that DOX significantly downregulated endogenous H2S production and endogenous synthetase cystathionine γ-lyase (CSE) expression and obviously stimulated the apoptosis in H9C2 cells. The supplement of H2S donor sodium hydrosulfide (NaHS) or overexpression of CSE inhibited DOX-induced H9C2 cell apoptosis. DOX enhanced the activities of caspase family members in cardiomyocytes, while NaHS attenuated DOX-enhanced caspase-3, caspase-2, and caspase-9 activities by 223.1%, 73.94%, and 52.29%, respectively. Therefore, taking caspase-3 as a main target, we demonstrated that NaHS or CSE overexpression alleviated the cleavage of caspase-3, suppressed caspase-3 activity, and inhibited the cleavage of poly ADP-ribose polymerase (PARP). Mechanistically, we found that H2S persulfidated caspase-3 in H9C2 cells and human recombinant caspase-3 protein, while the thiol-reducing agent dithiothreitol (DTT) abolished H2S-induced persulfidation of caspase-3 and thereby prevented the antiapoptotic effect of H2S on caspase-3 in H9C2 cells. The mutation of caspase-3 C148S and C170S failed to block caspase-3 persulfidation by H2S in H9C2 cells. However, caspase-3 C163S mutation successfully abolished the effect of H2S on caspase-3 persulfidation and the corresponding protection of H9C2 cells. Collectively, these findings indicate that endogenous H2S persulfidates caspase-3 at cysteine 163, inhibiting its activity and cardiomyocyte apoptosis. Sufficient endogenous H2S might be necessary for the protection against myocardial cell apoptosis induced by DOX. The results of the study might open new avenues with respect to the therapy of DOX-stimulated cardiomyopathy.

The Expression and Activity of Rhodanese, 3-Mercaptopyruvate Sulfurtransferase, Cystathionine γ-Lyase in the Most Frequently Chosen Cellular Research Models.

This paper provides information concerning the activity and expression levels of three sulfurtransferases (STRs): rhodanese (TST, EC: 2.8.1.1), 3-mercaptopyruvate sulfurtransferase (MPST, EC: 2.8.1.2) and cystathionine γ-lyase (CTH, EC: 4.4.1.1) in various cell lines. Since very limited data are available in the scientific literature on this subject, the available data are included in this paper. These shortages often force the researchers to carry out their own screening tests that allow them to choose an appropriate model for their further studies. This work supplements the existing deficiencies in this area and presents the activity and expression of STRs in the eight most frequently chosen cell lines: the mouse mammary gland cell line (NMuNG, ATCC: CRL-1636), mouse mammary gland tumor (4T1, ATCC: CRL-2539), mouse fibroblast (MEF, ATCC: SCRC-1008), mouse melanoma (B16-F1, ATCC: CRL-6323), human colorectal adenocarcinoma (Caco-2, ATCC: HTB-37), human embryonic kidney (HEK-293, ATCC: CRL-1573), human osteosarcoma (MG-63, ATCC: CRL-1427) and rat myocardium (H9c2, ATCC: CRL-1446). Changes in STRs activity are directly related to the bioavailability of cysteine and the sulfane sulfur level, and thus the present authors also measured these parameters, as well as the level of glutathione (its reduced (GSH) and oxidized (GSSG) form) and the [GSH]/[GSSG] ratio that determines the antioxidant capacity of the cells. STRs demonstrate diverse functionality and clinical relevance; therefore, we also performed an analysis of genetic variation of STRs genes that revealed a large number of polymorphisms. Although STRs still provide challenges in several fields, responding to them could not only improve the understanding of various diseases, but may also provide a way to treat them.

Mapping the Endothelial Cell S-Sulfhydrome Highlights the Crucial Role of Integrin Sulfhydration in Vascular Function.

BACKGROUND: In vascular endothelial cells, cysteine metabolism by the cystathionine γ lyase (CSE), generates hydrogen sulfide-related sulfane sulfur compounds (H2Sn), that exert their biological actions via cysteine S-sulfhydration of target proteins. This study set out to map the "S-sulfhydrome" (ie, the spectrum of proteins targeted by H2Sn) in human endothelial cells. METHODS: Liquid chromatography with tandem mass spectrometry was used to identify S-sulfhydrated cysteines in endothelial cell proteins and ß3 integrin intraprotein disulfide bond rearrangement. Functional studies included endothelial cell adhesion, shear stress-induced cell alignment, blood pressure measurements, and flow-induced vasodilatation in endothelial cell-specific CSE knockout mice and in a small collective of patients with endothelial dysfunction. RESULTS: Three paired sample sets were compared: (1) native human endothelial cells isolated from plaque-free mesenteric arteries (CSE activity high) and plaque-containing carotid arteries (CSE activity low); (2) cultured human endothelial cells kept under static conditions or exposed to fluid shear stress to decrease CSE expression; and (3) cultured endothelial cells exposed to shear stress to decrease CSE expression and treated with solvent or the slow-releasing H2Sn donor, SG1002. The endothelial cell "S-sulfhydrome" consisted of 3446 individual cysteine residues in 1591 proteins. The most altered family of proteins were the integrins and focusing on ß3 integrin in detail we found that S-sulfhydration affected intraprotein disulfide bond formation and was required for the maintenance of an extended-open conformation of the ß leg. ß3 integrin S-sulfhydration was required for endothelial cell mechanotransduction in vitro as well as flow-induced dilatation in murine mesenteric arteries. In cultured cells, the loss of S-sulfhydration impaired interactions between ß3 integrin and Gα13 (guanine nucleotide-binding protein subunit α 13), resulting in the constitutive activation of RhoA (ras homolog family member A) and impaired flow-induced endothelial cell realignment. In humans with atherosclerosis, endothelial function correlated with low H2Sn generation, impaired flow-induced dilatation, and failure to detect ß3 integrin S-sulfhydration, all of which were rescued after the administration of an H2Sn supplement. CONCLUSIONS: Vascular disease is associated with marked changes in the S-sulfhydration of endothelial cell proteins involved in mediating responses to flow. Short-term H2Sn supplementation improved vascular reactivity in humans highlighting the potential of interfering with this pathway to treat vascular disease.

Bioenergetic effects of hydrogen sulfide suppress soluble Flt-1 and soluble endoglin in cystathionine gamma-lyase compromised endothelial cells.

Endothelial dysfunction is a hallmark of preeclampsia, a life-threatening complication of pregnancy characterised by hypertension and elevated soluble Fms-Like Tyrosine Kinase-1 (sFlt-1). Dysregulation of hydrogen sulfide (H2S) by inhibition of cystathionine γ-lyase (CSE) increases sFlt-1 and soluble endoglin (sEng) release. We explored whether compromise in CSE/H2S pathway is linked to dysregulation of the mitochondrial bioenergetics and oxidative status. We investigated whether these effects were linked to CSE-induced sFlt-1 and sEng production in endothelial cells. Here, we demonstrate that CSE/H2S pathway sustain endothelial mitochondrial bioenergetics and loss of CSE increases the production of mitochondrial-specific superoxide. As a compensatory effect, low CSE environment enhances the reliance on glycolysis. The mitochondrial-targeted H2S donor, AP39, suppressed the antiangiogenic response and restored the mitochondrial bioenergetics in endothelial cells. AP39 revealed that upregulation of sFlt-1, but not sEng, is independent of the mitochondrial H2S metabolising enzyme, SQR. These data provide new insights into the molecular mechanisms for antiangiogenic upregulation in a mitochondrial-driven environment. Targeting H2S to the mitochondria may be of therapeutic benefit in the prevention of endothelial dysfunction associated with preeclampsia.

Cystathionine γ-lyase-H2S facilitates mandibular defect healing via inducing osteogenic differentiation of bone marrow mesenchymal stem cells.

OBJECTIVE: To investigate the effects of endogenous hydrogen sulfide (H2S) synthase, cystathionine-γ-lyase (CSE), on the healing of mandibular defect and the osteogenic differentiation of human mandibular bone marrow mesenchymal stem cells (HM-BMMSCs). METHODS: Sixty 8-week male C57BL/6 wild-type (WT) mice and CSE knockout (CSE-/-) mice were divided into WT group, CSE-/- group and CSE-/- + GYY4137 (a slow-releasing H2S donor) group. Mandibular defect healing in each group was identified by micro-CT. The histological staining and immunohistochemical staining were adopted to evaluate bone regeneration and reconstruction of mandibular defect. HM-BMMSCs were extracted and cultured for osteogenic induction, which were divided into control group, PAG (a CSE inhibitor) group, GYY4137 group and PAG + GYY4137 group. The mineralization of HM-BMMSCs in each group was determined by alkaline phosphatase (ALP) staining and alizarin red staining. Moreover, mRNA expressions of ALP and Runt-related transcription factor 2 (RUNX2) were detected by RT-PCR. RESULTS: Mandibular defect healing in CSE-/- mice was undesirable. When exogenous H2S were supplemented to CSE-/- mice, the new bone mass increased with higher degrees of bone mineralization and bone maturity. Bone mineral density (BMD), bone volume fraction (BV/TV) and bone trabecular thickness (Tb.Th) also significantly increased. in vitro experiments showed that PAG attenuated ALP activity and mineralized nodule formation ability in HM-BMMSCs, and repressed mRNA expressions of ALP and RUNX2. All these osteogenic indexes of HM-BMMSCs were reversed after exogenous H2S was supplemented. CONCLUSION: It is demonstrated that CSE deficiency thwarts the healing of mandibular defect. Blocking the synthesis of H2S inhibits the osteogenic differentiation of HM-BMMSCs, thereby affects bone healing.

Epigenetic Factors in Late-Onset Alzheimer's Disease: MTHFR and CTH Gene Polymorphisms, Metabolic Transsulfuration and Methylation Pathways, and B Vitamins.

DNA methylation and other epigenetic factors are important in the pathogenesis of late-onset Alzheimer's disease (LOAD). Methylenetetrahydrofolate reductase (MTHFR) gene mutations occur in most elderly patients with memory loss. MTHFR is critical for production of S-adenosyl-l-methionine (SAM), the principal methyl donor. A common mutation (1364T/T) of the cystathionine-γ-lyase (CTH) gene affects the enzyme that converts cystathionine to cysteine in the transsulfuration pathway causing plasma elevation of total homocysteine (tHcy) or hyperhomocysteinemia-a strong and independent risk factor for cognitive loss and AD. Other causes of hyperhomocysteinemia include aging, nutritional factors, and deficiencies of B vitamins. We emphasize the importance of supplementing vitamin B12 (methylcobalamin), vitamin B9 (folic acid), vitamin B6 (pyridoxine), and SAM to patients in early stages of LOAD.

The interaction of IGF-1/IGF-1R and hydrogen sulfide on the proliferation of mouse primary vascular smooth muscle cells.

Hydrogen sulfide (H2S) is mostly produced by cystathionine-gamma-lyase (CSE) in vascular system and it inhibits the proliferation of vascular smooth muscle cells (SMCs). Insulin-like growth factor-1 (IGF-1), via its receptor (IGF-1R), exerts multiple physiological and pathophysiological effects on the vasculature, including stimulating SMC proliferation and migration, and inhibiting SMC apoptosis. Since H2S and IGF-1/IGF-1R have opposite effects on SMC proliferation, it becomes imperative to better understand the interaction of these two signaling mechanisms on SMC proliferation. SMCs isolated from small mesenteric arteries of CSE knockout (KO) and wild-type (WT) mice were used in the present study. The effects of IGF-1 and H2S on SMC proliferation were evaluated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and bromodeoxyuridine (BrdU) assays. Protein expression was determined by western blot, and H2S-induced protein S-sulfhydration was assessed with a modified biotin switch assay. We found that IGF-1 dose-dependently increased the proliferation of both WT-SMCs and KO-SMCs, and this effect was more significant in KO-SMCs. Supplement of sodium hydrosulfide (NaHS) inhibited IGF-1-induced cell proliferation, while this effect was abolished by blocking IGF-1/IGF-1R signaling with picropodophyllin (PPP) or knocking out of the expression of IGF-1R. H2S significantly down-regulates the expression of IGF-1R, stimulates IGF-1R S-sulfhydration, and attenuates the binding of IGF-1 with IGF-1R. This study provides novel insight on the involvement of IGF-1/IGF-1R in H2S-inhibited SMC proliferation and suggests H2S-based innovative treatment strategies for proliferative cardiovascular diseases such as atherosclerosis.

Transcriptional control of amino acid homeostasis is disrupted in Huntington's disease.

Disturbances in amino acid metabolism, which have been observed in Huntington's disease (HD), may account for the profound inanition of HD patients. HD is triggered by an expansion of polyglutamine repeats in the protein huntingtin (Htt), impacting diverse cellular processes, ranging from transcriptional regulation to cognitive and motor functions. We show here that the master regulator of amino acid homeostasis, activating transcription factor 4 (ATF4), is dysfunctional in HD because of oxidative stress contributed by aberrant cysteine biosynthesis and transport. Consistent with these observations, antioxidant supplementation reverses the disordered ATF4 response to nutrient stress. Our findings establish a molecular link between amino acid disposition and oxidative stress leading to cytotoxicity. This signaling cascade may be relevant to other diseases involving redox imbalance and deficits in amino acid metabolism.

N-acetylcysteine modulates glutamatergic dysfunction and depressive behavior in Huntington's disease.

Glutamatergic dysfunction has been implicated in the pathogenesis of depressive disorders and Huntington's disease (HD), in which depression is the most common psychiatric symptom. Synaptic glutamate homeostasis is regulated by cystine-dependent glutamate transporters, including GLT-1 and system xc- In HD, the enzyme regulating cysteine (and subsequently cystine) production, cystathionine-γ-lygase, has recently been shown to be lowered. The aim of the present study was to establish whether cysteine supplementation, using N-acetylcysteine (NAC) could ameliorate glutamate pathology through the cystine-dependent transporters, system xc- and GLT-1. We demonstrate that the R6/1 transgenic mouse model of HD has lower basal levels of cystine, and showed depressive-like behaviors in the forced-swim test. Administration of NAC reversed these behaviors. This effect was blocked by co-administration of the system xc- and GLT-1 inhibitors CPG and DHK, showing that glutamate transporter activity was required for the antidepressant effects of NAC. NAC was also able to specifically increase glutamate in HD mice, in a glutamate transporter-dependent manner. These in vivo changes reflect changes in glutamate transporter protein in HD mice and human HD post-mortem tissue. Furthermore, NAC was able to rescue changes in key glutamate receptor proteins related to excitotoxicity in HD, including NMDAR2B. Thus, we have shown that baseline reductions in cysteine underlie glutamatergic dysfunction and depressive-like behavior in HD and these changes can be rescued by treatment with NAC. These findings have implications for the development of new therapeutic approaches for depressive disorders.

Metabolic changes of H2S in smokers and patients of COPD which might involve in inflammation, oxidative stress and steroid sensitivity.

Oxidative stress and inflammation play crucial role in the pathogenesis of chronic obstructive pulmonary disease (COPD). Most patients with COPD show a poor response to corticosteroids. Hydrogen sulfide (H2S ) has been implicated in the pathogenesis of COPD, but its expression and effects in lung tissue from COPD patients are not clear. In peripheral lung tissue samples from 24 patients, we found that compared with nonsmokers, the protein level of cystathionine-γ-lyase (CSE) was decreased in smokers and COPD patients. CSE mRNA increased but cystathionine-ß-synthase (CBS) mRNA decreased in COPD patients. H2S donors increased glutathione and superoxide dismutase in CS exposed U937 cells and inhibited CS-induced TNF-α and IL-8 secretion. Dexamethasone alone had no effect on lipopolysaccharide (LPS) induced TNF-α release by alveolar macrophages from CS exposed rats, however the combination of dexamethasone and H2S donor significantly inhibited TNF-α release. Thus, H2S metabolism is altered in lung tissue of smokers and COPD patients. Supplementation of H2S protects against CS-induced oxidative stress and inflammation in macrophages and H2S on steroid sensitivity deserves further investigation.

Erastin sensitizes glioblastoma cells to temozolomide by restraining xCT and cystathionine-γ-lyase function.

Glioblastoma multiforme (GBM) is one of the most common encephalic malignant tumors. Due to a high recurrence rate and a lack of effective treatments, the average survival rate remains low. Temozolomide (TMZ), a class of alkylating agent, is widely used as a first-line therapeutic drug during the adjuvant treatment for GBM patients. However, most patients exhibit a palpable resistance to TMZ treatment. Additionally, the underlying mechanism remains to be clarified. In this study, glutathione (GSH) and reactive oxygen species (ROS) levels were found to be closely associated with the sensitivity of GBM cells to TMZ. We also found that TMZ markedly induced xCT, the subunit of glutamate/cystine transporter system xc- expression, which together with the GSH synthesis was increased while the TMZ-inducible ROS level was decreased in GBM cells. In addition, the cystathionine γ-lyase (CTH) acivity, a key enzyme in the transsulfuration pathway was enhanced by TMZ, which insured a cysteine supply and GSH synthesis in a compensatory manner when xCT was blocked. Thus, the individual inhibition of xCT by siRNA and a pharmacological inhibitor (sulfasalazine) only partially inhibited GSH synthesis and moderately enhanced the GBM cell sensitivity to TMZ. However, the TMZ­induced cytotoxicity was markedly increased along with a marked decrease in GSH levels as result of co-treatment with erastin, which inhibited cysteine uptake from xCT transporter and suppressed CTH activity, leading to impaired transformation from methionine to cysteine. In conclusion, to GBM therapy with a drug combination of TMZ and erastin may be beneficial.

Involvement of threonine deaminase FgIlv1 in isoleucine biosynthesis and full virulence in Fusarium graminearum.

In this study we characterized FgIlv1, a homologue of the Saccharomyces cerevisiae threonine dehydratase (TD) from the important Fusarium head blight fungus Fusarium graminearum. TD catalyzes the first step in the biosynthesis pathway of isoleucine (Ile) for conversion of threonine (Thr) to 2-ketobutyrate (2-KB). The FgILV1 deletion mutant ΔFgIlv1-3 was unable to grow on minimal medium or fructose gelatin agar which lacked Ile. Exogenous supplementation of Ile or 2-KB but not Thr rescued the mycelial growth defect of ΔFgIlv1-3, indicating the involvement of FgIlv1 in the conversion of Thr to 2-KB in Ile biosynthesis. Additionally, exogenous supplementation of Methionine (Met) could also rescue the mycelial growth defect of ΔFgIlv1-3, indicating a crosstalk between Ile biosynthesis and Met catabolism in F. graminearum. Deletion of FgILV1 also caused defects in conidial formation and germination. In addition, ΔFgIlv1-3 displayed decreased virulence on wheat heads and a low level of deoxynivalenol (DON) production in wheat kernels. Taken together, results of this study indicate that FgIlv1 is an essential component in Ile biosynthesis and is required for various cellular processes including mycelial and conidial morphogenesis, DON biosynthesis, and full virulence in F. graminearum. Our data indicate the potential of targeting Ile biosynthesis for anti-FHB management.

Decreased cystathionine-γ-lyase (CSE) activity in livers of type 1 diabetic rats and peripheral blood mononuclear cells (PBMC) of type 1 diabetic patients.

The liver plays a major role in the formation of H2S, a novel signaling molecule. Diabetes is associated with lower blood levels of H2S. This study investigated the activities of cystathionine-γ-lyase (CSE, the enzyme that catalyzes H2S formation) in livers of type 1 diabetic (T1D) animals and in peripheral blood mononuclear cells (PBMC) isolated from T1D patients. T1D is associated with both hyperketonemia (acetoacetate and ß-hydroxybutyrate) and hyperglycemia. This study also examined the role of hyperglycemia and hyperketonemia per se in decreased CSE activity using U937 monocytes and PBMC isolated from healthy subjects. Livers from streptozotocin-treated T1D rats demonstrated a significantly higher reactive oxygen species production, lower CSE protein expression and activity, and lower H2S formation compared with those of controls. Studies with T1D patients showed a decrease in CSE protein expression and activity in PBMC compared with those of age-matched normal subjects. Cell culture studies demonstrated that high glucose (25 mm) and/or acetoacetate (4 mm) increased reactive oxygen species, decreased CSE mRNA expression, protein expression, and enzymatic activity, and reduced H2S levels; however, ß-hydroxybutyrate treatment had no effect. A similar effect, which was also observed in PBMC treated with high glucose alone or along with acetoacetate, was prevented by vitamin D supplementation. Studies with CSE siRNA provide evidence for a relationship between impaired CSE expression and reduced H2S levels. This study demonstrates for the first time that both hyperglycemia and hyperketonemia mediate a reduction in CSE expression and activity, which can contribute to the impaired H2S signaling associated with diabetes.

Cystathionine γ-lyase deficiency mediates neurodegeneration in Huntington's disease.

Huntington's disease is an autosomal dominant disease associated with a mutation in the gene encoding huntingtin (Htt) leading to expanded polyglutamine repeats of mutant Htt (mHtt) that elicit oxidative stress, neurotoxicity, and motor and behavioural changes. Huntington's disease is characterized by highly selective and profound damage to the corpus striatum, which regulates motor function. Striatal selectivity of Huntington's disease may reflect the striatally selective small G protein Rhes binding to mHtt and enhancing its neurotoxicity. Specific molecular mechanisms by which mHtt elicits neurodegeneration have been hard to determine. Here we show a major depletion of cystathionine γ-lyase (CSE), the biosynthetic enzyme for cysteine, in Huntington's disease tissues, which may mediate Huntington's disease pathophysiology. The defect occurs at the transcriptional level and seems to reflect influences of mHtt on specificity protein 1, a transcriptional activator for CSE. Consistent with the notion of loss of CSE as a pathogenic mechanism, supplementation with cysteine reverses abnormalities in cultures of Huntington's disease tissues and in intact mouse models of Huntington's disease, suggesting therapeutic potential.

[Change in plasma H2S level and therapeutic effect of H2S supplementation in tubulointerstitial fibrosis among rats with unilateral ureteral obstruction].

OBJECTIVE: To observe the level in plasma hydrogen sulfide (H2S) and the expression of cystathionine beta-synthase (CBS) and cystathionine gamma-lyase (CSE) (two key synthetases for endogenous H2S generation in the kidney) in obstructed kidney tissue among rats with tubulointerstitial fibrosis (TIF) induced by unilateral ureteral obstruction (UUO), and to explore the role of H2S in TIF. METHODS: Ninety-six male Sprague-Dawley rats were randomly divided into sham-operated, model, low-dose NaHS and high-dose NaHS groups (n=24 each). TIF was induced by UUO in the model, low-dose NaHS and high-dose NaHS groups. The low-dose and high-dose NaHS groups were intraperitoneally injected with NaHS (1.4 and 7.0 µmol/kg respectively) twice daily immediately after operation, and the sham-operated and model groups were intraperitoneally injected with an identical volume of normal saline. In each group, 8 rats were randomly selected and sacrificed at 7, 14 or 21 days after operation. Plasma H2S concentration was measured by deproteinization. The obstructed kidney tissue was subjected to hematoxylin and eosin staining and Masson staining, and the renal tubulointerstitial injury was evaluated under a microscope. mRNA and protein expression of CBS and CSE in the obstructed kidney tissue was measured by RT-PCR and immunohistochemistry respectively. RESULTS: The degree of UUO-induced renal tubulointerstitial injury was negatively correlated with plasma H2S concentration in (r=-0.891, P<0.01). With H2S supplementation, renal tubulointerstitial injury was reduced (P<0.01), the expression of mRNA and protein of CBS and CSE in the kidney tissue and plasma H2S level were upregulated (P<0.01), and the degree of TIF was reduced (P<0.01). There were no significant differences in plasma H2S level and mRNA and protein expression of CBS and CSE between the low-dose and high-dose NaHS groups (P>0.05). CONCLUSIONS: H2S is involved in the development of UUO-induced TIF, and the CBS/H2S and CSE/H2S systems play key roles in this process. H2S supplementation can delay the progression of TIF.

H(2)S inhibits hyperglycemia-induced intrarenal renin-angiotensin system activation via attenuation of reactive oxygen species generation.

Decrease in endogenous hydrogen sulfide (H2S) was reported to participate in the pathogenesis of diabetic nephropathy (DN). This study is aimed at exploring the relationship between the abnormalities in H2S metabolism, hyperglycemia-induced oxidative stress and the activation of intrarenal renin-angiotensin system (RAS). Cultured renal mesangial cells (MCs) and streptozotocin (STZ) induced diabetic rats were used for the studies. The expressions of angiotensinogen (AGT), angiotensin converting enzyme (ACE), angiotensin II (Ang II) type I receptor (AT1), transforming growth factor-ß1 (TGF-ß1) and collagen IV were measured by real time PCR and Western blot. Reactive oxygen species (ROS) production was assessed by fluorescent probe assays. Cell proliferation was analyzed by 5'-bromo-2'-deoxyuridine incorporation assay. Ang II concentration was measured by an enzyme immunoassay. AGT, ACE and AT1 receptor mRNA levels and Ang II concentration were increased in high glucose (HG) -treated MCs, the cell proliferation rate and the production of TGF-ß1 and of collagen IV productions were also increased. The NADPH oxidase inhibitor diphenylenechloride iodonium (DPI) was able to reverse the HG-induced RAS activation and the changes in cell proliferation and collagen synthesis. Supplementation of H2S attenuated HG-induced elevations in ROS and RAS activation. Blockade on H2S biosynthesis from cystathione-γ-lyase (CSE) by DL-propargylglycine (PPG) resulted in effects similar to that of HG treatment. In STZ-induced diabetic rats, the changes in RAS were also reversed by H2S supplementation without affecting blood glucose concentration. These data suggested that the decrease in H2S under hyperglycemic condition leads to an imbalance between oxidative and reductive species. The increased oxidative species results in intrarenal RAS activation, which, in turn, contributes to the pathogenesis of renal dysfunction.

Dysregulation of cystathionine γ-lyase (CSE)/hydrogen sulfide pathway contributes to ox-LDL-induced inflammation in macrophage.

Hydrogen sulfide (H2S), mainly produced by cystathionine γ-lyase (CSE) in vascular system, emerges as a novel gasotransmitter exerting anti-inflammatory and anti-atherosclerotic effects. Alterations of CSE/H2S pathway may thus be involved in atherosclerosis pathogenesis. However, the underlying mechanisms are poorly understood. The present study showed that the levels of CSE mRNA and protein expression, as well as H2S production were decreased in ox-LDL-treated macrophage. CSE overexpression reduced the ox-LDL-stimulated tumor necrosis factor-α (TNF-α) generation in Raw264.7 and primary macrophage while CSE knockdown enhanced it. Exogenous supplementation of H2S with NaHS and Na2S also decreased the production of TNF-α and intercellular adhesion molecule-1 (ICAM-1) in ox-LDL-stimulated macrophage, and alleviated the adhesion of macrophage to endothelial monolayer. Cysteine, a CSE preferential substrate for H2S biosynthesis, produced similar effects on the pro-inflammatory cytokine generation, which were reversed by CSE inhibitors PAG and BCA, respectively. Moreover, NaHS and Na2S attenuated the phosphorylation and degradation of IκBα and p65 nuclear translocation, as well as JNK activation caused by ox-LDL. The JNK inhibitor suppressed the NF-κB transcription activity in ox-LDL-treated cells. Furthermore, inhibitors of NF-κB (PDTC), ERK (U0126 and PD98059) and JNK (SP600125) partially blocked the suppression by ox-LDL on the CSE mRNA levels. Taken together, the findings demonstrate that ox-LDL may down-regulate the CSE/H2S pathway, which plays an anti-inflammatory role in ox-LDL-stimulated macrophage by suppressing JNK/NF-κB signaling. The study reveals new therapeutic strategies for atherosclerosis, based on modulating CSE/H2S pathway.

17ß-estradiol induces vasorelaxation by stimulating endothelial hydrogen sulfide release.

Estrogen exerts vascular protective effects, but the underlying mechanisms remain to be understood fully. In recent years, hydrogen sulfide (H(2)S) has increasingly been recognized as an important signaling molecule in the cardiovascular system. Vascular H(2)S is produced from L-cysteine, catalyzed by cystathionine γ-lyase (CSE). In our study, apolipoprotein E (ApoE)-deficient mice were ovariectomized and implanted with placebo (OVX mice) or 17ß-estradiol (E(2)) pellets (OVX + E(2) mice). Compared with OVX mice, OVX + E(2) mice showed increased plasma H(2)S levels (P = 0.012) and decreased aortic lesion area (P = 0.028). These effects were largely reversed when supplementing with the irreversible CSE inhibitor DL-propargylglycine (PPG) in the OVX + E(2) + PPG mice. Meanwhile, the nitric oxide and prostacyclin-resistant responses to cumulative application of acetylcholine (ACh) were studied among all the three groups of femoral arteries. Compared with the arteries in the OVX group, the vasodilator sensitivity of arteries to ACh was increased in the OVX + E(2) group and attenuated in the OVX + E(2) + PPG group. E(2) and estrogen receptor (ER) α agonist 4',4″,4'″-(4-propyl-[1H]-pyrazole-1,3,5-triyl) trisphenol rapidly increased H(2)S release in human endothelial cells, but not partially selective ERß agonist 2,3-bis-(4-hydroxyphenyl)-propionitrile. These effects were inhibited by ER antagonist ICI 182780 or by protein kinase G (PKG) inhibitor KT5823. Furthermore, endothelial PKG activity was increased by E(2) (P = 0.003) and E(2)-induced vasodilation was inhibited by KT5823 (P = 0.009). In conclusion, the endothelial CSE/H(2)S pathway is activated by E(2) through PKG, which leads to vasodilation. These actions may be relevant to estrogen's anti-atherogenic effect.

Vitamin D up-regulates glucose transporter 4 (GLUT4) translocation and glucose utilization mediated by cystathionine-γ-lyase (CSE) activation and H2S formation in 3T3L1 adipocytes.

A scientific explanation for the beneficial role of vitamin D supplementation in the lowering of glycemia in diabetes remains to be determined. This study examined the biochemical mechanism by which vitamin D supplementation regulates glucose metabolism in diabetes. 3T3L1 adipocytes were treated with high glucose (HG, 25 mm) in the presence or absence of 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) (25, 50 nm), the active form of vitamin D. 1,25(OH)(2)D(3) treatment caused significant up-regulation of GLUT4 total protein expression and its translocation to cell surface, and an increase in glucose uptake as well as glucose utilization in HG-treated cells. 1,25(OH)(2)D(3) also caused cystathionine-γ-lyase (CSE) activation and H(2)S formation in HG-treated adipocytes. The effect of 1,25(OH)(2)D(3) on GLUT4 translocation, glucose utilization, and H(2)S formation was prevented by propargylglycine, an inhibitor of CSE that catalyzes H(2)S formation. Studies using antisense CSE also demonstrated the inhibition of GLUT4 translocation as well as glucose uptake and utilization in 1,25(OH)(2)D(3)-supplemented CSE-siRNA-transfected adipocytes compared with controls. 1,25(OH)(2)D(3) treatment along with insulin enhanced GLUT4 translocation and glucose utilization compared with either insulin or 1,25(OH)(2)D(3) alone in HG-treated adipocytes. 1,25(OH)(2)D(3) supplementation also inhibited monocyte chemoattractant protein-1 and stimulated adiponectin secretion in HG-treated adipocytes, and this positive effect was prevented in propargylglycine-treated or CSE-knockdown adipocytes. This is the first report to demonstrate that 1,25(OH)(2)D(3) up-regulates GLUT4 translocation and glucose utilization and decreases inflammatory markers, which is mediated by CSE activation and H(2)S formation in adipocytes. This study provides evidence for a novel molecular mechanism by which 1,25(OH)(2)D(3) can up-regulate the GLUT4 translocation essential for maintenance of glucose metabolism.