Cystathionine Γ-lyase Research Articles

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.

Hydrogen sulfide ameliorates lipopolysaccharide-induced anxiety-like behavior by inhibiting checkpoint kinase 1 activation in the hippocampus of mice

Hydrogen sulfide (H2S), an endogenous gasotransmitter, exhibits the anxiolytic roles through its anti-inflammatory effects, although its underlying mechanisms remain largely elusive. Emerging evidence has documented that cell cycle checkpoint kinase 1 (Chk1)-regulated DNA damage plays an important role in the neurodegenerative diseases; however, there are few relevant reports on the research of Chk1 in neuropsychiatric diseases. Here, we aimed to investigate the regulatory role of H2S on Chk1 in lipopolysaccharide (LPS)-induced anxiety-like behavior focusing on inflammasome activation in the hippocampus. Cystathionine γ-lyase (CSE, a H2S-producing enzyme) knockout (CSE−/−) mice displayed anxiety-like behavior and activation of inflammasome-mediated inflammatory responses, manifesting by the increase levels of interleukin-1β (IL-1β), IL-6, and ionized calcium-binding adaptor molecule-1 (Iba-1, microglia marker) expression in the hippocampus. Importantly, expression of p-Chk1 and γ-H2AX (DNA damage marker) levels were also increased in the hippocampus of CSE−/− mice. LPS treatment decreased the expression of CSE and CBS while increased p-Chk1 and γ-H2AX levels and inflammasome-activated neuroinflammation in the hippocampus of mice. Moreover, p-Chk1 and γ-H2AX protein levels and cellular immunoactivity were significantly increased while CSE and CBS were markedly decreased in cultured BV2 cells followed by LPS treatment. Treatment of mice with GYY4137, a donor of H2S, inhibited LPS-induced increased in p-Chk1 and γ-H2AX levels, mitigated inflammasome activation and inflammatory responses as well as amelioration of anxiety-like behavior. Notably, SB-218078, a selective Chk1 inhibitor treatment attenuated the effect of LPS on inflammasome activation and inflammatory responses and the induction of anxiety-like behavior. Finally, STAT3 knockdown with AAV-STAT3 shRNA alleviated LPS-induced anxiety-like behavior and inhibited inflammasome activation in the hippocampus, and blockade of NLRP3 with MCC950 attenuated neuroinflammation induction and ameliorated LPS-induced anxiety-like behavior. Overall, this study indicates that downregulation of Chk1 activity by H2S activation may be considered as a valid strategy for preventing the progression of LPS-induced anxiety-like behavior.

Vasodilator Responses of Perivascular Adipose Tissue-Derived Hydrogen Sulfide Stimulated with L-Cysteine in Pregnancy Hypertension-Induced Endothelial Dysfunction in Rats.

Endothelium-derived nitric oxide (NO)-induced vasodilation is impaired in pregnancy hypertension. However, the role of perivascular adipose tissue (PVAT)-derived hydrogen sulfide (H2S), as an alternative for counteracting vascular dysfunction, is incompletely clear in hypertensive disorders of pregnancy. Therefore, PVAT-derived H2S-induced vasodilation was investigated in pregnancy hypertension-induced endothelial dysfunction. Non-pregnant (Non-Preg) and pregnant (Preg) rats were submitted (or not) to the deoxycorticosterone (DOCA)-salt protocol and assigned as follows (n = 10/group): Non-Preg, Non-Preg+DOCA, Preg, and Preg+DOCA groups. Systolic blood pressure (SBP), angiogenesis-related factors, determinant levels of H2S (PbS), NO (NOx), and oxidative stress (MDA) were assessed. Vascular changes were recorded in thoracic aortas with PVAT and endothelium (intact and removed layers). Vasorelaxation responses to the substrate (L-cysteine) for the H2S-producing enzyme cystathionine-γ-lyase (CSE) were examined in the absence and presence of CSE-inhibitor DL-propargylglycine (PAG) in thoracic aorta rings pre-incubated with cofactor for CSE (pyridoxal-5 phosphate: PLP) and pre-contracted with phenylephrine. Hypertension was only found in the Preg+DOCA group. Preg+DOCA rats showed angiogenic imbalances and increased levels of MDA. PbS, but not NOx, showed increased levels in the Preg+DOCA group. Pre-incubation with PLP and L-cysteine elevated determinants of H2S in PVAT and placentas of Preg-DOCA rats, whereas no changes were found in the aortas without PVAT. Aortas of Preg-DOCA rats showed that PVAT-derived H2S-dependent vasodilation was greater compared to endothelium-derived H2S, whereas PAG blocked these responses. PVAT-derived H2S endogenously stimulated with the amino acid L-cysteine may be an alternative to induce vasorelaxation in endothelial dysfunction related to pregnancy hypertension.

Hydrogen sulfide inhibits gene expression associated with aortic valve degeneration by inducing NRF2-related pro-autophagy effect in human aortic valve interstitial cells.

Aortic valve stenosis (AS) is the most common valvular heart disease but there are currently no effective medical treatments that can delay disease progression due to a lack of knowledge of the precise pathophysiology. The expression of sulfide: quinone oxidoreductase (SQOR) and nuclear factor erythroid 2-related factor 2 (NRF2) was decreased in the aortic valve of AS patients. However, the role of SQOR and NRF2 in the pathophysiology of AS has not been found. We investigated the effects of hydrogen sulfide (H2S)-releasing compounds on diseased aortic valve interstitial cells (AVICs) to explain the cellular mechanism of SQOR and elucidate the medical value of H2S for AS treatment. Sodium hydrosulfide (NaHS) treatment increased the expression of SQOR and NRF2 gene and consequently induced the NRF2 target genes, such as NAD(P)H quinone dehydrogenase 1 and cystathionine γ-lyase. In addition, NaHS dose-dependently decreased the expression level of fibrosis and inflammation-related genes (MMP9, TNF-α, IL6) and calcification-related genes (ALP, osteocalcin, RUNX2, COL1A1) in human AVICs. Furthermore, NaHS activated the AMPK-mTOR pathway and inhibited the PI3K-AKT pathway, resulting in a pro-autophagy effect in human AVICs. An NRF2 inhibitor, brusatol, attenuated NaHS-induced AMPK activation and decreased the autophagy markers Beclin-1 and LC3AB, suggesting that the mechanism of action of H2S is related to NRF2. In conclusion, H2S decreased gene expression levels related to aortic valve degeneration and activated AMPK-mTOR-mediated pro-autophagy function associated with NRF2 in human AVICs. Therefore, H2S could be a potential therapeutic target for the development of AS treatment.

Disrupted Binding of Cystathionine γ-Lyase to p53 Promotes Endothelial Senescence.

Advanced age is unequivocally linked to the development of cardiovascular disease; however, the mechanisms resulting in reduced endothelial cell regeneration remain poorly understood. Here, we investigated novel mechanisms involved in endothelial cell senescence that impact endothelial cell transcription and vascular repair after injury. Native endothelial cells were isolated from young (20±3.4 years) and aged (80±2.3 years) individuals and subjected to molecular analyses to assess global transcriptional and metabolic changes. In vitro studies were conducted using primary human and murine endothelial cells. A murine aortic re-endothelialization model was used to examine endothelial cell regenerative capacity in vivo. RNA sequencing of native endothelial cells revealed that aging resulted in p53-mediated reprogramming to express senescence-associated genes and suppress glycolysis. Reduced glucose uptake and ATP contributed to attenuated assembly of the telomerase complex, which was required for endothelial cell proliferation. Enhanced p53 activity in aging was linked to its acetylation on K120 due to enhanced activity of the acetyltransferase MOZ (monocytic leukemic zinc finger). Mechanistically, p53 acetylation and translocation were, at least partially, attributed to the loss of the vasoprotective enzyme, CSE (cystathionine γ-lyase). CSE physically anchored p53 in the cytosol to prevent its nuclear translocation and CSE absence inhibited AKT (Protein kinase B)-mediated MOZ phosphorylation, which in turn increased MOZ activity and subsequently p53 acetylation. In mice, the endothelial cell-specific deletion of CSE activated p53, induced premature endothelial senescence, and arrested vascular repair after injury. In contrast, the adeno-associated virus 9-mediated re-expression of an active CSE mutant retained p53 in the cytosol, maintained endothelial glucose metabolism and proliferation, and prevented endothelial cell senescence. Adenoviral overexpression of CSE in native endothelial cells from aged individuals maintained low p53 activity and reactivated telomerase to revert endothelial cell senescence. Aging-associated impairment of vascular repair is partly determined by the vasoprotective enzyme CSE.

THU263 Intestinal Production of H2S And Its Regulation Of GLP-2

Abstract Disclosure: J. Hammond: None. G. Yang: None. J. Gagnon: None. Background: Hydrogen sulfide (H2S) is an endogenously produced gasotransmitter that regulates a variety of physiological processes including hormone regulation. In individuals with Crohn’s disease or colitis, H2S is elevated. Whether this H2S plays a role in gut disease development or protection is not clear. Glucagon-Like Peptide-2 (GLP-2) is a gut health-promoting peptide hormone produced in the enteroendocrine L-cells located primarily in the distal GI tract. In the gut, GLP-2 increases cell proliferation, enhances barrier function, and decreases inflammation. Furthermore, GLP-2 administration has been shown to be protective in both Crohn’s and Colitis. Finally, our previous work has shown a role for H2S in the regulation of glucagon-like peptides. Hypothesis: We hypothesize that local H2S production in the distal GI tract and in enteroendocrine cells can regulate GLP-2 secretion and downstream gut physiology.Methods: GLP-2 secretion and gut physiology were examined in mice receiving exogenous H2S injections or lacking the H2S-producing enzyme cystathionine gamma-lyase (CSE). Additionally, H2S production and GLP-2 secretion were examined from mouse enteroendocrine L-cells (GLUTag). Results: CSE KO mice had significantly reduced intestinal H2S production. A slight increase in circulating GLP-2 was found in CSE KO mice. H2S was also produced directly from enteroendocrine cells which was partially blocked using CSE enzyme inhibitors. When enteroendocrine L-cells were treated with H2S donors (NaHS and GYY 4137), GLP-2 secretion was significantly reduced (with no effect on cell viability). Ongoing work will explore the direct effects of H2S administration in mice on circulating GLP-2, and any changes that occur in the gut epithelium as a result. Discussion/Conclusions: Our work indicates that the enteroendocrine cell can produce the gasotransmitter H2S, and that H2S donors can suppress GLP-2 secretion. These findings suggest that GLP-2 may play a role in the interplay between H2S and gastric health. These results could provide support for the potential use of H2S drugs in the treatment of GI diseases. Presentation: Thursday, June 15, 2023

Dual targeting of H2S synthesizing enzymes; cystathionine β-synthase and cystathionine γ-lyase by miR-939-5p effectively curbs triple negative breast cancer

IntroductionHydrogen sulfide (H2S) has been recently scrutinized for its critical role in aggravating breast cancer (BC) tumorigenicity. Several cancers aberrantly express H2S synthesizing enzymes; Cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE). However, their levels and interdependence in BC require further studies. ObjectivesFirstly, this study aimed to demonstrate a comparative expression profile of H2S synthesizing enzymes in BC vs normal tissue. Moreover, to investigate the reciprocal relationship between CBS and CSE and highlight the importance of dual targeting. Finally, to search for a valid dual repressor of the H2S synthesizing enzymes that could cease H2S production and reduce TNBC pathogenicity. MethodsPairwise analysis of tumor vs. normal tissues of 40 BC patients was carried out. The TNBC cell line MDA-MB-231 was transfected with oligonucleotides to study the H2S mediated molecular mechanisms. In silico screening was performed to identify dual regulator(s) for CBS and CSE. Gene expression analysis was performed using qRT-PCR and was confirmed on protein level using Western blot. TNBC hallmarks were evaluated using MTT, migration, and clonogenicity assays. H2S levels were detected using a AzMc fluorescent probe. ResultsBC tissues exhibited elevated levels of both CBS and CSE. Interestingly, upon CBS knockdown, CSE levels increased compensating for H2S production in TNBC cells, underlining the importance of dually targeting both enzymes in TNBC. In silico screening suggested miR-939-5p as a regulator of both CBS and CSE with high binding scores. Low expression levels of miR-939-5p were found in BC tissues, especially the aggressive subtypes. Ectopic expression of miR-939-5p significantly repressed CBS and CSE transcript and protein levels, diminished H2S production and attenuated TNBC hallmarks. Moreover, it improved the immune surveillance potency of TNBC cells through up regulating the NKG2D ligands, MICB and ULBP2 and reducing the immune suppressive cytokine IL-10. ConclusionThis study sheds light on the reciprocal relationship between CBS and CSE and on the importance of their dual targeting, particularly in TNBC. It also postulates miR-939-5p as a potent dual repressor for CBS and CSE overcoming their redundancy in H2S production, a mechanism that can potentially attenuate TNBC oncogenicity and improves the immunogenic response.

Discovery of a cystathionine γ-lyase (CSE) selective inhibitor targeting active-site pyridoxal 5′-phosphate (PLP) via Schiff base formation

D,L-Propargylglycine (PAG) has been widely used as a selective inhibitor to investigate the biological functions of cystathionine γ-lyase (CSE), which catalyzes the formation of reactive sulfur species (RSS). However, PAG also inhibits other PLP (pyridoxal-5′-phosphate)-dependent enzymes such as methionine γ-lyase (MGL) and L-alanine transaminase (ALT), so highly selective CSE inhibitors are still required. Here, we performed high-throughput screening (HTS) of a large chemical library and identified oxamic hydrazide 1 as a potent inhibitor of CSE (IC50 = 13 ± 1 μM (mean ± S.E.)) with high selectivity over other PLP-dependent enzymes and RSS-generating enzymes. Inhibitor 1 inhibited the enzymatic activity of human CSE in living cells, indicating that it is sufficiently membrane-permeable. X-Ray crystal structure analysis of the complex of rat CSE (rCSE) with 1 revealed that 1 forms a Schiff base linkage with the cofactor PLP in the active site of rCSE. PLP in the active site may be a promising target for development of selective inhibitors of PLP-dependent enzymes, including RSS-generating enzymes such as cystathionine β-synthase (CBS) and cysteinyl-tRNA synthetase 2 (CARS2), which have unique substrate binding pocket structures.

ICI 182,780 Attenuates Selective Upregulation of Uterine Artery Cystathionine β-Synthase Expression in Rat Pregnancy.

Endogenous hydrogen sulfide (H2S) produced by cystathionine β-synthase (CBS) and cystathionine-γ lyase (CSE) has emerged as a novel uterine vasodilator contributing to pregnancy-associated increases in uterine blood flow, which safeguard pregnancy health. Uterine artery (UA) H2S production is stimulated via exogenous estrogen replacement and is associated with elevated endogenous estrogens during pregnancy through the selective upregulation of CBS without altering CSE. However, how endogenous estrogens regulate uterine artery CBS expression in pregnancy is unknown. This study was conducted to test a hypothesis that endogenous estrogens selectively stimulate UA CBS expression via specific estrogen receptors (ER). Treatment with E2β (0.01 to 100 nM) stimulated CBS but not CSE mRNA in organ cultures of fresh UA rings from both NP and P (gestational day 20, GD20) rats, with greater responses to all doses of E2β tested in P vs. NP UA. ER antagonist ICI 182,780 (ICI, 1 µM) completely attenuated E2β-stimulated CBS mRNA in both NP and P rat UA. Subcutaneous injection with ICI 182,780 (0.3 mg/rat) of GD19 P rats for 24 h significantly inhibited UA CBS but not mRNA expression, consistent with reduced endothelial and smooth muscle cell CBS (but not CSE) protein. ICI did not alter mesenteric and renal artery CBS and CSE mRNA. In addition, ICI decreased endothelial nitric oxide synthase mRNA in UA but not in mesenteric or renal arteries. Thus, pregnancy-augmented UA CBS/H2S production is mediated by the actions of endogenous estrogens via specific ER in pregnant rats.

Hydrogen sulfide promotes migration of trophoblast cells by a Rho GTPase mediated actin cytoskeleton reorganization

IntroductionPreeclampsia (PE) arises due to defective spiral artery remodelling which may be due to deficient migration of trophoblast cells. Migration of human endothelial cells has been shown to be promoted via Hydrogen sulphide(H2S)/Rho GTPase Rac1 axis. This novel role of H2S and its downstream processes have not yet been studied in the development and function of the placental trophoblast cells. MethodsPlacental tissues were obtained post-delivery from consented preeclamptic and normotensive mothers (n = 60). The protein expression levels of cystathionine-gamma-lyase (CSE) and cystathionine-beta-synthase (CBS) along with its downstream migratory molecules were compared in both the arms. The pro-migratory role of H2S was investigated in a first trimester placental cell line. ResultsH2S promoted the migration of trophoblast cells in a Rho GTPase dependent manner mediated by actin cytoskeleton reorganization. The reduced levels of H2S producing enzymes in the PE placentae along with decreased levels of Rho GTPases (Rac1 and Rho A) corroborate the results of PAG and AOAA treatment in down regulating the Rho GTPases in the in vitro grown placental cultures. Reduction of the migratory potential of trophoblastic cells caused due to hypoxia/reoxygenation was rescued by upregulating the H2S expression with the use of NaHS as a H2S donor. DiscussionExogenous H2S increases the migratory potential of the placental cells in culture conditions and also post hypoxia/reoxygenation injury. H2S as a gaso-transmitter holds a great potential as a therapeutic agent. Its long-term effects need to be investigated using model systems (rat/mouse) of PE following it up with clinical regulatory trials.

Cystathionine Gamma Lyase Is Regulated by Flow and Controls Smooth Muscle Migration in Human Saphenous Vein.

The saphenous vein is the conduit of choice for bypass grafting. Unfortunately, the hemodynamic stress associated with the arterial environment of the bypass vein graft leads to the development of intimal hyperplasia (IH), an excessive cellular growth and collagen deposition that results in restenosis and secondary graft occlusion. Hydrogen sulfide (H2S) is a ubiquitous redox-modifying gasotransmitter that inhibits IH. H2S is produced via the reverse trans-sulfuration pathway by three enzymes: cystathionine γ-lyase (CSE), cystathionine β-synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (3-MST). However, the expression and regulation of these enzymes in the human vasculature remains unclear. Here, we investigated the expression of CSE, CBS and 3-MST in segments of native human saphenous vein and large arteries. Furthermore, we evaluated the regulation of these enzymes in vein segments cultured under static, venous (7 mmHg pressure) or arterial (100 mmHg pressure) pressure. CSE was expressed in the media, neointima and intima of the vessels and was negatively regulated by arterial shear stress. Adenoviral-mediated CSE overexpression or RNA interference-mediated CSE knock-down revealed that CSE inhibited primary human VSMC migration but not proliferation. We propose that high shear stress in arteriovenous bypass grafts inhibits CSE expression in both the media and endothelium, which may contribute to increased VSMC migration in the context of IH.

Characterisation of the hydrogen sulfide system in early diabetic kidney disease.

A deficiency in hydrogen sulfide has been implicated in the development and progression of diabetic chronic kidney disease. The purpose of this study was to determine the effect of diabetes on the H2S system in early-stage diabetic kidney disease. We characterised gene and protein expression profile of the enzymes that regulate H2S production and degradation, and H2S production capacity, in the kidney from 10-week-old C57BL6J db/db mice (n=6), in age-matched heterozygous controls (n=7), and in primary endothelial cells (HUVECs) exposed to high glucose. In db/db mice renal H2S levels were significantly reduced (p=0.009). Protein expression of the H2S production enzymes was differentially affected by diabetes: Cystathionine β-synthase (CBS) was significantly lower in both db/db mice and high glucose treated HUVECS (p<0.0001; p= 0.0318) whereas 3-Mercatopyruvate sulfurtransferase (3-MST) expression was higher in the db/db kidney (p<0.0001), yet lower in the HUVECs (p=0.0001). Diabetes had no effect on the expression of Cystathionine γ-lyase (CSE) in the db/db kidney (p=ns), but was associated with reduced expression in the HUVECs (p=0.0004). Protein expression of degradation enzyme Sulfide quinone reductase (SQOR) was significantly higher in db/db kidney (p=0.048) and lower in the high glucose treated HUVECs (p= 0.008). Immunofluorescence studies revealed differential localization of the H2S enzymes in the kidney, including both tubular and vascular localisation, suggestive of functionally distinct actions in the kidney. The results of this study provide foundational knowledge for future research looking at the H2S system in both kidney physiology and the aetiology of chronic diabetic kidney disease.

Pharmacological Inhibition and Genetic Deletion of Cystathionine Gamma-Lyase in Mice Protects against Organ Injury in Sepsis: A Key Role of Adhesion Molecules on Endothelial Cells.

Hydrogen sulfide (H2S), synthesized by cystathionine gamma-lyase (Cth), contributes to the inflammatory response observed in sepsis. This study examines the effect of Cth-derived H2S in adhesion molecules on endothelial cells of vital organs in mice in a cecal ligation puncture (CLP)-induced model of sepsis, using two different and complementary approaches: Cth gene deletion and pharmacological inhibition. Our findings revealed a decreased level of H2S-synthesizing activity (via Cth) in both Cth-/- mice and PAG-treated wild-type (WT) mice following CLP-induced sepsis. Both treatment groups had reduced MPO activity and expression of chemokines (MCP-1 and MIP-2α), adhesion molecules (ICAM-1 and VCAM-1), ERK1/2 phosphorylation, and NF-κB in the liver and lung compared with in CLP-WT mice. Additionally, we found that PAG treatment in Cth-/- mice had no additional effect on the expression of ERK1/2 phosphorylation, NF-κB, or the production of chemokines and adhesion molecules in the liver and lung compared to Cth-/- mice following CLP-induced sepsis. The WT group with sepsis had an increased immunoreactivity of adhesion molecules on endothelial cells in the liver and lung than the WT sham-operated control. The Cth-/-, PAG-treated WT, and Cth-/- groups of mice showed decreased immunoreactivity of adhesion molecules on endothelial cells in the liver and lung following sepsis. Inhibition of H2S production via both approaches reduced adhesion molecule expression on endothelial cells and reduced liver and lung injury in mice with sepsis. In conclusion, this study demonstrates that H2S has an important role in the pathogenesis of sepsis and validates PAG use as a suited tool for investigating the Cth/H2S-signalling axis in sepsis.

Inhibition of cystathionine-gamma lyase dampens vasoconstriction in mouse and human intracerebral arterioles.

In extracerebral vascular beds cystathionine-gamma lyase (CSE) activity plays a vasodilatory role but the role of this hydrogen sulfide (H2 S) producing enzyme in the intracerebral arterioles remain poorly understood. We hypothesized a similar function in the intracerebral arterioles. Intracerebral arterioles were isolated from wild type C57BL/6J mouse (9-12 months old) brains and from human brain biopsies. The function (contractility and secondary dilatation) of the intracerebral arterioles was tested ex vivo by pressure myography using a perfusion set-up. Reverse transcription polymerase chain reaction was used for detecting CSE expression. CSE is expressed in human and mouse intracerebral arterioles. CSE inhibition with L-propargylglycine (PAG) significantly dampened the K+ -induced vasoconstriction in intracerebral arterioles of both species (% of maximum contraction: in human control: 45.4 ± 2.7 versus PAG: 27 ± 5.2 and in mouse control: 50 ± 1.5 versus PAG: 33 ± 5.2) but did not affect the secondary dilatation. This effect of PAG was significantly reversed by the H2 S donor sodium hydrosulfide (NaSH) in human (PAG + NaSH: 38.8 ± 7.2) and mouse (PAG + NaSH: 41.7 ± 3.1) arterioles, respectively. The endothelial NO synthase (eNOS) inhibitor, Nω-Nitro-l-arginine methyl ester (L-NAME), and the inhibitor of soluble guanylate cyclase (sGC), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) reversed the effect of PAG on the K+ -induced vasoconstriction in the mouse arterioles and attenuated the K+ -induced secondary dilatation significantly. CSE contributes to the K+ -induced vasoconstriction via a mechanism involving H2 S, eNOS, and sGC whereas the secondary dilatation is regulated by eNOS and sGC but not by CSE.

Deficiency of hydrogen sulfide production and pregnancy rate in an experimental model: Association with preterm delivery.

Pro-inflammatory phenomena drive preterm delivery (PTD). Hydrogen sulfide is a gasotransmitter with anti-inflammatory properties produced through the activity of the enzyme cystathionine-γ-lyase (CSE), and its impact was studied in models of normal delivery and PTD in mice. Female CSE+/+ and CSE-/- mice were mated with male CSE+/+ mice; mating was done with drinking water unsupplemented and supplemented with cysteine. The pregnancy rate was monitored. PTD was induced by the intraperitoneal injection of bacterial lipopolysaccharide (LPS) on day 14.5 of pregnancy. Mice were sacrificed for tissue collection and splenocyte isolation after 6 and 12h. Isolated splenocytes were stimulated for the production of tumor necrosis factor-alpha (TNFα), interleukin (IL)-10 and interferon-gamma (IFNγ); TNFα and vascular endothelial growth factor (VEGF) were measured in the fetuses and the placenta. The successful pregnancy rate was lower in CSE-/- mice and it was restored with cysteine supplementation. CSE deficiency was associated with higher tissue concentrations of TNFα in the fetuses, attenuated IL-10 responses and higher IFNγ production from splenocytes. CSE deficiency was not associated with PTD. Following PTD induction, CSE-/- mice did not show attenuated IL-10 responses but the production of TNFα and IFNγ was lowered over-time; placental VEGF was also increased over-time. CSE deficiency has an unfavorable impact on pregnancy. H2 S deficiency through CSE does not drive PTD but mediates pro-inflammatory phenomena in fetuses.

Allicin-Loaded Intelligent Hydrogel Coating Improving Vascular Implant Performance.

Coronary atherosclerosis is closely related to inflammation and oxidative stress. Owing to poor biocompatibility, lack of personalized treatment, and late toxic side effects, traditional drug-eluting stent intervention, releasing antiproliferative drugs, can delay endothelial repair and cause late thrombosis. The inflammation caused by atherosclerosis results in an acidic microenvironment and oxidative stress, which can be considered as triggers for precise and intelligent treatment. Here, we used catechol hyaluronic acid (C-HA) and cystamine (Cys) to prepare C-HA-Cys hydrogel coatings by amide reaction. The H2S-releasing donor allicin was loaded in the hydrogel to form an intelligent biomimetic coating. The disulfide bond of Cys made the cross-linked network redox-responsive to the inflammation and oxidative stress in the microenvironment by releasing the drug and H2S intelligently to combat the side effects of stent implantation. This study evaluated the hemocompatibility, anti-inflammatory capacity, vascular wall cytocompatibility, and in vivo histocompatibility of this intelligent hydrogel coating. Furthermore, the effect of H2S released from the coating on atherosclerosis-related signaling pathways such as CD31 and cystathionine γ-lyase (CSE), CD36, and ACAT-1 was investigated. Our results indicate that the C-HA-Cys-Allicin hydrogel coating could be manufactured on the surface of vascular interventional devices to achieve a precise response to the microenvironment of the lesion to release drug, which can attain the purpose of prevention of in-stent restenosis and ensure the effectiveness and safety of the application of interventional devices.

H2S regulation of iron homeostasis by IRP1 improves vascular smooth muscle cell functions

Either H2S or iron is essential for cellular processes. Abnormal metabolism of H2S and iron has increased risk for cardiovascular diseases. The aim of the present study is to examine the mutual interplay of iron and H2S signals in regulation of vascular smooth muscle cell (SMC) functions. Here we found that deficiency of cystathionine gamma-lyase (CSE, a major H2S-producing enzyme in vascular system) induced but NaHS (a H2S donor) administration attenuated iron accumulation in aortic tissues from angiotensin II-infused mice. In vitro, iron overload induced labile iron levels, promoted cell proliferation, disrupted F-actin filaments, and inhibited protein expressions of SMC-specific markers (αSMA and calponin) more significantly in SMCs from CSE knockout mice (KO-SMCs) than the cells from wild-type mice (WT-SMCs), which could be reversed by exogenously applied NaHS. In contrast, KO-SMCs were more vulnerable to iron starvation-induced cell death. Either iron overload or NaHS did not affect elastin level and gelatinolytic activity. We further found that H2S induced more aconitase activity of iron regulatory protein 1 (IRP1) but inhibited its RNA binding activity accompanied with increased protein levels of ferritin and ferriportin, which would contribute to the lower level of labile iron level inside the cells. In addition, iron was able to suppress CSE-derived H2S generation, while iron also non-enzymatically induced H2S release from cysteine. This study reveals the mutual interaction between iron and H2S signals in regulating SMC phenotypes and functions; CSE/H2S system would be a target for preventing iron metabolic disorder-related vascular diseases.

T-type calcium channel modulation by hydrogen sulfide in neuropathic pain conditions.

Neuropathic pain can appear as a direct or indirect nerve damage lesion or disease that affects the somatosensory nervous system. If the neurons are damaged or indirectly stimulated, immune cells contribute significantly to inflammatory and neuropathic pain. After nerve injury, peripheral macrophages/spinal microglia accumulate around damaged neurons, producing endogenous hydrogen sulfide (H2S) through the cystathionine-γ-lyase (CSE) enzyme. H2S has a pronociceptive modulation on the Cav3.2 subtype, the predominant Cav3 isoform involved in pain processes. The present review provides relevant information about H2S modulation on the Cav3.2 T-type channels in neuropathic pain conditions. We have discussed that the dual effect of H2S on T-type channels is concentration-dependent, that is, an inhibitory effect is seen at low concentrations of 10µM and an augmentation effect on T-current at 100µM. The modulation mechanism of the Cav3.2 channel by H2S involves the direct participation of the redox/Zn2+ affinity site located in the His191 in the extracellular loop of domain I of the channel, involving a group of extracellular cysteines, comprising C114, C123, C128, and C1333, that can modify the local redox environment. The indirect interaction pathways involve the regulation of the Cav3.2 channel through cytokines, kinases, and post-translational regulators of channel expression. The findings conclude that the CSE/H2S/Cav3.2 pathway could be a promising therapeutic target for neuropathic pain disorders.

Ethanol inhibition of undifferentiated rat neural progenitor cell replication can be prevented by chlorogenic acid via the NFATc4/CSE signaling pathway.

Prenatal ethanol exposure hinders oxidative stress-mediated neuroblast/neural progenitor cell proliferation by inhibiting G1-S transition, a process vital to neocortical development. We previously showed that ethanol elicits this redox imbalance by repressing cystathionine γ-lyase (CSE), the rate-limiting enzyme in the transsulfuration pathway in fetal brain and cultured cerebral cortical neurons. However, the mechanism by which ethanol impacts the CSE pathway in proliferating neuroblasts is not known. We conducted experiments to define the effects of ethanol on CSE regulation and the molecular signaling events that control this vital pathway. This enabled us to develop an intervention to prevent the ethanol-associated cytostasis. Spontaneously immortalized undifferentiated E18 rat neuroblasts from brain cerebral cortex were exposed to ethanol to mimic an acute consumption pattern in humans. We performed loss- and gain-of-function studies to evaluate whether NFATc4 is a transcriptional regulator of CSE. The neuroprotective effects of chlorogenic acid (CGA) against the effects of ethanol were assessed using ROS and GSH/GSSG assays as measures of oxidative stress, transcriptional activation of NFATc4, and expression of NFATc4 and CSE by qRT-PCR and immunoblotting. Ethanol treatment of E18-neuroblast cells elicited oxidative stress and significantly reduced CSE expression with a concomitant decrease in NFATc4 transcriptional activation and expression. In parallel, inhibition of the calcineurin/NFAT pathway by FK506 exaggerated ethanol-induced CSE loss. In contrast, NFATc4 overexpression prevented loss of ethanol-induced CSE. CGA increased and activated NFATc4, amplified CSE expression, rescued ethanol-induced oxidative stress, and averted the cytostasis of neuroblasts by rescuing cyclin D1 expression. These findings demonstrate that ethanol can perturb CSE-dependent redox homeostasis by impairing the NFATc4 signaling pathway in neuroblasts. Notably, ethanol-associated impairments were rescued by genetic or pharmacological activation of NFATc4. Furthermore, we found a potential role for CGA in mitigating the ethanol-related neuroblast toxicity with a compelling connection to the NFATc4/CSE pathway.

Transsulfuration pathway: a targeting neuromodulator in Parkinson's disease.

The transsulfuration pathway (TSP) is a metabolic pathway involving sulfur transfer from homocysteine to cysteine. Transsulfuration pathway leads to many sulfur metabolites, principally glutathione, H2S, taurine, and cysteine. Key enzymes of the TSP, such as cystathionine β-synthase and cystathionine γ-lyase, are essential regulators at multiple levels in this pathway. TSP metabolites are implicated in many physiological processes in the central nervous system and other tissues. TSP is important in controlling sulfur balance and optimal cellular functions such as glutathione synthesis. Alterations in the TSP and related pathways (transmethylation and remethylation) are altered in several neurodegenerative diseases, including Parkinson's disease, suggesting their participation in the pathophysiology and progression of these diseases. In Parkinson's disease many cellular processes are comprised mainly those that regulate redox homeostasis, inflammation, reticulum endoplasmic stress, mitochondrial function, oxidative stress, and sulfur content metabolites of TSP are involved in these damage processes. Current research on the transsulfuration pathway in Parkinson's disease has primarily focused on the synthesis and function of certain metabolites, particularly glutathione. However, our understanding of the regulation of other metabolites of the transsulfuration pathway, as well as their relationships with other metabolites, and their synthesis regulation in Parkinson´s disease remain limited. Thus, this paper highlights the importance of studying the molecular dynamics in different metabolites and enzymes that affect the transsulfuration in Parkinson's disease.