Skeletal muscle cystathionine γ-lyase deficiency promotes obesity and insulin resistance and results in hyperglycemia and skeletal muscle injury upon HFD in mice.

OBJECTIVES: The objective of this study was to investigate whether skeletal muscle cystathionine γ-lyase (CTH) contributes to high-fat diet (HFD)-induced metabolic disorders using skeletal muscle Cth knockout (CthΔskm) mice. METHODS: The CthΔskm mice and littermate Cth-floxed (Cthf/f) mice were fed with either HFD or chow diet for 13 weeks. Metabolomics and transcriptome analysis were used to assess the impact of CTH deficiency in skeletal muscle. RESULTS: Metabolomics coupled with transcriptome showed that CthΔskm mice displayed impaired energy metabolism and some signaling pathways linked to insulin resistance (IR) in skeletal muscle although the mice had normal insulin sensitivity. HFD led to reduced CTH expression and impaired energy metabolism in skeletal muscle in Cthf/f mice. CTH deficiency and HFD had some common pathways enriched in the aspects of amino acid metabolism, carbon metabolism, and fatty acid metabolism. CthΔskm+HFD mice exhibited increased body weight gain, fasting blood glucose, plasma insulin, and IR, and reduced glucose transporter 4 and CD36 expression in skeletal muscle compared to Cthf/f+HFD mice. Impaired mitochondria and irregular arrangement in myofilament occurred in CthΔskm+HFD mice. Omics analysis showed differential pathways enriched between CthΔskm mice and Cthf/f mice upon HFD. More severity in impaired energy metabolism, reduced AMPK signaling, and increased oxidative stress and ferroptosis occurred in CthΔskm+HFD mice compared to Cthf/f+HFD mice. DISCUSSION: Our results indicate that skeletal muscle CTH expression dysregulation contributes to metabolism disorders upon HFD.

Altered circadian dynamics of Per2 after cystathionine-ß-synthase and/or cystathionine-γ-lyase pharmacological inhibition in serum-shocked NIH-3T3 cells.

Circadian clock genes are found in almost every cell that has a nucleus; they regulate the rhythmic nature of all processes that are cyclical. Among the genes controlled by the circadian clock, there are numerous factors that regulate key processes in the functioning of the cell. Disturbances in the functioning of the circadian clock are associated with numerous disorders. A recent study has shown the key role of H2S in regulating circadian rhythm. In this study, we investigated the in vitro effect of pharmacological inhibition of cystathionine-ß-synthase (CBS) and/or cystathionine-γ-lyase (CSE) on the circadian dynamics of Per2 expression in serum-shocked NIH-3T3 cells. Alternatively, Cbs and Cse were knocked down by transfection with siRNA. The 48-h treatment of serum-shocked NIH-3T3 cells with 1 mM dl-propargylglycine (PAG), a specific CSE inhibitor, significantly decreased the amplitude and baseline expression of Per2. During exposure to an effective CBS and CSE inhibitor (aminooxyacetic acid [AOAA]), the amplitude of oscillation and baseline expression of Per2 significantly increased. Incubation of NIH-3T3 cells with both inhibitors also significantly increased the amplitude and baseline expression of Per2 messenger RNA (mRNA). siCbs or siCse knockdowan significantly reduced the baseline and amplitude of oscillation of Per2. In conclusion, we showed that CBS/CSE/H2S pathway participates in the regulation of the circadian clock system. PAG and AOAA, change the general expression and dynamics of Per2 genes, but the increase of amplitude and overall Per2 mRNA level due to exposure to AOAA is probably caused by factors other than CBS and CSE activity.

Homocysteine Hypothesis on the Impaired Peripheral but Not Central Nervous System Oxytocin Responses in Cystathionine γ-Lyase-Deficient Dam Mice.

An elevated plasma homocysteine level is an independent risk factor for cardiovascular diseases, neurological disorders, and pregnancy complications. We recently demonstrated partial lactation failure in cystathionine γ-lyase-deficient (Cth-/-) dam mice and their defective oxytocin responses in peripheral tissues: uterine (ex vivo) and mammary gland (in vivo). We reasoned that elevated levels of circulatory homocysteine in Cth-/- dam mice counteract with oxytocin-dependent milk ejection from the mammary gland. Based on our observation that those mice displayed normal maternal behaviors against their pups and adult Cth-/- male mice exhibited normal social behaviors against adult wild-type female mice, both of which are regulated by oxytocin in the central nervous system (CNS), we conducted the present study to investigate the amino acid profiles, including total homocysteine, in both blood and cerebrospinal fluid (CSF) of wild-type and Cth-/- female mice before pregnancy and at day 1 of lactation (L1). Serum levels of total homocysteine in wild-type and Cth-/- L1 dam mice were 9.44 and 188 µmol/L, respectively, whereas their CSF levels were below 0.21 (limit of quantification) and 3.62 µmol/L, respectively. Their CSF/serum level ratio was the lowest (1/51.9) among all 20 proteinogenic amino acids, sulfur-containing amino acids, and citrulline/ornithine in Cth-/- mice. Therefore, we hypothesize that the blood-brain barrier protects the CNS from high levels of circulatory homocysteine in Cth-/- dam mice, thereby conferring normal oxytocin-dependent maternal behaviors.

Cystathionine-γ-lyase (CSE) deficiency increases erythropoiesis and promotes mitochondrial electron transport via the upregulation of coproporphyrinogen III oxidase and consequent stimulation of heme biosynthesis.

BACKGROUND: Hydrogen sulfide (H2S) is an endogenous gasotransmitter produced by mammalian cells. The current study investigated the potential role of H2S in the regulation of heme biosynthesis using mice deficient in cystathionine gamma-lyase (CSE), one of the three major mammalian H2S-producing enzymes. METHODS: Wild-type and global CSE-/- mice, as well as mitochondria prepared from their liver were used. In vivo, arterial and venous blood gases were measured, and survival of the mice to severe global hypoxia was monitored. Ex vivo, expression of various heme biosynthetic enzymes including coproporphyrinogen oxidase (CPOX) was measured, and mitochondrial function was evaluated using Extracellular Flux Analysis. Urine samples were collected to measure the oxidized porphyrinogen intermediates. The in vivo/ex vivo studies were complemented with mitochondrial bioenergetic studies in hepatocytes in vitro. Moreover, the potential effect of H2S on the CPOX promoter was studied in cells expressing a CPOX promoter construct system. RESULTS: The main findings are as follows: (1) CSE-/- mice exhibit elevated red blood cell counts and red blood cell mean corpuscular volumes compared to wild-type mice; (2) these changes are associated with elevated plasma and liver heme levels and (3) these alterations are likely due to an induction of CPOX (the sixth enzyme involved in heme biosynthesis) in CSE-/- mice. (4) Based on in vitro promoter data the promoter activation of CPOX is directly influenced by H2S, the product of CSE. With respect to the potential functional relevance of these findings, (5) the increased circulating red blood cell numbers do not correspond to any detectable alterations in blood gas parameters under resting conditions, (6) nor do they affect the hypoxia tolerance of the animals in an acute severe hypoxia model. However, there may be a functional interaction between the CSE system and the CPOX system in terms of mitochondrial bioenergetics: (7) CSE-/- hepatocytes and mitochondria isolated from them exhibit increased oxidative phosphorylation parameters, and (8) this increase is partially blunted after CPOX silencing. Although heme is essential for the biosynthesis of mitochondrial electron chain complexes, and CPOX is required for heme biosynthesis, (9) the observed functional mitochondrial alterations are not associated with detectable changes in mitochondrial electron transport chain protein expression. CONCLUSIONS: The CSE system regulates the expression of CPOX and consequent heme synthesis. These effects in turn, do not influence global oxygen transport parameters, but may regulate mitochondrial electron transport.

Preeclampsia-Like Features and Partial Lactation Failure in Mice Lacking Cystathionine γ-Lyase-An Animal Model of Cystathioninuria.

Elevated plasma homocysteine levels are considered as a risk factor for cardiovascular diseases as well as preeclampsia-a pregnancy disorder characterized by hypertension and proteinuria. We previously generated mice lacking cystathionine γ-lyase (Cth) as cystathioninuria models and found them to be with cystathioninemia/homocysteinemia. We investigated whether Cth-deficient (Cth-/-) pregnant mice display any features of preeclampsia. Cth-/- females developed normally but showed mild hypertension (~10 mmHg systolic blood pressure elevation) in late pregnancy and mild proteinuria throughout development/pregnancy. Cth-/- dams had normal numbers of pups and exhibited normal maternal behavior except slightly lower breastfeeding activity. However, half of them could not raise their pups owing to defective lactation; they could produce/store the first milk in their mammary glands but not often provide milk to their pups after the first ejection. The serum oxytocin levels and oxytocin receptor expression in the mammary glands were comparable between wild-type and Cth-/- dams, but the contraction responses of mammary gland myoepithelial cells to oxytocin were significantly lower in Cth-/- dams. The contraction responses to oxytocin were lower in uteruses isolated from Cth-/- mice. Our results suggest that elevated homocysteine or other unknown factors in preeclampsia-like Cth-/- dams interfere with oxytocin that regulates milk ejection reflex.

Cystathionine γ-lyase deficiency enhances airway reactivity and viral-induced disease in mice exposed to side-stream tobacco smoke.

BACKGROUND: Environmental tobacco smoke (ETS) is a known risk factor for severe respiratory syncytial virus (RSV) infections, yet the mechanisms of ETS/RSV comorbidity are largely unknown. Cystathionine γ-lyase regulates important physiological functions of the respiratory tract. METHODS: We used mice genetically deficient in the cystathionine γ-lyase enzyme (CSE), the major H2S-generating enzyme in the lung to determine the contribution of H2S to airway disease in response to side-stream tobacco smoke (TS), and to TS/RSV co-exposure. RESULTS: Following a 2-week period of exposure to TS, CSE-deficient mice (KO) showed a dramatic increase in airway hyperresponsiveness (AHR) to methacholine challenge, and greater airway cellular inflammation, compared with wild-type (WT) mice. TS-exposed CSE KO mice that were subsequently infected with RSV exhibited a more severe clinical disease, airway obstruction and AHR, enhanced viral replication, and lung inflammation, compared with TS-exposed RSV-infected WT mice. TS-exposed RSV-infected CSE KO mice had also a significant increase in the number of neutrophils in bronchoalveolar lavage fluid and increased levels of inflammatory cytokines and chemokines. CONCLUSION: This study demonstrates the critical contribution of the H2S-generating pathway to airway reactivity and disease following exposure to ETS alone or in combination with RSV infection.

Cystathionine γ Lyase Sulfhydrates the RNA Binding Protein Human Antigen R to Preserve Endothelial Cell Function and Delay Atherogenesis.

BACKGROUND: Hydrogen sulfide (H2S), generated by cystathionine γ lyase (CSE), is an important endogenous regulator of vascular function. The aim of the present study was to investigate the control and consequences of CSE activity in endothelial cells under physiological and proatherogenic conditions. METHODS: Endothelial cell CSE knockout mice were generated, and lung endothelial cells were studied in vitro (gene expression, protein sulfhydration, and monocyte adhesion). Mice were crossed onto the apolipoprotein E-deficient background, and atherogenesis (partial carotid artery ligation) was monitored over 21 days. CSE expression, H2S bioavailability, and amino acid profiling were also performed with human material. RESULTS: The endothelial cell-specific deletion of CSE selectively increased the expression of CD62E and elevated monocyte adherence in the absence of an inflammatory stimulus. Mechanistically, CD62E mRNA was more stable in endothelial cells from CSE-deficient mice, an effect attributed to the attenuated sulfhydration and dimerization of the RNA-binding protein human antigen R. CSE expression was upregulated in mice after partial carotid artery ligation and in atheromas from human subjects. Despite the increase in CSE protein, circulating and intraplaque H2S levels were reduced, a phenomenon that could be attributed to the serine phosphorylation (on Ser377) and inhibition of the enzyme, most likely resulting from increased interleukin-1ß. Consistent with the loss of H2S, human antigen R sulfhydration was attenuated in atherosclerosis and resulted in the stabilization of human antigen R-target mRNAs, for example, CD62E and cathepsin S, both of which are linked to endothelial cell activation and atherosclerosis. The deletion of CSE from endothelial cells was associated with the accelerated development of endothelial dysfunction and atherosclerosis, effects that were reversed on treatment with a polysulfide donor. Finally, in mice and humans, plasma levels of the CSE substrate l-cystathionine negatively correlated with vascular reactivity and H2S levels, indicating its potential use as a biomarker for vascular disease. CONCLUSIONS: The constitutive S-sulfhydration of human antigen R (on Cys13) by CSE-derived H2S prevents its homodimerization and activity, which attenuates the expression of target proteins such as CD62E and cathepsin S. However, as a consequence of vascular inflammation, the beneficial actions of CSE-derived H2S are lost owing to the phosphorylation and inhibition of the enzyme.

Cystathionine γ-lyase deficiency aggravates obesity-related insulin resistance via FoxO1-dependent hepatic gluconeogenesis.

Hepatic gluconeogenesis makes a significant contribution to the pathogenesis of obesity and its related insulin resistance. Cystathionine γ-lyase (CSE; also cystathionase), a principal hydrogen sulfide (H2S)-synthesizing enzyme in the liver, is involved in glucose and lipid metabolism disorders. However, the roles and precise mechanisms of CSE/H2S in obesity and its related insulin resistance remain obscure. Here we show that CSE knockout exacerbated high-fat diet-induced mouse obesity as well as its related insulin resistance. Further study elucidated that the inhibition of insulin and AMPK signaling pathways by CSE deficiency resulted in nuclear accumulation of Forkhead box protein O1 and subsequently promoted hepatic gluconeogenesis. These phenomena can be reversed by NaHS supplementation. However, in wild-type mice, NaHS treatment ameliorates high fat diet-induced obesity and metabolism disorders, indicating that maintaining an appropriate level of H2S is critical for its mutual change of positive and negative effects of obesity-associated insulin resistance. Our study reveals a double-edged sword effect and a novel mechanism for CSE/H2S in obesity associated with insulin resistance and provides evidence for CSE/H2S as a promising therapeutic potential target for obesity-related insulin resistance.-Guo, W., Li, D., You, Y., Li, W., Hu, B., Zhang, S., Miao, L., Xian, M., Zhu, Y., Shen, X. Cystathionine γ-lyase deficiency aggravates obesity-related insulin resistance via FoxO1-dependent hepatic gluconeogenesis.

Endogenous Hydrogen Sulfide Ameliorates NOX4 Induced Oxidative Stress in LPS-Stimulated Macrophages and Mice.

BACKGROUND/AIMS: Sepsis is a severe and complicated syndrome that is characterized by dysregulation of host inflammatory responses and organ failure. Cystathionine-γ-lyase (CSE)/ hydrogen sulfide (H2S) has potential anti-inflammatory activities in a variety of inflammatory diseases. NADPH oxidase 4 (Nox4), a member of the NADPH oxidases, is the major source of reactive oxygen species (ROS) and its expression is increased in sepsis, but its function in CSE-mediated anti-inflammatory activities remains unknown. METHODS: Macrophages were either transfected with CSE, Nox4 siRNA or transduced with lentiviral vector encoding CSE or Nox4, and then stimulated with lipopolysaccharide (LPS). The expression of inflammatory mediators and signaling pathway activation were measured by quantitative PCR (qPCR), ELISA, and immunoblotting. LPS-induced shock severity in WT, Nox4 knockdown and CSE knockout (CSE-/-) mice was assessed. RESULTS: Here we showed that CSE and Nox4 were upregulated in macrophage and mouse in response to LPS. After LPS stimulation, the inflammatory responses were significantly ameliorated by lentiviral Nox4 shRNA knockdown, but were exacerbated by lentiviral overexpressing Nox4. Furthermore, Nox4 mediated inflammation through PI3K/Akt and p-p38 mitogen-activated protein kinase signal pathway. Notably, CSE knockout served to amplify the inflammatory cascade by increasing Nox4-ROS signaling activation in septic mice and macrophage. Similarly, the enhanced production of inflammatory mediators by macrophages was reduced by CSE overexpression. CONCLUSION: Thus, we demonstrated that CSE/H2S attenuated LPS-induced sepsis against oxidative stress and inflammation damage probably largely through mediated Nox4 pathway.

The Capture of Cadmium by Reactive Polysulfides Attenuates Cadmium-Induced Adaptive Responses and Hepatotoxicity.

Cadmium (Cd) is an environmental electrophile that modifies protein nucleophiles, thereby modulating cellular signaling and toxicity. While reactive persulfides/polysulfides exhibit relatively high nucleophilic properties, their roles in the altered gene expression and toxicity caused by Cd remain unclear. Exposing primary mouse hepatocytes to Cd caused heat shock protein 70 (HSP70) and metallothionein (MT)-I/II to be upregulated and cytotoxicity to occur. These effects were blocked in the presence of polysulfide sodium tetrasulfide (Na2S4). Electrospray ionization mass spectrometry analysis indicated that cadmium sulfide (CdS) and cadmium thiosulfate (CdS2O3) were produced when Cd reacted with Na2S4. Authentic CdS did not cause cellular signaling responses to be activated or hepatotoxic effects, while CdS2O3 had effects similar to those of Cd. HSP70 and MT-I/II upregulation and hepatotoxicity caused by exposure to Cd were significantly enhanced by the deletion of cystathionine γ-lyase (CSE), which catalyzes the formation of reactive persulfides/polysulfides. Deleting CSE also exacerbated Cd-mediated liver injury, whereas little hepatic damage was found when CdS or Na2S4 along with Cd was administered. Overall, the results suggest that the persulfide/polysulfide-mediated formation of sulfur adducts of Cd such as CdS rather than CdS2O3 is, at least in part, involved in decreasing the level of Cd-mediated activation of cellular signaling and toxicity.

Hydrogen sulfide-producing cystathionine γ-lyase is critical in the progression of kidney fibrosis.

Cystathionine γ-lyase (CSE), the last key enzyme of the transsulfuration pathway, is involved in the production of hydrogen sulfide (H2S) and glutathione (GSH), which regulate redox balance and act as important antioxidant molecules. Impairment of the H2S- and GSH-mediated antioxidant system is associated with the progression of chronic kidney disease (CKD), characterized by kidney fibrosis and dysfunction. Here, we evaluated the role of CSE in the progression of kidney fibrosis after unilateral ureteral obstruction (UUO) using mice deficient in the Cse gene. UUO of wild-type mice reduced the expression of H2S-producing enzymes, CSE, cystathionine ß-synthase, and 3-mercaptopyruvate sulfurtransferase in the obstructed kidneys, resulting in decreased H2S and GSH levels. Cse gene deletion lowered H2S and GSH levels in the kidneys. Deleting the Cse gene exacerbated the decrease in H2S and GSH levels and increase in superoxide formation and oxidative damage to proteins, lipids, and DNA in the kidneys after UUO, which were accompanied by greater kidney fibrosis, deposition of extracellular matrixes, expression of α-smooth muscle actin, tubular damage, and infiltration of inflammatory cells. Furthermore, Cse gene deletion exacerbated mitochondrial fragmentation and apoptosis of renal tubule cells after UUO. The data provided herein constitute in vivo evidence that Cse deficiency impairs renal the H2S- and GSH-producing activity and exacerbates UUO-induced kidney fibrosis. These data propose a novel therapeutic approach against CKD by regulating CSE and the transsulfuration pathway.

Endogenous hydrogen sulfide-mediated MAPK inhibition preserves endothelial function through TXNIP signaling.

Mounting evidence demonstrated deficient cystathionine-γ-lyase (CSE)/H2S implicated the development of cardiovascular disease. The present study aimed to evaluating the favorable action of CSE derived H2S on endothelial function in CSE-/- mice. CSE-/- mice exhibited attenuated endothelium-dependent relaxations, coupled with reduction of endothelial nitric oxide synthase (eNOS) phosphorylation at site of Ser1177, increase of thioredoxin interacting protein (TXNIP) level and MAPK phosphorylation, which were corrected by sodium hydrosulfide chronic treatment for 8 weeks. Impaired relaxations to ACh and upregulated TXNIP of CSE-/- mice aorta were partially corrected by p38 inhibitor, extracellular regulated protein kinase (ERK) inhibitor and c-Jun N-terminal kinase (JNK) inhibitor and totally corrected by combined treatment. Pharmacological inhibition of CSE with DL-propargylglycine (PPG) in vivo and ex vivo induced endothelial dysfunction. PPG stimulated the phosphorylation of p38, JNK and ERK in human umbilical vein endothelial cells (HUVECs). MAPK inhibition by combined treatment of p38, JNK and ERK inhibitors normalized the endothelial changes of eNOS phosphorylation and TXNIP protein level in CSE-/- mice aorta and PPG-treated HUVECs. NaHS offered similar effect with MAKP inhibitors. TXNIP siRNA prevented against endothelial function by PPG and TXNIP overexpression mimics the detrimental effect of PPG treatment on endothelial function, whereas MAPK inhibitor or NaHS has no beneficial effect. In a word, Endogenous CSE/H2S benefits against endothelial dysfunction through suppressing MAPK/TXNIP cascade. CSE deficiency and consequently lowered endogenous H2S level should be considered as risk factors and biomarkers for endothelial dysfunction.

Age-Dependent Allergic Asthma Development and Cystathionine Gamma-Lyase Deficiency.

AIMS: The pathogenic mechanisms for the higher prevalence of allergic asthma in children than in adults have not been settled. The aim of the present study is to examine whether the age-dependent development of allergic asthma is caused by age-dependent expression of cystathionine gamma-lyase (CSE), a key enzyme that catalyzes the production of hydrogen sulfide (H2S). RESULTS: Allergic asthma was induced with ovalbumin in wild-type (WT) and CSE knock-out (KO) mice at young and old ages. CSE expression and H2S production were lower in immune cells of young WT mice than in those of old WT mice. Coincidentally, more severe asthmatic symptoms with a greater type-2 immunoreaction were found in young WT mice than old WT mice. H2S supplementation reversed the asthmatic symptoms. Lower expression levels of CSE proteins were also found in human umbilical cord blood mononuclear cells in comparison with that of peripheral blood mononuclear cells from adult people. The age-dependent asthma propensity vanished in CSE-KO mice, but these mice developed more severe asthma than WT mice. More splenocytes were differentiated to type-2 cytokine-generating cells in young WT mice and in CSE-KO mice at all ages. This differentiation was inhibited by H2S donors. GATA3 translocation to the nucleus and type-2 immunoreaction of splenocytes were inhibited after GATA3 was S-sulfhydrated by H2S. Innovation and Conclusion: For the first time, this study demonstrated that lower abundance of CSE expression and H2S production enhances type-2 immunoreaction and renders a higher incidence of allergic asthma at a young age. As such, H2S level may be a biomarker for asthma development and a H2S-based strategy can be perceived for asthma prevention and treatment. Antioxid. Redox Signal. 27, 931-944.

Cystathionine-gamma-lyase deficient mice are protected against the development of multiorgan failure and exhibit reduced inflammatory response during burn.

Considering the role of H2S in critical illness, the aim of this study was to compare the outcome of burn in wild-type mice and in mice deficient in CSE, one of the principal mammalian H2S-generating enzymes. Animals were subjected to scald burn. Outcome variables included indices of organ injury, clinical chemistry parameters and plasma levels of inflammatory mediators. Plasma levels of H2S significantly increased in response to burn in wild-type mice, but remained unchanged in CSE-/- mice. Expression of the three H2S-producing enzymes (CSE, CBS and 3-MST) in the lung and liver, and the capacity of tissue homogenates to produce H2S, however, was not affected by burn. In CSE deficient mice there was a significant amelioration of burn-induced accumulation of myeloperoxidase levels in heart, lung, liver and kidney and significantly lower degree of malon dialdehyde accumulation in the heart, lung and kidney than in wild-type mice. CSE deficient mice, compared to wild-type mice, showed a significant attenuation of the burn-induced elevation in circulating alkaline aminotransferase and blood urea nitrogen and creatinine levels, indicative of protective effects of CSE deficiency against burn-induced hepatic, and renal functional impairment. Multiple burn-induced inflammatory mediators (TNF-α, IL-1ß, IL-4, IL-6, IL-10 and IL-12) were significantly lower in the plasma of CSE-/- animals after burn than in the plasma of wild-type controls subjected to burns. In conclusion, CSE deficiency improves organ function and attenuates the inflammatory response in a murine model of burn.

Microvascular Endothelial Dysfunction in Obesity Is Driven by Macrophage-Dependent Hydrogen Sulfide Depletion.

OBJECTIVE: The function of perivascular adipose tissue as an anticontractile mediator in the microvasculature is lost during obesity. Obesity results in inflammation and recruitment of proinflammatory macrophages to the perivascular adipose tissue that is paralleled by depletion of the vasorelaxant signaling molecule hydrogen sulfide (H2S) in the vessel. The current objective was to assess the role of macrophages in determining vascular [H2S] and defining how this impinged on vasodilation. APPROACH AND RESULTS: Contractility and [H2S] were measured in mesenteric resistance arterioles from lean and obese mice by using pressure myography and confocal microscopy, respectively. Vasodilation was impaired and smooth muscle and endothelial [H2S] decreased in vessels from obese mice compared with those from lean controls. Coculturing vessels from lean mice with macrophages from obese mice, or macrophage-conditioned media, recapitulated obese phenotypes in vessels. These effects were mediated by low molecular weight species and dependent on macrophage inducible nitric oxide synthase activity. CONCLUSIONS: The inducible nitric oxide synthase activity of perivascular adipose tissue-resident proinflammatory macrophages promotes microvascular endothelial dysfunction by reducing the bioavailability of H2S in the vessel. These findings support a model in which vascular H2S depletion underpins the loss of perivascular adipose tissue anticontractile function in obesity.

Cystathionine γ-Lyase Deficiency Exacerbates CCl4-Induced Acute Hepatitis and Fibrosis in the Mouse Liver.

AIMS: The present study examined the role of cystathionine γ-lyase (CSE) in carbon tetrachloride (CCl4)-induced liver damage. RESULTS: A CSE gene knock-out and luciferase gene knock-in (KI) mouse model was constructed to study the function of CSE and to trace its expression in living status. CCl4 or lipopolysaccharide markedly downregulated CSE expression in the liver of mice. CSE-deficient mice showed increased serum alanine aminotransferase and aspartate aminotransferase levels, and liver damage after CCl4 challenge, whereas albumin and endogenous hydrogen sulfide (H2S) levels decreased significantly. CSE knockout mice showed increased serum homocysteine levels, upregulation of inflammatory cytokines, and increased autophagy and IκB-α degradation in the liver in response to CCl4 treatment. The increase in pro-inflammatory cytokines, including tumor necrosis factor-alpha in CSE-deficient mice after CCl4 challenge, was accompanied by a significant increase in liver tissue hydroxyproline and α-smooth muscle actin and histopathologic changes in the liver. However, H2S donor pretreatment effectively attenuated most of these imbalances. INNOVATION: Here, a CSE knock-out and luciferase KI mouse model was established for the first time to study the transcriptional regulation of CSE expression in real time in a non-invasive manner, providing information on the effects and potential mechanisms of CSE on CCl4-induced liver injury. CONCLUSION: CSE deficiency increases pro-inflammatory cytokines in the liver and exacerbates acute hepatitis and liver fibrosis by reducing H2S production from L-cysteine in the liver. The present data suggest the potential of an H2S donor for the treatment of liver diseases such as toxic hepatitis and fibrosis. Antioxid. Redox Signal. 27, 133-149.

The Role of Cystathionine-γ-Lyase In Blunt Chest Trauma in Cigarette Smoke Exposed Mice.

Pretraumatic cigarette smoke (CS) exposure aggravates posttraumatic acute lung injury (ALI). Cystathionine-γ-lyase (CSE) protects against ALI and CS exposure-induced chronic obstructive lung disease (COPD). Therefore, we tested the hypothesis whether genetic CSE knockout (CSE) would aggravate posttraumatic ALI after CS exposure. After 3 to 4 weeks of CS exposure, anesthetized wild-type (WT) and CSE mice underwent blunt chest trauma, surgical instrumentation and 4 h of lung-protective mechanical ventilation. We measured hemodynamics, lung mechanics, gas exchange, metabolism, and acid-base status together with blood and tissue cytokine and chemokine levels, tissue expression of mediator proteins, parameters of oxidative and nitrosative stress, and histology. CSE mice without CS exposure showed higher cytokine and chemokine levels, and this was further enhanced by CS exposure, particularly in males. CS exposure in WT mice aggravated posttraumatic alveolar membrane thickening, dystelectasis, and inflammatory cell accumulation, which was associated with higher thoracopulmonary compliance. Pretraumatic CS exposure in CSE mice produced a similar response, except for less alveolar membrane thickening, most likely due to lung hyperinflation. CS-exposed WT mice showed the most pronounced metabolic acidosis, while CS exposure in CSE mice resulted in the lowest blood glucose levels. Urinary output and anesthesia rate were highest in male CS-exposed CSE animals. In conclusion, in murine acute-on-chronic pulmonary disease, CSE knockout aggravated posttraumatic inflammation, which was further worsened upon pretraumatic CS exposure, and this effect was particularly pronounced in males. Hence, maintaining CSE expression is critically important for stress adaptation during ALI and CS-induced COPD, most likely in a gender-dependent manner.

Effect of endotoxemia in mice genetically deficient in cystathionine-γ-lyase, cystathionine-ß-synthase or 3-mercaptopyruvate sulfurtransferase.

Hydrogen sulfide (H2S) has been proposed to exert pro- as well as anti-inflammatory effects in various models of critical illness. In this study, we compared bacterial lipopolysaccharide (LPS)­induced changes in inflammatory mediator production, indices of multiple organ injury and survival in wild­type (WT) mice and in mice with reduced expression of one of the three H2S­producing enzymes, cystathionine-γ-lyase (CSE), cystathionine-ß-synthase (CBS) or 3-mercaptopyruvate sulfurtransferase (3MST). Mice were injected intraperitoneally (i.p.) with LPS (10 mg/kg). After 6 h, the animals were sacrificed, blood and organs were collected and the following parameters were evaluated: blood urea nitrogen (BUN) levels in blood, myeloperoxidase (MPO) and malondialdehyde (MDA) in the lung, cytokine levels in plasma and the expression of the three H2S­producing enzymes (CBS, CSE and 3MST) in the spleen, lung, liver and kidney. LPS induced a tissue­dependent upregulation of some of the H2S­producing enzymes in WT mice (upregulation of CBS in the spleen, upregulation of 3MST in the liver and upregulation of CBS, CSE and 3MST in the lung). Moreover, LPS impaired glomerular function, as evidenced by increased BUN levels. Renal impairment was comparable in the CSE­/­ and Δ3MST mice after LPS challenge; however, it was attenuated in the CBS+/­ mice. MPO levels (an index of neutrophil infiltration) and MDA levels (an index of oxidative stress) in lung homogenates were significantly increased in response to LPS; these effects were similar in the WT, CBS+/­, CSE­/­ and Δ3MST mice; however, the MDA levels tended to be lower in the CBS+/­ and CSE­/­ mice. LPS induced significant increases in the plasma levels of multiple cytokines [tumor necrosis factor (TNF)α, interleukin (IL)­1ß, IL­6, IL­10, IL­12 and interferon (IFN)γ] in plasma; TNFα, IL­10 and IL­12 levels tended to be lower in all three groups of animals expressing lower levels of H2S­producing enzymes. The survival rates after the LPS challenge did not show any significant differences between the four animal groups tested. Thus, the findings of this study indicate that a deficiency in 3MST does not significantly affect endotoxemia, while a deficiency in CBS or CSE slightly ameliorates the outcome of LPS-induced endotoxemia in vivo.

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.

Hydrogen Sulfide Is an Antiviral and Antiinflammatory Endogenous Gasotransmitter in the Airways. Role in Respiratory Syncytial Virus Infection.

Hydrogen sulfide (H2S) is an endogenous gaseous transmitter whose role in the pathophysiology of several lung diseases has been increasingly appreciated. Our recent studies in vitro have shown, we believe for the first time, that H2S has an important antiviral and antiinflammatory activity in respiratory syncytial virus (RSV) infection, the leading cause of bronchiolitis and viral pneumonia in children. Our objective was to evaluate the therapeutic potential of GYY4137, a novel slow-releasing H2S donor, for the prevention and treatment of RSV-induced lung disease, as well as to investigate the role of endogenous H2S in a mouse model of RSV infection. Ten- to 12-week-old BALB/c mice treated with GYY4137, or C57BL/6J mice genetically deficient in the cystathionine γ-lyase enzyme, the major H2S-generating enzyme in the lung, were infected with RSV and assessed for viral replication, clinical disease, airway hyperresponsiveness, and inflammatory responses. Our results show that intranasal delivery of GYY4137 to RSV-infected mice significantly reduced viral replication and markedly improved clinical disease parameters and pulmonary dysfunction compared with the results in vehicle-treated control mice. The protective effect of the H2S donor was associated with a significant reduction of viral-induced proinflammatory mediators and lung cellular infiltrates. Furthermore, cystathionine γ-lyase-deficient mice showed significantly enhanced RSV-induced lung disease and viral replication compared with wild-type animals. Overall, our results indicate that H2S exerts a novel antiviral and antiinflammatory activity in the context of RSV infection and represent a potential novel pharmacological approach for ameliorating virus-induced lung disease.