Hydrogen Sulfide Modulates Endothelial-Mesenchymal Transition in Heart Failure.

BACKGROUND: Hydrogen sulfide is a critical endogenous signaling molecule that exerts protective effects in the setting of heart failure. Cystathionine γ-lyase (CSE), 1 of 3 hydrogen-sulfide-producing enzyme, is predominantly localized in the vascular endothelium. The interaction between the endothelial CSE-hydrogen sulfide axis and endothelial-mesenchymal transition, an important pathological process contributing to the formation of fibrosis, has yet to be investigated. METHODS: Endothelial-cell-specific CSE knockout and Endothelial cell-CSE overexpressing mice were subjected to transverse aortic constriction to induce heart failure with reduced ejection fraction. Cardiac function, vascular reactivity, and treadmill exercise capacity were measured to determine the severity of heart failure. Histological and gene expression analyses were performed to investigate changes in cardiac fibrosis and the activation of endothelial-mesenchymal transition. RESULTS: Endothelial-cell-specific CSE knockout mice exhibited increased endothelial-mesenchymal transition and reduced nitric oxide bioavailability in the myocardium, which was associated with increased cardiac fibrosis, impaired cardiac and vascular function, and worsened exercise performance. In contrast, genetic overexpression of CSE in endothelial cells led to increased myocardial nitric oxide, decreased endothelial-mesenchymal transition and cardiac fibrosis, preserved cardiac and endothelial function, and improved exercise capacity. CONCLUSIONS: Our data demonstrate that endothelial CSE modulates endothelial-mesenchymal transition and ameliorate the severity of pressure-overload-induced heart failure, in part, through nitric oxide-related mechanisms. These data further suggest that endothelium-derived hydrogen sulfide is a potential therapeutic for the treatment of heart failure with reduced ejection fraction.

Host cystathionine-γ lyase derived hydrogen sulfide protects against Pseudomonas aeruginosa sepsis.

Hydrogen sulfide (H2S) has recently been recognized as a novel gaseous transmitter with several anti-inflammatory properties. The role of host- derived H2S in infections by Pseudomonas aeruginosa was investigated in clinical and mouse models. H2S concentrations and survival was assessed in septic patients with lung infection. Animal experiments using a model of severe systemic multidrug-resistant P. aeruginosa infection were performed using mice with a constitutive knock-out of cystathionine-γ lyase (Cse) gene (Cse-/-) and wild-type mice with a physiological expression (Cse+/+). Experiments were repeated in mice after a) treatment with cyclophosphamide; b) bone marrow transplantation (BMT) from a Cse+/+ donor; c) treatment with H2S synthesis inhibitor aminooxyacetic acid (ΑΟΑΑ) or propargylglycine (PAG) and d) H2S donor sodium thiosulfate (STS) or GYY3147. Bacterial loads and myeloperoxidase activity were measured in tissue samples. The expression of quorum sensing genes (QS) was determined in vivo and in vitro. Cytokine concentration was measured in serum and incubated splenocytes. Patients survivors at day 28 had significantly higher serum H2S compared to non-survivors. A cut- off point of 5.3 µΜ discriminated survivors with sensitivity 92.3%. Mortality after 28 days was 30.9% and 93.7% in patients with H2S higher and less than 5.3 µΜ (p = 7 x 10-6). In mice expression of Cse and application of STS afforded protection against infection with multidrug-resistant P. aeruginosa. Cyclophosphamide pretreatment eliminated the survival benefit of Cse+/+ mice, whereas BMT increased the survival of Cse-/- mice. Cse-/- mice had increased pathogen loads compared to Cse+/+ mice. Phagocytic activity of leukocytes from Cse-/- mice was reduced but was restored after H2S supplementation. An H2S dependent down- regulation of quorum sensing genes of P.aeruginosa could be demonstrated in vivo and in vitro. Endogenous H2S is a potential independent parameter correlating with the outcome of P. aeruginosa. H2S provides resistance to infection by MDR bacterial pathogens.

Hippocampal neural stem cells and microglia response to experimental inflammatory bowel disease (IBD).

Inflammatory bowel disease (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), is a disease associated with dysbiosis, resulting in compromised intestinal epithelial barrier and chronic mucosal inflammation. Patients with IBD present with increased incidence of psychiatric disorders and cognitive impairment. Hippocampus is a brain region where adult neurogenesis occurs with functional implications in mood control and cognition. Using a well-established model of experimental colitis based on the administration of dextran sodium sulfate (DSS) in the drinking water, we sought to characterize the short and long-term effects of colitis on neurogenesis and glia responses in the hippocampus. We show that acute DSS colitis enhanced neurogenesis but with deficits in cell cycle kinetics of proliferating progenitors in the hippocampus. Chronic DSS colitis was characterized by normal levels of neurogenesis but with deficits in the migration and integration of newborn neurons in the functional circuitry of the DG. Notably, we found that acute DSS colitis-induced enhanced infiltration of the hippocampus with macrophages and inflammatory myeloid cells from the periphery, along with elevated frequencies of inflammatory M1-like microglia and increased release of pro-inflammatory cytokines. In contrast, increased percentages of tissue-repairing M2-like microglia, along with elevated levels of the anti-inflammatory cytokine, IL-10 were observed in the hippocampus during chronic DSS colitis. These findings uncover key effects of acute and chronic experimental colitis on adult hippocampal neurogenesis and innate immune cell responses, highlighting the potential mechanisms underlying cognitive and mood dysfunction in patients with IBD.

Mercaptopyruvate acts as endogenous vasodilator independently of 3-mercaptopyruvate sulfurtransferase activity.

Hydrogen sulfide (H2S) is produced by the action of cystathionine-ß-synthase (CBS), cystathionine-γ-lyase (CSE) or 3-mercaptopyruvate sulfurtransferase (3-MST). 3-MST converts 3-mercaptopyruvate (MPT) to H2S and pyruvate. H2S is recognized as an endogenous gaseous mediator with multiple regulatory roles in mammalian cells and organisms. In the present study we demonstrate that MPT, the endogenous substrate of 3-MST, acts also as endogenous H2S donor. Colorimetric, amperometric and fluorescence based assays demonstrated that MPT releases H2S in vitro in an enzyme-independent manner. A functional study was performed on aortic rings harvested from C57BL/6 (WT) or 3-MST-knockout (3-MST-/-) mice with and without endothelium. MPT relaxed mouse aortic rings in endothelium-independent manner and at the same extent in both WT and 3-MST-/- mice. N5-(1-Iminoethyl)-l-ornithine dihydrochloride (L-NIO, an inhibitor of endothelial nitric oxide synthase) as well as 1H-[1,2,4]oxadiazolo [4,3-a]quinoxalin-1-one (ODQ, a soluble guanylyl cyclase inhibitor) did not affect MPT relaxant action. Conversely, hemoglobin (as H2S scavenger), as well as glybenclamide (an ATP-dependent potassium channel blocker) markedly reduced MPT-induced relaxation. The functional data clearly confirmed a non enzymatic vascular effect of MPT. In conclusion, MPT acts also as an endogenous H2S donor and not only as 3-MST substrate. MPT could, thus, be further investigated as a means to increase H2S in conditions where H2S bioavailability is reduced such as hypertension, coronary artery disease, diabetes or urogenital tract disease.

Reduced adipose tissue H2S in obesity.

Hydrogen sulfide (H2S) is an endogenously produced signaling molecule synthesized by cystathionine γ-lyase (CSE), cystathionine ß-synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (3-MST). Given that H2S exerts significant effects on bioenergetics and metabolism, the goal of the current study was to determine the expression of H2S-producing enzymes in adipose tissues in models of obesity and metabolic disruption. Mice fed a western diet expressed lower mRNA levels of all three enzymes in epididymal fat (EWAT), while only CSE and 3-MST were reduced in brown adipose tissue (BAT). At the protein level 3-MST was reduced in all fat depots studied. Using db/db mice, a genetic model of obesity, we found that CSE, CBS and 3-MST mRNA were reduced in white fat, while only CSE was reduced in BAT. CBS and CSE protein levels were suppressed in all three fat depots. In a model of age-related weight gain, no reduction in the mRNA of any of the enzymes was noted. Smaller amounts of 3-MST protein were found in EWAT, while both CSE and 3-MST were reduced in BAT. Tissue levels of H2S were lower in WAT in HFD mice; both WAT and BAT contained lower H2S amounts in db/db animals. Taken together, our data suggest that obesity is associated with a decreased expression of H2S-synthesizing enzymes and reduced H2S levels in adipose tissues of mice. We propose that the reduction in H2S may contribute to the metabolic response associated with obesity. Further work is needed to determine whether restoring H2S levels may have a beneficial effect on obesity-associated metabolic alterations.

Regulation and role of endogenously produced hydrogen sulfide in angiogenesis.

Recent studies have implicated endogenously produced H2S in the angiogenic process. On one hand, pharmacological inhibition and silencing of the enzymes involved in H2S synthesis attenuate the angiogenic properties of endothelial cells, including proliferation, migration and tube-like structure network formation. On the other hand, enhanced production of H2S by substrate supplementation or over-expression of H2S-producing enzymes leads to enhanced angiogenic responses in cultured endothelial cells. Importantly, H2S up-regulates expression of the key angiogenic factor vascular endothelial growth factor (VEGF) and contributes to the angiogenic signaling in response to VEGF. The signaling pathways mediating H2S-induced angiogenesis include mitogen-activated protein kinases, phosphoinositide-3 kinase, nitric oxide/cGMP-regulated cascades and ATP-sensitive potassium channels. Endogenously produced H2S has also been shown to facilitate neovascularization in prototypical model systems in vivo, and to contribute to wound healing, post-ischemic angiogenesis in the heart and other tissues, as well as in tumor angiogenesis. Targeting of H2S synthesizing enzymes might offer novel therapeutic opportunities for angiogenesis-related diseases.

Loss of cardiac mitochondrial complex I persulfidation impairs NAD+ homeostasis in aging.

Protein persulfidation is a significant post-translational modification that involves addition of a sulfur atom to the cysteine thiol group and is facilitated by sulfide species. Persulfidation targets reactive cysteine residues within proteins, influencing their structure and/or function across various biological systems. This modification is evolutionarily conserved and plays a crucial role in preventing irreversible cysteine overoxidation, a process that becomes prominent with aging. While, persulfidation decreases with age, its levels in the aged heart and the functional implications of such a reduction in cardiac metabolism remain unknown. Here we interrogated the cardiac persulfydome in wild-type adult mice and age-matched mice lacking the two sulfide generating enzymes, namely cystathionine gamma lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3MST). Our findings revealed that cardiac persulfidated proteins in wild type hearts are less abundant compared to those in other organs, with a primary involvement in mitochondrial metabolic processes. We further focused on one specific target, NDUFB7, which undergoes persulfidation by both CSE and 3MST derived sulfide species. In particular, persulfidation of cysteines C80 and C90 in NDUFB7 protects the protein from overoxidation and maintains the complex I activity in cardiomyocytes. As the heart ages, the levels of CSE and 3MST in cardiomyocytes decline, leading to reduced NDUFB7 persulfidation and increased cardiac NADH/NAD+ ratio. Collectively, our data provide compelling evidence for a direct link between cardiac persulfidation and mitochondrial complex I activity, which is compromised in aging.

An optimized protocol for chromatin immunoprecipitation from murine inguinal white adipose tissue.

Chromatin immunoprecipitation (ChIP) protocols have been used to reveal protein-DNA interactions of various cell types and tissues; however, optimization is required for each specific type of sample. Here, we present a ChIP protocol from murine inguinal white adipose tissue. We describe steps for tissue harvesting, crosslinking, chromatin extraction, shearing, immunoprecipitation, and purification. We then detail procedures for analysis including library preparation, sequencing, and qRT-PCR validation. For complete details on the use and execution of this protocol, please refer to Antonia Katsouda et al. (2022).1.

CTH/MPST double ablation results in enhanced vasorelaxation and reduced blood pressure via upregulation of the eNOS/sGC pathway.

Hydrogen sulfide (H2S), a gasotransmitter with protective effects in the cardiovascular system, is endogenously generated by three main enzymatic pathways: cystathionine gamma lyase (CTH), cystathionine beta synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (MPST) enzymes. CTH and MPST are the predominant sources of H2S in the heart and blood vessels, exhibiting distinct effects in the cardiovascular system. To better understand the impact of H2S in cardiovascular homeostasis, we generated a double Cth/Mpst knockout (Cth/Mpst -/- ) mouse and characterized its cardiovascular phenotype. CTH/MPST-deficient mice were viable, fertile and exhibited no gross abnormalities. Lack of both CTH and MPST did not affect the levels of CBS and H2S-degrading enzymes in the heart and the aorta. Cth/Mpst -/- mice also exhibited reduced systolic, diastolic and mean arterial blood pressure, and presented normal left ventricular structure and fraction. Aortic ring relaxation in response to exogenously applied H2S was similar between the two genotypes. Interestingly, an enhanced endothelium-dependent relaxation to acetylcholine was observed in mice in which both enzymes were deleted. This paradoxical change was associated with upregulated levels of endothelial nitric oxide synthase (eNOS) and soluble guanylate cyclase (sGC) α1 and ß1 subunits and increased NO-donor-induced vasorelaxation. Administration of a NOS-inhibitor, increased mean arterial blood pressure to a similar extent in wild-type and Cth/Mpst -/- mice. We conclude that chronic elimination of the two major H2S sources in the cardiovascular system, leads to an adaptive upregulation of eNOS/sGC signaling, revealing novel ways through which H2S affects the NO/cGMP pathway.

Cystathionine gamma-lyase (CTH) inhibition attenuates glioblastoma formation.

PURPOSE: Glioblastoma (GBM) is the most common type of adult brain tumor with extremely poor survival. Cystathionine-gamma lyase (CTH) is one of the main Hydrogen Sulfide (H2S) producing enzymes and its expression contributes to tumorigenesis and angiogenesis but its role in glioblastoma development remains poorly understood. METHODS: and Principal Results: An established allogenic immunocompetent in vivo GBM model was used in C57BL/6J WT and CTH KO mice where the tumor volume and tumor microvessel density were blindly measured by stereological analysis. Tumor macrophage and stemness markers were measured by blinded immunohistochemistry. Mouse and human GBM cell lines were used for cell-based analyses. In human gliomas, the CTH expression was analyzed by bioinformatic analysis on different databases. In vivo, the genetic ablation of CTH in the host led to a significant reduction of the tumor volume and the protumorigenic and stemness transcription factor sex determining region Y-box 2 (SOX2). The tumor microvessel density (indicative of angiogenesis) and the expression levels of peritumoral macrophages showed no significant changes between the two genotypes. Bioinformatic analysis in human glioma tumors revealed that higher CTH expression is positively correlated to SOX2 expression and associated with worse overall survival in all grades of gliomas. Patients not responding to temozolomide have also higher CTH expression. In mouse or human GBM cells, pharmacological inhibition (PAG) or CTH knockdown (siRNA) attenuates GBM cell proliferation, migration and stem cell formation frequency. MAJOR CONCLUSIONS: Inhibition of CTH could be a new promising target against glioblastoma formation.

MPST sulfurtransferase maintains mitochondrial protein import and cellular bioenergetics to attenuate obesity.

Given the clinical, economic, and societal impact of obesity, unraveling the mechanisms of adipose tissue expansion remains of fundamental significance. We previously showed that white adipose tissue (WAT) levels of 3-mercaptopyruvate sulfurtransferase (MPST), a mitochondrial cysteine-catabolizing enzyme that yields pyruvate and sulfide species, are downregulated in obesity. Here, we report that Mpst deletion results in fat accumulation in mice fed a high-fat diet (HFD) through transcriptional and metabolic maladaptation. Mpst-deficient mice on HFD exhibit increased body weight and inguinal WAT mass, reduced metabolic rate, and impaired glucose/insulin tolerance. At the molecular level, Mpst ablation activates HIF1α, downregulates subunits of the translocase of outer/inner membrane (TIM/TOM) complex, and impairs mitochondrial protein import. MPST deficiency suppresses the TCA cycle, oxidative phosphorylation, and fatty acid oxidation, enhancing lipid accumulation. Sulfide donor administration to obese mice reverses the HFD-induced changes. These findings reveal the significance of MPST for white adipose tissue biology and metabolic health and identify a potential new therapeutic target for obesity.

Generation and Characterization of a CRISPR/Cas9 -Induced 3-mst Deficient Zebrafish.

3-mercaptopyruvate sulfurtransferase (3-MST) is an enzyme capable of synthesizing hydrogen sulfide (H2S) and polysulfides. In spite of its ubiquitous presence in mammalian cells, very few studies have investigated its contribution to homeostasis and disease development, thus the role of 3-MST remains largely unexplored. Here, we present a clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated protein-9 (Cas9) induced 3-mst mutant zebrafish line, which will allow the study of 3-MST's role in several biological processes. The 3-mst zebrafish orthologue was identified using a bioinformatic approach and verified by its ability to produce H2S in the presence of 3-mercaptopyruvate (3-MP). Its expression pattern was analyzed during zebrafish early development, indicating predominantly an expression in the heart and central nervous system. As expected, no detectable levels of 3-Mst protein were observed in homozygous mutant larvae. In line with this, H2S levels were reduced in 3-mst-/- zebrafish. Although the mutants showed no obvious morphological deficiencies, they exhibited increased lethality under oxidative stress conditions. The elevated levels of reactive oxygen species, detected following 3-mst deletion, are likely to drive this phenotype. In line with the increased ROS, we observed accelerated fin regenerative capacity in 3-mst deficient zebrafish. Overall, we provide evidence for the expression of 3-mst in zebrafish, confirm its important role in redox homeostasis and indicate the enzyme's possible involvement in the regeneration processes.

Cardiovascular phenotype of mice lacking 3-mercaptopyruvate sulfurtransferase.

RATIONALE: Hydrogen sulfide (H2S) is a physiological mediator that regulates cardiovascular homeostasis. Three major enzymes contribute to the generation of endogenously produced H2S, namely cystathionine γ-lyase (CSE), cystathionine ß-synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (3-MST). Although the biological roles of CSE and CBS have been extensively investigated in the cardiovascular system, very little is known about that of 3-MST. In the present study we determined the importance of 3-MST in the heart and blood vessels, using a genetic model with a global 3-MST deletion. RESULTS: 3-MST is the most abundant transcript in the mouse heart, compared to CSE and CBS. 3-MST was mainly localized in smooth muscle cells and cardiomyocytes, where it was present in both the mitochondria and the cytosol. Levels of serum and cardiac H2S species were not altered in adult young (2-3 months old) 3-MST-/- mice compared to WT animals. No significant changes in the expression of CSE and CBS were observed. Additionally, 3-MST-/- mice had normal left ventricular structure and function, blood pressure and vascular reactivity. Interestingly, genetic ablation of 3-MST protected mice against myocardial ischemia reperfusion injury, and abolished the protection offered by ischemic pre- and post-conditioning. 3-MST-/- mice showed lower expression levels of thiosulfate sulfurtransferase, lower levels of cellular antioxidants and elevated basal levels of cardiac reactive oxygen species. In parallel, 3-MST-/- mice showed no significant alterations in endothelial NO synthase or downstream targets. Finally, in a separate cohort of older 3-MST-/- mice (18 months old), a hypertensive phenotype associated with cardiac hypertrophy and NO insufficiency was observed. CONCLUSIONS: Overall, genetic ablation of 3-MST impacts on the mouse cardiovascular system in an age-dependent manner. Loss of 3-MST exerts a cardioprotective role in young adult mice, while with aging it predisposes them to hypertension and cardiac hypertrophy.

CD8+ T cells in beige adipogenesis and energy homeostasis.

Although accumulation of lymphocytes in the white adipose tissue (WAT) in obesity is linked to insulin resistance, it remains unclear whether lymphocytes also participate in the regulation of energy homeostasis in the WAT. Here, we demonstrate enhanced energy dissipation in Rag1-/- mice, increased catecholaminergic input to subcutaneous WAT, and significant beige adipogenesis. Adoptive transfer experiments demonstrated that CD8+ T cell deficiency accounts for the enhanced beige adipogenesis in Rag1-/- mice. Consistently, we identified that CD8-/- mice also presented with enhanced beige adipogenesis. The inhibitory effect of CD8+ T cells on beige adipogenesis was reversed by blockade of IFN-γ. All together, our findings identify an effect of CD8+ T cells in regulating energy dissipation in lean WAT, mediated by IFN-γ modulation of the abundance of resident immune cells and of local catecholaminergic activity. Our results provide a plausible explanation for the clinical signs of metabolic dysfunction in diseases characterized by altered CD8+ T cell abundance and suggest targeting of CD8+ T cells as a promising therapeutic approach for obesity and other diseases with altered energy homeostasis.