Cystathionine Γ-lyase Knockout Research Articles

Hydrogen Sulfide Regulates Erectile Function through Stimulation of Antioxidant Defense in the Corpus Cavernosum

Hydrogen sulfide (H2S) is a gaseous cell signaling molecule with vasodilatory properties. Diminished H2S production has been implicated in the pathology of several cardiovascular diseases. H2S may protect against oxidative stress through scavenging of reactive oxygen species (ROS) or through stimulation of the Nrf2-mediated cellular antioxidant defense systems. We have previously demonstrated that mice fed a western diet for 12 weeks develop elevated penile ROS levels and impaired erectile function, which coincides with diminished corpus cavernosal expression of cystathionine γ-lyase (CSE), a major H2S producing enzyme. The objective of this study was to investigate the role of H2S in the regulation of erectile function and penile redox balance. We hypothesized that H2S levels will be positively associated with cellular antioxidant defense and erectile function. Young male C57Bl/6J mice (n = 120) were fed a control diet (CD) or western diet (WD) ad libitum for 18 weeks. For the final six weeks of the dietary intervention, half of the mice were switched to an equivalent diet containing the H2S pro-drug SG1002 at approximately 20 mg/kg/day. Separately, male CSE knockout mice (n = 26) and wild type (WT) littermate controls (n = 26) were fed a standard diet and studied at 6 months. Following the interventions, erectile function was assessed by measuring intracavernosal pressure (ICP) and mean arterial pressure (MAP) during cavernous nerve stimulation (1, 2, 4 V). In separate mice, penile in vivo ROS production was measured via microdialysis. Relative expression of 18 genes related to cellular antioxidant defense were assessed from naïve corpus cavernosum tissue by qRT-PCR. Group differences were determined by two-way ANOVA, with an α-level of 0.05. Erectile function was impaired in CSE KO mice (2V ICP/MAP - WT: 0.70±0.039 vs. KO: 0.55±0.043; p<0.05). Erectile function was similarly impaired in the untreated WD-fed mice (CD: 0.68±0.035 vs. WD: 0.49±0.012; p<0.05), while SG1002 induced a 54% functional restoration in WD mice (WD+SG1002: 0.59±0.019; p<0.05). Penile production of the ROS hydrogen peroxide and superoxide were elevated in CSE KO mice (WT: 0.73±0.04 vs. KO: 1.19±0.13; p<0.01). Penile ROS were also elevated in the WD mice (CD: 0.71±0.07 vs. WD: 1.09±0.15; p<0.01), which were normalized by SG1002 treatment (WD+SG1002: 0.83±0.07; p<0.01 vs. WD). Five antioxidant genes (Gclc, Gpx1, Gpx4, Prdx3, and Hmox1) that were stimulated by SG1002 treatment were also downregulated in CSE KO cavernosal tissues. Conversely, Txnip, a negative regulator of the thioredoxin antioxidant system was suppressed by SG1002 treatment and increased in CSE KO tissues. These results indicate that H2S is a key regulator of the cellular antioxidant systems and ROS balance in the penis, which may also regulate erectile function. Grant number K01DK115540 from the National Institutes of Health. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Hydrogen sulfide regulates macrophage polarization and necroptosis to accelerate diabetic skin wound healing

Hydrogen sulfide (H2S), recognized as the third gasotransmitter, plays a pivotal role in the pathophysiological processes of various diseases. Cystathionine γ-lyase (CSE) is the main enzyme for H2S production in the skin. However, effects and mechanisms of H2S in diabetic skin wound healing remain unclear. Our findings revealed a decrease in plasma H2S content in diabetic patients with skin wounds. CSE knockout (KO) diabetic mice resulted in delayed wound healing, reduced blood perfusion, and CD31 expression around the wounds. It also led to increased infiltration of inflammatory cells and M1-type macrophages, decreased collagen levels, α-smooth muscle actin (α-SMA), and proliferating cell nuclear antigen (PCNA) expression. Additionally, there were enhanced expressions of necroptosis related proteins, including receptor interacting protein kinase 1 (RIPK1), RIPK3 and mixed lineage kinase domain like protein (MLKL). In comparison, sodium hydrosulfide (NaHS), H2S donor, accelerated skin wound healing in leptin receptor deficiency (db/db) mice. This acceleration was accompanied by increased blood perfusion and CD31 expression, reduced infiltration of inflammatory cells and M1-type macrophages, elevated collagen levels, α-SMA, and PCNA expressions, and decreased necroptosis-related protein expressions together with nuclear factor-κB (NF-κB) p65 phosphorylation. In conclusion, H2S regulates macrophage polarization and necroptosis, contributing to the acceleration of diabetic skin wound healing. These findings offer a novel strategy for the treatment of diabetic skin wounds.

Supersulfides to Regulate Electrophilic Stress

Environmental electrophiles modify thiol groups of proteins in organs, disrupting cellular functions carried out by the modified proteins and increasing the risk of various diseases. The transcription factor NF-E2-related factor 2 (Nrf2) plays a crucial role in detoxifying electrophiles by forming glutathione adducts and subsequently excreting them into extracellular spaces. Supersulfides such as cysteine persulfides (CysSSH) produced by cystathionine γ-lyase (CSE) capture environmental electrophiles through sulfur adduct formation. However, the Nrf2 and CSE contributions to blocking environmental electrophile-mediated toxicity have yet to be evaluated. Therefore, we assessed the individual and combined roles of Nrf2 and CSE in suppressing toxicity induced by environmental electrophiles using Nrf2 knockout (KO), CSE KO, and Nrf2/CSE double KO (DKO) mice. Our findings indicate that CSE/Nrf2 DKO mice are more sensitive to environmental electrophiles compared to their single KO counterparts, highlighting the distinct mechanisms through which both pathways mitigate the toxic effects of reactive electrophiles. Moreover, diverse metabolites produced by symbiotic gut bacteria in the human body are known to exert various effects on host organ functions beyond the intestinal tract. We observed reduced blood supersulfide levels in mice lacking gut microflora compared to normal mice. Furthermore, we identified intestinal bacteria belonging to the families Ruminococcaceae and Lachnospiraceae as high CysSSH-producing bacteria. This suggests that the gut microbiota serves as a source of in vivo supersulfide molecules. These findings suggest that supersulfide derived from gut bacteria may act protectively against environmental electrophilic exposure in the host.

Hydrogen sulfide inhibits lipopolysaccharide-based neuroinflammation-induced astrocyte polarization after cerebral ischemia/reperfusion injury

The effect of lipopolysaccharide (LPS)-based neuroinflammation following cerebral ischemia/reperfusion (I/R) on the genotypic transformation of reactive astrocytes and its relationship with endogenous hydrogen sulfide (H2S) were investigated in present study. We found that LPS promoted the cerebral I/R-induced A1 astrocytes proliferation in mouse hippocampal tissues and deteriorated the reduction of hydrogen sulfide (H2S) content in mouse sera, H2S donor NaHS could inhibit A1 astrocytes proliferation. Similarly, knockout of cystathionine γ-lyase (CSE), one of endogenous H2S synthases, likewise up-regulated the cerebral I/R-induced A1 astrocytes proliferation, which could also be blocked by NaHS. Besides, supplement with H2S promoted the A2 astrocytes proliferation in hippocampal tissues of CSE knockout (CSE KO) mice or LPS-treated mice following cerebral I/R. In the oxygen glucose deprivation/reoxygenation (OGD/R) model of astrocytes, H2S also promoted the transformation of astrocytes into A2 subtype. Moreover, we found that H2S could up-regulate the expression of α-subunit of large-conductance Ca2+-activated K+ (BKCa) channels in astrocytes, and the channel opener BMS-191011 likewise promoted the transformation of astrocyte into A2 subtype. In conclusion, H2S inhibits the proliferation of A1 astrocytes induced by LPS-based neuroinflammation following cerebral I/R and promotes the transformation of astrocytes into A2 subtype, which may be related to up-regulation of BKCa channels.

Hydrogen Sulfide Modulates Endothelial-Mesenchymal Transition in Heart Failure.

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. 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. 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. 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.

CSE/H2S ameliorates colitis in mice via protection of enteric glial cells and inhibition of the RhoA/ROCK pathway.

The enteric glial cells (EGCs) participate in the homeostasis of the gastrointestinal tract, and RhoA/ROCK signaling pathway plays a vital role in colonic tight junctions. Hydrogen sulfide (H2S) has been reported to alleviate colitis. However, the effect and mechanism of endogenous H2S on colitis remain unclear. This study established a Cystathionine-γ-lyase (CSE) knockout mouse model, a significant source of H2S production in the gut. The role of CSE-produced H2S on EGCs and the RhoA/ROCK signaling pathway was investigated in experimental colitis using CSE knockout (KO) and wild-type (WT) mice. CSE gene knockout animals presented with disease progression, more deteriorated clinical scores, colon shortening, and histological damage. EGCs dysfunction, characterized by decreased expression of the glial fibrillary acidic protein (GFAP), C3, and S100A10, was observed in the colon of WT and KO mice, especially in KO mice. RhoA/ROCK pathway was significantly upregulated in colon of colitis mice, which was more evident in KO mice. Pretreatment with NaHS, an exogenous H2S donor, significantly ameliorated mucosal injury and inhibited the expression of proinflammatory factors. Furthermore, we found that NaHS promoted the transformation of EGCs from “A1” to “A2” type, with decreased expression of C3 and increased expression of S100A10. These findings suggest that CSE/H2S protects mice from colon inflammation, which may be associated with preserving EGCs function by promoting EGCs transformation and inhibiting the RhoA/ROCK pathway.

Hydrogen Sulfide Ameliorated High Choline-Induced Cardiac Dysfunction by Inhibiting cGAS-STING-NLRP3 Inflammasome Pathway.

Although it is an essential nutrient, high choline intake directly or indirectly via its metabolite is associated with increased risk of cardiovascular disease, the mechanism of which remains to be elucidated. The present study was performed to investigate whether hydrogen sulfide (H2S) was involved in high choline-induced cardiac dysfunction and explore the potential mechanisms. We found that ejection fraction (EF) and fractional shortening (FS), the indicators of cardiac function measured by echocardiography, were significantly decreased in mice fed a diet containing 1.3% choline for 4 months as compared to the control, while applying 3,3-dimethyl-1-butanol (DMB) to suppress trimethylamine N-oxide (TMAO, a metabolite of choline) generation ameliorated the cardiac function. Subsequently, we found that feeding choline or TMAO significantly increased the protein levels of cyclic GMP-AMP (cGAMP) synthase (cGAS), stimulator of interferon genes (STING), NOD-like receptor protein 3 (NLRP3), caspase-1, and interleukin-1β (IL-1β) as compared to the control, which indicated the activation of cGAS-STING-NLRP3 inflammasome axis. Moreover, the protein expression of cystathionine γ-lyase (CSE), the main enzyme for H2S production in the cardiovascular system, was significantly increased after dietary supplementation with choline, but the plasma H2S levels were significantly decreased. To observe the effect of endogenous H2S, CSE knockout (KO) mice were used, and we found that the EF, FS, and plasma H2S levels in WT mice were significantly decreased after dietary supplementation with choline, while there was no difference between CSE KO + control and CSE KO + choline group. To observe the effect of exogenous H2S, mice were intraperitoneally injected with sodium hydrosulfide (NaHS, a H2S donor) for 4 months, and we found that NaHS improved the cardiac function and reduced the protein levels of cGAS, STING, NLRP3, caspase-1, and IL-1β in mice receiving dietary choline. In conclusion, our studies revealed that high choline diet decreased plasma H2S levels and induced cardiac dysfunction via cGAS-STING-NLRP3 inflammasome axis while H2S treatment could restore the cardiac function by inhibiting cGAS-STING-NLRP3 inflammasome axis.

Deficiency of cystathionine gamma-lyase promotes aortic elastolysis and medial degeneration in aged mice

Enzymatic degradation of elastin by matrix metalloproteinases (MMPs) leads to the permanent dilation of aortic wall and constitutes the most prominent characters of aortic aneurysm and aging-related medial degeneration. Hydrogen sulfide (H2S) as a gasotransmitter exhibits a wide variety of cardio-protective functions through its anti-inflammatory and anti-oxidative actions. Cystathionine gamma-lyase (CSE) is a main H2S-generating enzyme in cardiovascular system. The regulatory roles of CSE/H2S system on elastin homeostasis and blood vessel degeneration have not yet been explored. Here we found that aged CSE knockout mice had severe aortic dilation and elastic degradation in abdominal aorta and were more sensitive to angiotensin II-induced aortic elastolysis and medial degeneration. Administration of NaHS would protect the mice from angiotensin II-induced inflammation, gelatinolytic activity, elastin fragmentation, and aortic dilation. In addition, human aortic aneurysm samples had higher inflammatory infiltration and lower expression of CSE. In cultured smooth muscle cells (SMCs), TNFα-induced MMP2/9 hyperactivity and elastolysis could be attenuated by exogenously applied NaHS or CSE overexpression while further deteriorated by complete knockout of CSE. It was further found that H2S inhibited MMP2 transcription by posttranslational modification of Sp1 via S-sulfhydration. H2S also directly suppressed MMP hyperactivity by S-sulfhydrating the cysteine switch motif. Taken together, this study revealed the involvement of CSE/H2S system in the pathogenesis of aortic elastolysis and medial degeneration by maintaining the inactive form of MMPs, suggesting that CSE/H2S system can be a target for the prevention of age-related medial degeneration and treatment of aortic aneurysm.

Hepatocellular cystathionine γ lyase/hydrogen sulfide attenuates nonalcoholic fatty liver disease by activating farnesoid X receptor.

Hydrogen sulfide (H2 S) plays a protective role in NAFLD. However, whether cystathionine γ lyase (CSE), a dominant H2 S generating enzyme in hepatocytes, has a role in the pathogenesis of NAFLD is currently unclear. We showed that CSE protein expression is dramatically downregulated, especially in fibrotic areas, in livers from patients with NAFLD. In high-fat diet (HFD)-induced NAFLD mice or an oleic acid-induced hepatocyte model, the CSE/H2 S pathway is also downregulated. To illustrate a regulatory role for CSE in NAFLD, we generated a hepatocyte-specific CSE knockout mouse (CSELKO ). Feeding an HFD to CSELKO mice, they showed more hepatic lipid deposition with increased activity of the fatty acid de novo synthesis pathway, increased hepatic insulin resistance, and higher hepatic gluconeogenic ability compared to CSELoxp control mice. By contrast, H2 S donor treatment attenuated these phenotypes. Furthermore, the protection conferred by H2 S was blocked by farnesoid X receptor (FXR) knockdown. Consistently, serum deoxycholic acid and lithocholic acid (FXR antagonists) were increased, and tauro-β-muricholic acid (FXR activation elevated) was reduced in CSELKO . CSE/H2 S promoted a post-translation modification (sulfhydration) of FXR at Cys138/141 sites, thereby enhancing its activity to modulate expression of target genes related to lipid and glucose metabolism, inflammation, and fibrosis. Sulfhydration proteomics in patients' livers supported the CSE/H2 S modulation noted in the CSELKO mice. FXR sulfhydration is a post-translational modification affected by hepatic endogenous CSE/H2 S that may promote FXR activity and attenuate NAFLD. Hepatic CSE deficiency promotes development of nonalcoholic steatohepatitis. The interaction between H2 S and FXR may be amenable to therapeutic drug treatment in NAFLD.

Abstract 12371: Genetic Deletion of Cystathionine Gamma Lyase in Endothelial Cells Exacerbates Cardiovascular Dysfunction in Heart Failure With Preserved Ejection Fraction

Background: Endothelial dysfunction has been suggested as a central pathophysiological mechanism in heart failure with preserved ejection fraction (HFpEF). We reported that Cystathionine Gamma Lyase (CSE), one of the 3 hydrogen sulfide producing enzymes, expressed in endothelial cells is critical for maintaining normal endothelial function and protecting against myocardial ischemia/reperfusion injury. However, the role of endothelial CSE in HFpEF remains unknown. Method: Endothelial specific CSE knockout mice (EC-CSE KO, n=15) and CSE flox/flox control animals (n=6) were fed a high-fat diet (60% kcal from fat) and administered L-NAME (0.5 g/L in drinking water) for 18 weeks to induce HFpEF. Body weight and cardiac function (echocardiography and doppler imaging) were monitored every 4 weeks. Invasive left ventricular hemodynamic procedures were performed and vascular reactivity was measured in isolated thoracic aorta at 18 weeks. Results: Baseline body weight, ejection fraction (EF), the ratios of E/A and E/E’ were not different between EC-CSE KO and controls. At 12 & 16 weeks following HFD and L-NAME, E/E’ ratios were significantly higher in EC-CSE KO compared to controls (p < 0.05), with normal EF in both groups. At 18 weeks of HFpEF, a 33.3% of mortality was observed in the EC-CSE KO group while no mortality was shown in control. In addition, left ventricular end-diastolic pressure (LVEDP) was higher (p < 0.01) and aortic vasorelaxation maximal response to acetylcholine was lower (p < 0.01) in EC-CSE KO compared to controls. Conclusion: Genetic deletion of CSE in vascular endothelial cells exacerbates cardiac diastolic dysfunction, promotes vascular dysfunction, and increases mortality in HFpEF. Studies are currently underway to define the protective role of endothelial CSE in HFpEF.

Abstract 13455: Endothelium-Derived Hydrogen Sulfide Modulates Endothelial-Mesenchymal Transition in Heart Failure

Introduction: Hydrogen sulfide (H 2 S) is a critical endogenous signaling molecule that exerts protective effects in the setting of heart failure (HF). Cystathionine γ-lyase (CSE), one of three H 2 S producing enzyme, is predominantly localized in the vascular endothelium. The interaction between endothelial cell derived H 2 S and endothelial-mesenchymal transition (EndoMT), an important pathological process contributing to the formation of fibrosis, has yet to be investigated. Methods: Endothelial cell specific CSE knockout (EC-CSE KO) and overexpressing (EC-CSE Tg) mice were subjected to transverse aortic constriction (TAC) to induce HF with reduced ejection fraction (HFrEF). Cardiac function, vascular reactivity, and treadmill exercise capacity following TAC were measured to determine the severity of HF. Histological analysis was performed to investigate changes in cardiac fibrosis. Vimentin and Von Willebrand factor co-immunostaining were utilized to investigate EndoMT activation. Results: EC-CSE KO mice exhibited reduced circulating H 2 S level, which was accompanied by increased EndoMT in the myocardium, resulting in increased perivascular fibrosis in the heart, impaired cardiac and vascular function, and worsened exercise performance. In contrast, genetic overexpression of CSE in endothelial cells elevated circulating and myocardial H 2 S, led to decreased EndoMT and subsequent fibrosis, preserved cardiac and endothelial function, and improved exercise capacity. Conclusions: Our data demonstrate that endothelial CSE modulates endothelial-mesenchymal transition to ameliorate the severity of pressure overload - induced heart failure. These data further suggest that endothelium-derived H 2 S is a potential therapeutic for the treatment of heart failure with reduced ejection fraction.

CSE-Derived H2S Inhibits Reactive Astrocytes Proliferation and Promotes Neural Functional Recovery after Cerebral Ischemia/Reperfusion Injury in Mice Via Inhibition of RhoA/ROCK2 Pathway.

The effect of cystathionine-γ-lyase (CSE)-derived hydrogen sulfide (H2S) on the reactive proliferation of astrocytes and neural functional recovery over 30 d after acute cerebral ischemia and reperfusion (I/R) was determined by applying wild-type (WT) and CSE knockout (KO) mice. The changes of glial fibrillary acidic protein (GFAP) expression in hippocampal tissues was tested. Besides, we assessed the changes of mice spatial learning memory ability, neuronal damage, RhoA, Rho kinase 2 (ROCK2), and myelin basic protein (MBP) expressions in hippocampal tissues. The results revealed that cerebral I/R resulted in obvious increase of GFAP expression in hippocampal tissues. Besides, we found the neuronal damage, learning, and memory deficits of mice induced by cerebral I/R as well as revealed the upregulation of RhoA and ROCK2 expressions and reduced MBP expression in hipppcampal tissues of mice following cerebral I/R. Not surprisingly, the GFAP expression and cerebral injury as well as the upregulation of the RhoA/ROCK2 pathway were more remarkable in CSE KO mice, compared with those in WT mice over 30 d following acute cerebral I/R, which could be blocked by NaHS treatment, a donor of exogenous H2S. In addition, the ROCK inhibitor Fasudil also inhibited the reactive proliferation of astrocytes and ameliorated the recovery of neuronal function over 30 d after cerebral I/R. For the purpose of further confirmation of the role of H2S on the astrocytes proliferation following cerebral I/R, the immunofluorescence double staining: bromodeoxyuridine (BrdU) and GFAP was evaluated. There was a marked upregulation of BrdU-labeled cells coexpressed with GFAP in hippocampal tissues at 30 d after acute cerebral I/R; however, the increment of astrocytes proliferation could be ameliorated by both NaHS and Fasudil. These findings indicated that CSE-derived H2S could inhibit the reactive proliferation of astrocytes and promote the recovery of mice neural functional deficits induced by a cerebral I/R injury via inhibition of the RhoA/ROCK2 signal pathway.

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

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

Hydrogen Sulfide Is a Regulator of Hemoglobin Oxygen-Carrying Capacity via Controlling 2,3-BPG Production in Erythrocytes.

Hydrogen sulfide (H2S) is naturally synthesized in a wide range of mammalian tissues. Whether H2S is involved in the regulation of erythrocyte functions remains unknown. Using mice with a genetic deficiency in a H2S natural synthesis enzyme cystathionine-γ-lyase (CSE) and high-throughput metabolomic profiling, we found that levels of erythrocyte 2,3-bisphosphoglycerate (2,3-BPG), an erythroid-specific metabolite negatively regulating hemoglobin- (Hb-) oxygen (O2) binding affinity, were increased in CSE knockout (Cse−/−) mice under normoxia. Consistently, the 50% oxygen saturation (P50) value was increased in erythrocytes of Cse−/− mice. These effects were reversed by treatment with H2S donor GYY4137. In the models of cultured mouse and human erythrocytes, we found that H2S directly acts on erythrocytes to decrease 2,3-BPG production, thereby enhancing Hb-O2 binding affinity. Mouse genetic studies showed that H2S produced by peripheral tissues has a tonic inhibitory effect on 2,3-BPG production and consequently maintains Hb-O2 binding affinity in erythrocytes. We further revealed that H2S promotes Hb release from the membrane to the cytosol and consequently enhances bisphosphoglycerate mutase (BPGM) anchoring to the membrane. These processes might be associated with S-sulfhydration of Hb. Moreover, hypoxia decreased the circulatory H2S level and increased the erythrocyte 2,3-BPG content in mice, which could be reversed by GYY4137 treatment. Altogether, our study revealed a novel signaling pathway that regulates oxygen-carrying capacity in erythrocytes and highlights a previously unrecognized role of H2S in erythrocyte 2,3-BPG production.

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

In vascular endothelial cells, cysteine metabolism by the cystathionine γ lyase (CSE), generates hydrogen sulfide-related sulfane sulfur compounds (H2Sn), that exert their biological actions via cysteine S-sulfhydration of target proteins. This study set out to map the "S-sulfhydrome" (ie, the spectrum of proteins targeted by H2Sn) in human endothelial cells. Liquid chromatography with tandem mass spectrometry was used to identify S-sulfhydrated cysteines in endothelial cell proteins and β3 integrin intraprotein disulfide bond rearrangement. Functional studies included endothelial cell adhesion, shear stress-induced cell alignment, blood pressure measurements, and flow-induced vasodilatation in endothelial cell-specific CSE knockout mice and in a small collective of patients with endothelial dysfunction. Three paired sample sets were compared: (1) native human endothelial cells isolated from plaque-free mesenteric arteries (CSE activity high) and plaque-containing carotid arteries (CSE activity low); (2) cultured human endothelial cells kept under static conditions or exposed to fluid shear stress to decrease CSE expression; and (3) cultured endothelial cells exposed to shear stress to decrease CSE expression and treated with solvent or the slow-releasing H2Sn donor, SG1002. The endothelial cell "S-sulfhydrome" consisted of 3446 individual cysteine residues in 1591 proteins. The most altered family of proteins were the integrins and focusing on β3 integrin in detail we found that S-sulfhydration affected intraprotein disulfide bond formation and was required for the maintenance of an extended-open conformation of the β leg. β3 integrin S-sulfhydration was required for endothelial cell mechanotransduction in vitro as well as flow-induced dilatation in murine mesenteric arteries. In cultured cells, the loss of S-sulfhydration impaired interactions between β3 integrin and Gα13 (guanine nucleotide-binding protein subunit α 13), resulting in the constitutive activation of RhoA (ras homolog family member A) and impaired flow-induced endothelial cell realignment. In humans with atherosclerosis, endothelial function correlated with low H2Sn generation, impaired flow-induced dilatation, and failure to detect β3 integrin S-sulfhydration, all of which were rescued after the administration of an H2Sn supplement. Vascular disease is associated with marked changes in the S-sulfhydration of endothelial cell proteins involved in mediating responses to flow. Short-term H2Sn supplementation improved vascular reactivity in humans highlighting the potential of interfering with this pathway to treat vascular disease.

Decreased bioavailability of hydrogen sulfide links vascular endothelium and atrial remodeling in atrial fibrillation

Oxidative stress drives the pathogenesis of atrial fibrillation (AF), the most common arrhythmia. In the cardiovascular system, cystathionine γ-lyase (CSE) serves as the primary enzyme producing hydrogen sulfide (H2S), a mammalian gasotransmitter that reduces oxidative stress. Using a case control study design in patients with and without AF and a mouse model of CSE knockout (CSE-KO), we evaluated the role of H2S in the etiology of AF. Patients with AF (n = 51) had significantly reduced plasma acid labile sulfide levels compared to patients without AF (n = 65). In addition, patients with persistent AF (n = 25) showed lower plasma free sulfide levels compared to patients with paroxysmal AF (n = 26). Consistent with an important role for H2S in AF, CSE-KO mice had decreased atrial sulfide levels, increased atrial superoxide levels, and enhanced propensity for induced persistent AF compared to wild type (WT) mice. Rescuing H2S signaling in CSE-KO mice by Diallyl trisulfide (DATS) supplementation or reconstitution with endothelial cell specific CSE over-expression significantly reduced atrial superoxide, increased sulfide levels, and lowered AF inducibility. Lastly, low H2S levels in CSE KO mice was associated with atrial electrical remodeling including longer effective refractory periods, slower conduction velocity, increased myocyte calcium sparks, and increased myocyte action potential duration that were reversed by DATS supplementation or endothelial CSE overexpression. Our findings demonstrate an important role of CSE and H2S bioavailability in regulating electrical remodeling and susceptibility to AF.

Activation of the CaR-CSE/H2S pathway confers cardioprotection against ischemia-reperfusion injury

Ischemia-reperfusion (I/R) injury is a multifactorial process triggered when an organ is subjected to transiently reduced blood supply. The result is a cascade of pathological complications and organ damage due to the production of reactive oxygen species following reperfusion. The present study aims to evaluate the role of activated calcium-sensing receptor (CaR)-cystathionine γ-lyase (CSE)/hydrogen sulfide (H2S) pathway in I/R injury. Firstly, an I/R rat model with CSE knockout was constructed. Transthoracic echocardiography, TTC and HE staining were performed to determine the cardiac function of rats following I/R Injury, followed by TUNEL staining observation on apoptosis. Besides, with the attempt to better elucidate how CaR-CSE/H2S affects I/R, in-vitro culture of human coronary artery endothelial cells (HCAECs) was conducted with gadolinium chloride (GdCl3, a CaR agonist), H2O2, siRNA against CSE (siCSE), or W7 (a CaM inhibitor). The interaction between CSE and CaM was subsequently detected. Plasma oxidative stress indexes, H2S and CSE, and apoptosis-related proteins were all analyzed following cell apoptosis. We found that H2S elevation led to the improvement whereas CSE knockdown decreased cardiac function in rats with I/R injury. Moreover, oxidative stress injury in I/R rats with CSE knockout was aggravated, while the increased expression of H2S and CSE in the aortic tissues resulted in alleviated the oxidative stress injury. Moreover, increased H2S and CSE levels were found to inhibit cell apoptotic ability in the aortic tissues after I/R injury, thus attenuating oxidative stress injury, accompanied by inhibited expression of apoptosis-related proteins. In HCAECs following oxidative stress treatment, siCSE and CaM inhibitor were observed to reverse the protection of CaR agonist. Coimmunoprecipitation assay revealed the interaction between CSE and CaM. Taken together, all above-mentioned data provides evidence that activation of the CaR-CSE/H2S pathway may confer a potent protective effect in cardiac I/R injury.

Endothelial Cell Cystathionine γ-Lyase Expression Level Modulates Exercise Capacity, Vascular Function, and Myocardial Ischemia Reperfusion Injury.

BackgroundHydrogen sulfide (H2S) is an important endogenous physiological signaling molecule and exerts protective properties in the cardiovascular system. Cystathionine γ‐lyase (CSE), 1 of 3 H2S producing enzyme, is predominantly localized in the vascular endothelium. However, the regulation of CSE in vascular endothelium remains incompletely understood.Methods and ResultsWe generated inducible endothelial cell‐specific CSE overexpressed transgenic mice (EC‐CSE Tg) and endothelial cell‐specific CSE knockout mice (EC‐CSE KO), and investigated vascular function in isolated thoracic aorta, treadmill exercise capacity, and myocardial injury following ischemia‐reperfusion in these mice. Overexpression of CSE in endothelial cells resulted in increased circulating and myocardial H2S and NO, augmented endothelial‐dependent vasorelaxation response in thoracic aorta, improved exercise capacity, and reduced myocardial‐reperfusion injury. In contrast, genetic deletion of CSE in endothelial cells led to decreased circulating H2S and cardiac NO production, impaired endothelial dependent vasorelaxation response and reduced exercise capacity. However, myocardial‐reperfusion injury was not affected by genetic deletion of endothelial cell CSE.ConclusionsCSE‐derived H2S production in endothelial cells is critical in maintaining endothelial function, exercise capacity, and protecting against myocardial ischemia/reperfusion injury. Our data suggest that the endothelial NO synthase—NO pathway is likely involved in the beneficial effects of overexpression of CSE in the endothelium.

CD4 + T-Cell Endogenous Cystathionine γ Lyase–Hydrogen Sulfide Attenuates Hypertension by Sulfhydrating Liver Kinase B1 to Promote T Regulatory Cell Differentiation and Proliferation

Hydrogen sulfide (H2S) has antihypertension and anti-inflammatory effects, and its endogenous-generation key enzyme cystathionine γ lyase (CSE) is expressed in CD4+ T cells. However, the role of CD4+ T-cell endogenous CSE/H2S in the development of hypertension is unclear. Peripheral blood lymphocytes were isolated from hypertensive patients or spontaneously hypertensive rats, then H2S production and expression of its generation enzymes, cystathionine β synthase and CSE, were measured to determine the major H2S generation system changes in hypertension. Mice with CSE-specific knockout in T cells (conditional knockout, by CD4cre mice hybridization) and CD4 null mice were generated for investigating the pathophysiological relevance of the CSE/H2S system. In lymphocytes, H2S from CSE, but not cystathionine β synthase, responded to blood pressure changes, supported by lymphocyte CSE protein changes and a negative correlation between H2S production with systolic blood pressure and diastolic blood pressure, but positive correlation with the serum level of interleukin 10 (an anti-inflammatory cytokine). Deletion of CSE in T cells elevated BP (5-8 mm Hg) under the physiological condition and exacerbated angiotensin II-induced hypertension. In keeping with hypertension, mesenteric artery dilation impaired association with arterial inflammation, an effect attributed to reduced immunoinhibitory T regulatory cell (Treg) numbers in the blood and kidney, thus causing excess CD4+ and CD8+ T cell infiltration in perivascular adipose tissues and kidney. CSE knockout CD4+ T cell transfer into CD4 null mice, also showed the similar phenotypes' confirming the role of endogenous CSE/H2S action. Adoptive transfer of Tregs (to conditional knockout mice) reversed hypertension, vascular relaxation impairment, and immunocyte infiltration, which confirmed that conditional knockout-induced hypertension was attributable, in part, to the reduced Treg numbers. Mechanistically, endogenous CSE/H2S promoted Treg differentiation and proliferation by activating AMP-activated protein kinase. In part, it depended on activation of its upstream kinase, liver kinase B1, by sulfhydration to facilitate its substrate binding and phosphorylation. The constitutive sulfhydration of liver kinase B1 by CSE-derived H2S activates its target kinase, AMP-activated protein kinase, and promotes Treg differentiation and proliferation, which attenuates the vascular and renal immune-inflammation, thereby preventing hypertension.

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

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