Slow-releasing H2S Donor GYY4137 Research Articles

THE FUNCTION OF THE CYSTATHIONINE-GAMMA-LYASE/HYDROGEN SULFIDE AXIS IN THE PATHOGENESIS OF ULCERATIVE COLITIS

Abstract INTRODUCTION Current combination therapies in ulcerative colitis (UC) target different inflammatory cascades, including tumor necrosis factor-alpha (TNF-alpha) signaling. However, most patients do not achieve long-term remission, suggesting that the molecular pathophysiology of UC is still poorly understood. Endogenously produced hydrogen sulfide (H2S) has emerged as a critical physiological mediator of gastrointestinal homeostasis, limiting mucosal inflammation and promoting tissue healing in response to epithelial injury. Inhibition of cystathionine-gamma-lyase (CSE)-dependent H2S production in animal models of UC has been shown to exacerbate colitis and delay tissue repair. Our research group has strong evidence revealing that the CSE/H2S axis regulates TNF-alpha signaling in colonic epithelial cells. Therefore, we hypothesize that decreased CSE/H2S pathway activity in colonic mucosa cells leads to dysregulation of TNF-alpha signaling and uncontrolled inflammatory/immune responses with aberrant wound repair mechanisms contributing to the pathogenesis of UC. RESULTS We found a significant decrease in CSE mRNA and protein expression in colonic biopsies from patients with UC. Using a preclinical animal model of epithelium injury-induced chronic colitis (DSS), we confirmed a sustained downregulation of CSE, as well as upregulation of TNF-alpha and tumor necrosis receptor 2 expressions in colonic mucosa samples. Treatment with the slow-releasing H2S-donor GYY4137 significantly reduced the severity of DSS-induced inflammation and improved mucosal integrity and restitution responses. In epithelial cell and colonoid cultures, the inhibition of CSE-dependent H2S synthesis decreased the activation of extracellular-signal-regulated-kinase-1/2 (ERK1/2), increased IL-6 and TNF-alpha mRNA levels and suppressed basic and TNF-alpha–mediated colonocyte migration. CONCLUSION These studies demonstrate that CSE regulates mucosal TNF-alpha signaling, pro- and anti-inflammatory responses as well as colonic epithelial repair. Adding the H2S donor GYY4137 reduces inflammation, suggesting the restoration of normal TNF-alpha signaling function. Acknowledgments: KM is supported by the DoD Career Development Award (W81XWH2010641) and ACS Research Scholar Grant (RSG–21–027–01–CSM).

Vasoactive Effects of Chronic Treatment with Fructose and Slow-Releasing H2S Donor GYY-4137 in Spontaneously Hypertensive Rats: The Role of Nitroso and Sulfide Signalization.

Increased fructose consumption induces metabolic-syndrome-like pathologies and modulates vasoactivity and the participation of nitric oxide (NO) and hydrogen sulfide (H2S). We investigated whether a slow-releasing H2S donor, GYY-4137, could exert beneficial activity in these conditions. We examined the effect of eight weeks of fructose intake on the blood pressure, biometric parameters, vasoactive responses, and NO and H2S pathways in fructose-fed spontaneously hypertensive rats with or without three weeks of GYY-4137 i.p. application. GYY-4137 reduced triacylglycerol levels and blood pressure, but not adiposity, and all were increased by fructose intake. Fructose intake generally enhanced endothelium-dependent vasorelaxation, decreased adrenergic contraction, and increased protein expression of interleukin-6 (IL-6), tumor necrosis factor alpha (TNFα), and concentration of conjugated dienes in the left ventricle (LV). Although GYY-4137 administration did not affect vasorelaxant responses, it restored disturbed contractility, LV oxidative damage and decreased protein expression of TNFα in fructose-fed rats. While the participation of endogenous H2S in vasoactive responses was not affected by fructose treatment, the expression of H2S-producing enzyme cystathionine β-synthase in the LV was increased, and the stimulation of the NO signaling pathway improved endothelial function in the mesenteric artery. On the other hand, chronic treatment with GYY-4137 increased the expression of H2S-producing enzyme cystathionine γ-lyase in the LV and stimulated the beneficial pro-relaxant and anti-contractile activity of endogenous H2S in thoracic aorta. Our results suggest that sulfide and nitroso signaling pathways could trigger compensatory vasoactive responses in hypertensive rats with metabolic disorder. A slow H2S-releasing donor could partially amend metabolic-related changes and trigger beneficial activity of endogenous H2S.

Hydrogen sulfide exacerbated periodontal inflammation and induced autophagy in experimental periodontitis

Hydrogen sulfide (H2S), the metabolite produced by gram-negative bacteria, is present in deep periodontal pockets of periodontitis patients at high concentrations. The harsh conditions in the diseased periodontium may stimulate a local autophagy response. However, how H2S participates in pathogenesis and whether H2S induces autophagy in periodontitis remain partially unknown. In this article, we determined the role of the slow-releasing H2S donor GYY4137 in experimental periodontitis and its possible regulation in autophagy involved. We found that GYY4137 dose-dependently decreased cell viability and increased the level of proinflammatory cytokines in LPS-stimulated human periodontal ligament cells (HPDLCs). Topically applied GYY4137 also exacerbated periodontal inflammation and alveolar bone loss in ligature-induced rats. Moreover, GYY4137 activated autophagy by upregulating the expression levels of the autophagy-related proteins LC3 and Beclin-1 and downregulating P62 in LPS-treated HPDLCs and inflamed periodontal tissues. Blocking autophagy with 3-methyladenine resulted in further increased expression of proinflammatory cytokines in LPS- and GYY4137-induced HPDLCs. Our results indicate that GYY4137 exerted proinflammatory effects and promoted autophagy in periodontitis, and the induced autophagy may function as a cytoprotective mechanism to prevent excessive inflammation.

H2S as a Therapeutic Adjuvant Against COVID-19: Why and How?

H2S as a Therapeutic Adjuvant Against COVID-19: Why and How?

Hydrogen Sulfide: A Potential Therapeutic Target in the Development of Diabetic Retinopathy.

PurposeHyperglycemia damages the retinal mitochondria, and the mitochondrial damage plays a central role in the development of diabetic retinopathy. Patients with diabetes also have higher homocysteine levels, and abnormalities in homocysteine metabolism result in decreased levels of hydrogen sulfide (H2S), an endogenous gasotransmitter signaling molecule with antioxidant properties. This study aimed to investigate the role of H2S in the development of diabetic retinopathy.MethodsStreptozotocin-induced diabetic mice were administered a slow releasing H2S donor GYY4137 for 6 months. The retina was used to measure H2S levels, and their retinal vasculature was analyzed for the histopathology characteristic of diabetic retinopathy and oxidative stress, mitochondrial damaging matrix metalloproteinase-9 (MMP-9), and mitochondrial integrity. These parameters were also measured in the isolated retinal endothelial cells incubated in high glucose medium containing GYY4137.ResultsAdministration of GYY4137 to diabetic mice ameliorated decrease in H2S and prevented the development of histopathology, characteristic of diabetic retinopathy. Diabetes-induced increase in oxidative stress, MMP-9 activation, and mitochondrial damage were also attenuated in mice receiving GYY4137. Results from isolated retinal endothelial cells confirmed the results obtained from diabetic mice.ConclusionsThus, supplementation of H2S donor prevents the development of diabetic retinopathy by ameliorating increase in oxidative stress and preserving the mitochondrial integrity. H2S donors may provide a novel therapeutic strategy to inhibit the development of diabetic retinopathy.

H2S promotes developmental brain angiogenesis via the NOS/NO pathway in zebrafish

BackgroundHydrogen sulphide (H2S) is considered as the third member of the gasotransmitter family, along with nitric oxide (NO) and carbon monoxide. H2S has been reported to induce angiogenesis by promoting...

Chronic Treatment With Hydrogen Sulfide Donor GYY4137 Mitigates Microglial and Astrocyte Activation in the Spinal Cord of Streptozotocin-Induced Diabetic Rats.

Long-term diabetic patients suffer immensely from diabetic neuropathy. This study was designed to investigate the effects of hydrogen sulfide (H2S) on peripheral neuropathy, activation of microglia, astrocytes, and the cascade secretion of proinflammatory cytokines in the streptozotocin (STZ)-induced peripheral diabetic neuropathy rat model. STZ-induced diabetic rats were treated with the water-soluble, slow-releasing H2S donor GYY4137 (50 mg/kg; i.p.) daily for 4 weeks. Antiallodynic/antihyperalgesic activities were evaluated using different tests and histopathological changes and the expression of proinflammatory cytokines in the spinal cord were examined. GYY4137 treatment produced neuroprotective effects in the spinal cord of diabetic animals and modulated their sensory deficits. The treatment decreased allodynia (p < 0.05) and mechanical hyperalgesia (p < 0.01) and restored thermal hyperalgesia (p < 0.001) compared with diabetic rats. The treatment decreased the microglial response and increased astrocyte counts in spinal cord gray and white matter compared with untreated diabetic rats. Proinflammatory cytokines were reduced in the treated group compared with diabetic rats. These results suggest that H2S has a potentially ameliorative effect on the neuropathic pain through the control of astrocyte activation and microglia-mediated inflammation, which may be considered as a possible treatment of peripheral nerve hypersensitivity in diabetic patients.

Upregulation of Tolerogenic Pathways by the Hydrogen Sulfide Donor GYY4137 and Impaired Expression of H2S-Producing Enzymes in Multiple Sclerosis.

The aim of this study was to examine the in vitro effects of the slow-releasing H2S donor GYY4137 on the immune cells involved in the pathogenesis of the central nervous system (CNS) autoimmune disease, multiple sclerosis (MS). GYY4137 specifically potentiated TGF-β expression and production in dendritic cells and significantly reduced IFN-γ and IL-17 production in the lymph node and spinal cord T cells obtained from mice immunized with CNS antigens. Both the proportion of FoxP3+ regulatory CD4+ T cells in the lymph node cells, and the percentage of IL-17+ CD4+ T cells in the spinal cord cells were reduced upon culturing with GYY4137. Interestingly, the peripheral blood mononuclear cells obtained from the MS patients had a lower expression of the H2S-producing enzyme, 3-mercaptopyruvate-sulfurtransferase (MPST), in comparison to those obtained from healthy donors. A significant inverse correlation between the expression of MPST and several pro-inflammatory factors was also observed. Further studies on the relevance of the observed results for the pathogenesis and therapy of MS are warranted.

H2S donor GYY4137 ameliorates paclitaxel-induced neuropathic pain in mice

Paclitaxel-induced neuropathic pain (PINP) is a dose-limiting side effect that largely affects the patient’s quality of life and may limit the use of the drug as a chemotherapeutic agent for treating metastatic breast cancer and other solid tumors. Recently, a putative role for the gaseous mediator hydrogen sulfide (H2S) in nociception modulation has been suggested. The aim of the present study was to investigate the potential efficacy of the slow release H2S donor GYY4137 to alleviate and prevent PINP. Female BALB/c mice that were intraperitoneally (i.p.) injected with paclitaxel (2 mg/kg) for 5 consecutive days developed thermal hyperalgesia, cold and mechanical allodynia and had reduced of H2S, generation in the spinal cord and paw skin. Treatment of mice with established thermal hyperalgesia with GYY4137 or the analgesic positive control drug gabapentin produced antihyperalgesic activities. The antihyperalgesic activity of GYY4137 was antagonized by the ATP sensitive potassium channels (KATP channels) blocker glibenclamide. Co-treatment with GYY4137 and paclitaxel prevented the paclitaxel-induced decrease in H2S, generation as well as the paclitaxel-induced thermal hyperalgesia, cold allodynia and mechanical allodynia. GYY4137 enhanced paclitaxel's anti-proliferative effects against the breast cancer cell line MCF-7. The present results suggest that GYY4137 alleviates paclitaxel-induced thermal hyperalgesia, via KATP channels. GYY4137 prevents PINP possibly by blocking the paclitaxel-induced reduction in the generation of H2S, in the tissues, while enhancing the anti-cancer activity of paclitaxel, and therefore warrants further research as a candidate for prevention of PINP in clinical settings.

H2S counteracts proinflammatory effects of LPS through modulation of multiple pathways in human cells.

Hydrogen sulfide donors reduce inflammatory signaling in vitro and in vivo. The biological effect mediated by H2S donors depends on the kinetics of the gas release from the donor molecule. However, the molecular mechanisms of H2S-induced immunomodulation were poorly addressed. Here, we studied the effect of two different hydrogen sulfide (H2S)-producing agents on the generation of the LPS-induced inflammatory mediators. Importantly, we investigated the transcriptomic changes that take place in human cells after the LPS challenge, combined with the pretreatment with a slow-releasing H2S donor-GYY4137. We investigated the effects of GYY4137 and sodium hydrosulfide on the release of proinflammatory molecules such as ROS, NO and TNF-α from LPS-treated human SH-SY5Y neuroblastoma and the THP-1 promonocytic cell lines. Transcriptomic and RT-qPCR studies using THP-1 cells were performed to monitor the effects of the GYY4137 on multiple signaling pathways, including various immune-related and proinflammatory genes after combined action of LPS and GYY4137. The GYY4137 and sodium hydrosulfide differed in the ability to reduce the production of the LPS-evoked proinflammatory mediators. The pre-treatment with GYY4137 resulted in a drastic down-regulation of many TNF-α effectors that are induced by LPS treatment in THP-1 cells. Furthermore, GYY4137 pretreatment of LPS-exposed cells ameliorates the LPS-mediated induction of multiple pro-inflammatory genes and decreases expression of immunoproteasome genes. Besides, in these experiments we detected the up-regulation of several important pathways that are inhibited by LPS. Based on the obtained results we believe that our transcriptomic analysis significantly contributes to the understanding of the molecular mechanisms of anti-inflammatory and cytoprotective activity of hydrogen sulfide donors, and highlights their potential against LPS challenges and other forms of inflammation.

The H2S-Releasing Naproxen Derivative ATB-346 and the Slow-Release H2S Donor GYY4137 Reduce Intestinal Inflammation and Restore Transit in Postoperative Ileus.

Objective: Intestinal inflammation triggers postoperative ileus (POI), commonly seen after abdominal surgery and characterized by impaired gastrointestinal transit; when prolonged, this leads to increased morbidity. Hydrogen sulfide (H2S) is recognized as an important mediator of many (patho)physiological processes, including inflammation, and is now investigated for anti-inflammatory application. Therefore, the aim of this study was to investigate the effect of the H2S-releasing naproxen derivative ATB-346, developed to reduce gastrointestinal injury by naproxen, and the slow-release H2S donor GYY4137 on intestinal inflammation and delayed gastrointestinal transit in murine POI.Methods: C57Bl6J mice were fasted for 6 h, anesthetized and after laparotomy, POI was induced by compressing the small intestine with two cotton applicators for 5 min (intestinal manipulation; IM). GYY4137 (50 mg/kg, intraperitoneally), ATB-346 (16 mg/kg, intragastrically) or naproxen (10 mg/kg, intragastrically) were administered 1 h before IM. At 24 h postoperatively, gastrointestinal transit was assessed via fluorescent imaging, and mucosa-free muscularis segments were prepared for later analysis. Inflammatory parameters and activity of inducible nitric oxide synthase (iNOS) and cyclo-oxygenase (COX)-2 were measured. Histological examination of whole tissue sections was done on hematoxylin-eosin stained slides.Results: Pre-treatment with GYY4137 (geometric center; GC: 7.6 ± 0.5) and ATB-346 (GC: 8.4 ± 0.3) prevented the delayed transit induced by IM (GC: 3.6 ± 0.5 vs. 9.0 ± 0.4 in non-operated controls) while naproxen only partially did (GC: 5.9 ± 0.5; n = 8 for all groups). GYY4137 and ATB-346 significantly reduced the IM-induced increase in muscular myeloperoxidase (MPO) activity and protein levels of interleukin (IL)-6, IL-1β and monocyte chemotactic protein 1; the reduction by naproxen was less pronounced and only reached significance for MPO activity and IL-6 levels. All treatments significantly reduced the increase in COX-2 activity caused by IM, whereas only GYY4137 significantly reduced the increase in iNOS activity. Naproxen treatment caused significant histological damage of intestinal villi. Conclusion: The study shows that naproxen partially prevents POI, probably through its inhibitory effect on COX-2 activity. Both ATB-346 and GYY4137 were more effective, the result with GYY4137 showing that H2S per se can prevent POI.

Alleviation of impaired reactivity in the corpus cavernosum of STZ-diabetic rats by slow-release H2S donor GYY4137.

GYY4137 is a novel hydrogen sulfide (H2S) releasing molecule with vasodilator activity. The objectives of this study were to investigate: (1) the pharmacological effect of GYY4137 on the reactivity of the corpus cavernosum (CC) from normal and diabetic rats; (2) the contribution of ATP-sensitive potassium (K-ATP) channels and nitric oxide (NO) pathway; (3) the reactivity to vasoactive agonists following ex vivo incubation of the diabetic rat CC with GYY4137. Longitudinal strips of CC from control and diabetic male Sprague-Dawley (SD) rats (n = 5-6 animals per group) were suspended in organ-baths. Responses to GYY4137, carbachol, or phenylephrine (PE) were determined by measurement of changes in isometric tension. The effects of acute incubation of the CC strips with L-NAME (NO synthase inhibitor) or glibenclamide (K-ATP channel inhibitor) on the relaxant responses to GYY4137 were examined. The effect of ex vivo incubation with GYY4137 (10-5 M) on the responses of CC to carbachol or PE was evaluated. We found that GYY4137 provoked relaxation in the CC strips, which was significantly reduced in the presence of L-NAME or glibenclamide. Ex vivo incubation of diabetic CC with GYY4137 resulted in a significant improvement in the vascular responses to the added agonists. We conclude that GYY4137 is a relaxant agonist in SD rats CC, and the response is mediated, at least in part, by NO and K-ATP channels. Brief incubation of diabetic CC with GYY4137 markedly improved the impaired vascular reactivity, thus raising the question whether chronic in vivo treatment of diabetic animals with GYY4137 would have any protective effect, which is worth further investigation.

Daily therapy with a slow-releasing H2S donor GYY4137 enables early functional recovery and ameliorates renal injury associated with urinary obstruction

ObjectivesTo assess the effects of slow-releasing H2S donor GYY4137 on post-obstructive renal function and injury following unilateral ureteral obstruction (UUO) by using the UUO and reimplantation (UUO-R) model in rats and to elucidate potential mechanisms by using an in vitro model of epithelial-mesenchymal transition (EMT). MethodsMale Lewis rats underwent UUO at the left ureterovesical junction. From post-operative day (POD) 1–13, rats received daily intraperitoneal (IP) injection of phosphate buffered saline (PBS, 1 mL) or GYY4137 (200 μmol/kg/day in 1 mL PBS, IP). On POD 14, the ureter was reimplanted back into the bladder, followed by a right nephrectomy. Urine and serum samples were collected to monitor renal function. On POD 30, the left kidney was removed and tissue sections were stained with H&E, TUNEL, CD68, CD206, myeloperoxidase, and Masson's trichrome to determine cortical thickness, apoptosis, inflammation, and fibrosis. In our in vitro model of EMT, NRK52E cells were treated with 10 ng/mL TGF-β1, 10 μM GYY4137 and/or 50 μM GYY4137. Western blot analysis was performed to determine the expression of E-cadherin, vimentin, Smad7 and TGF-β1 receptor II (TβRII). ResultsGYY4137 led to a moderate decrease in post-obstructive serum creatinine, cystatin C and FENa. We also observed a trend towards a decrease in post-obstructive proteinuria following GYY4137 treatment. Histologically, we observed a significant decrease in apoptosis, inflammation, and fibrosis. Furthermore, our in vitro studies demonstrate that in the presence of TGF-β1, GYY4137 significantly decreases vimentin and TβRII and significantly increases E-cadherin and Smad7. ConclusionsH2S may help to accelerate the recovery of renal function post-obstruction and attenuates renal injury associated with UUO. It is possible that H2S mitigates fibrosis by regulating the TGF-β1-mediated EMT pathway. Taken together, our data suggest that H2S may be a potential novel therapy for improving renal function and limiting renal injury associated with obstructive uropathy.

Abstract 2980: Cystathionine-beta-synthase (CBS)-derived hydrogen sulfide (H2S) supports proliferation, migration and bioenergetics in colon cancer-associated fibroblasts (CAFs)

Abstract Introduction: Our group has recently demonstrated that CBS, one of the H2S-producing enzymes, is abundantly overexpressed in human colorectal cancer (CRC) tissue specimens resulting in an increased H2S production. In order to extend our observations, we have now compared, using immunohistochemistry, CBS expression in carcinoma-associated fibroblasts (CAFs) compared to normal colonic fibroblasts (NCFs) in human colon adenocarcinoma samples. The overall goal of our studies is to characterize how the CBS/H2S axis contributes to the malignant phenotype of the CAFs in the tumor microenvironment. Methods: Human CAFs and NCFs were collected from CRC under an IRB approved protocol and cultured in vitro. Western blot analysis of both NCF and CAF was performed to determine CBS protein expression levels. SiRNA/shRNA-mediated silencing of CBS in CAFs was performed. A pharmacological inhibitor of CBS, aminooxyacetic acid (AOAA), in both CAF and NCF cultures was also utilized. Hydrogen sulfide (H2S) levels were measured using the fluorogenic probe 7-Azido-4-methylcoumarin. We also performed proliferation assays using a coulter counter in CAFs, NCFs, and CAFs subjected to siRNA/shRNA-mediated CBS silencing or AOAA. Control cells were transduced with shNT vectors. Cells were also treated with the slow-releasing H2S donor GYY4137. We also conducted cell migration assays in transwell chambers (migration of CAFs toward conditioned media (CM) collected either from HCT116 cells or normal colon mucosal cells (NCM356). Finally, the XF24 Extracellular Flux Analyzer (Seahorse) was used to measure bioenergetic function in CAFs subjected to siRNA/shRNA-mediated silencing of CBS or treated with AOAA. Results: CBS protein expression and H2S production in CAFs was increased compared to NCFs. The H2S donor, GYY 4137, increased the proliferation rate of CAFs but not of NCFs. CBS silencing or AOAA treatment reduced CAF migration toward conditioned media from HCT116 cells. Genetic or pharmacological inhibition of CBS in CAF cells suppressed key bioenergetic parameters including basal respiration, ATP production, maximal respiratory capacity, and spare respiratory capacity compared to NCF cells or CAF shNT cells. Conclusions: Thus, CBS/H2S axis serves as a mechanism to support CAFs’ function. In these cells, H2S contributes to maintaining their bioenergetic functions, which, in turn, promotes their proliferation and migration toward colon cancer cells. We hypothesize that these processes may contribute CRC progression. Citation Format: Katalin Modis, Manjit Maskey, Paul Johnson, Csaba Szabo, Mark R. Hellmich, Celia Chao. Cystathionine-beta-synthase (CBS)-derived hydrogen sulfide (H2S) supports proliferation, migration and bioenergetics in colon cancer-associated fibroblasts (CAFs) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2980. doi:10.1158/1538-7445.AM2017-2980

Hydrogen sulfide donors alleviate itch secondary to the activation of type-2 protease activated receptors (PAR-2) in mice

Hydrogen sulfide (H2S) has been highlighted as an endogenous signaling molecule and we have previously found that it can inhibit histamine-mediated itching. Pruritus is the most common symptom of cutaneous diseases and anti-histamines are the usual treatment; however, anti-histamine-resistant pruritus is common in some clinical settings. In this way, the involvement of mediators other than histamine in the context of pruritus requires new therapeutic targets. Considering that the activation of proteinase-activated receptor 2 (PAR-2) is involved in pruritus both in rodents and humans, in this study we investigated the effect of H2S donors on the acute scratching behavior mediated by PAR-2 activation in mice, as well as some of the possible pharmacological mechanisms involved. The intradermal injection of the PAR-2 peptide agonist SLIGRL-NH2 (8–80nmol) caused a dose-dependent scratching that was unaffected by intraperitoneal pre-treatment with the histamine H1 antagonist pyrilamine (30mg/kg). Co-injection of SLIGRL-NH2 (40nmol) with either the slow-release H2S donor GYY4137 (1 and 3nmol) or the spontaneous donor NaHS (1 and 0.3nmol) significantly reduced pruritus. Co-treatment with the KATP channel blocker glibenclamide (200nmol) or the nitric oxide (NO) donor sodium nitroprusside (10nmol) abolished the antipruritic effects of NaHS; however, the specific soluble guanylyl cyclase inhibitor ODQ (30μg) had no significant effects. The transient receptor potential ankyrin type 1 (TRPA1) antagonist HC-030031 (20μg) significantly reduced SLIGRL-NH2-induced pruritus; however pruritus induced by the TRPA1 agonist AITC (1000nmol) was unaffected by NaHS. Based on these data, we conclude that pruritus secondary to PAR-2 activation can be reduced by H2S, which acts through KATP channel opening and involves NO in a cyclic guanosine monophosphate (cGMP)-independent manner. Furthermore, TRPA1 receptors mediate the pruritus induced by activation of PAR-2, but H2S does not interfere with this pathway. These results provide additional support for the development of new therapeutical alternatives, mainly intended for treatment of pruritus in patients unresponsive to anti-histamines.

GYY4137, a Slow-Releasing Hydrogen Sulfide Donor, Ameliorates Renal Damage Associated with Chronic Obstructive Uropathy

Chronic obstructive uropathy can cause irreversible kidney injury, atrophy and inflammation, which can ultimately lead to fibrosis. Epithelial-mesenchymal transition is a key trigger of fibrosis that is caused by up-regulation of TGF-β1 (transforming growth factor-β1) and ANGII (angiotensin II). H2S is an endogenously produced gasotransmitter with cytoprotective properties. We sought to elucidate the effects of the slow-releasing H2S donor GYY4137 on chronic ureteral obstruction and evaluate the potential mechanisms. Following unilateral ureteral obstruction male Lewis rats were given daily intraperitoneal administration of phosphate buffered saline vehicle (obstruction group) or phosphate buffered saline plus 200μmol/kg GYY4137 (obstruction plus GYY4137 group) for 30 days. Urine and serum samples were collected to determine physiological parameters of renal function and injury. Kidneys were removed on postoperative day 30 to evaluate histopathology and protein expression. Epithelial-mesenchymal transition in LLC-PK1 pig kidney epithelial cells was induced with TGF-β1 and treated with GYY4137 to evaluate potential mechanisms via invitro scratch wound assays. H2S treatment decreased serum creatinine and the urine protein-to-creatinine excretion ratio after unilateral ureteral obstruction. In addition, H2S mitigated cortical loss, inflammatory damage and tubulointerstitial fibrosis. Tissues showed decreased expression of epithelial-mesenchymal transition markers upon H2S treatment. Epithelial-mesenchymal transition progression in LLC-PK1 was alleviated upon invitro administration of GYY4137. To our knowledge our findings demonstrate for the first time the protective effects of H2S in chronic obstructive uropathy. This may represent a potential therapeutic solution to ameliorate renal damage and improve the clinical outcomes of urinary obstruction.

GYY4137 attenuates remodeling, preserves cardiac function and modulates the natriuretic peptide response to ischemia

AimsMyocardial infarction followed by adverse left ventricular (LV) remodeling is the most frequent proximate cause of heart failure. Hydrogen sulfide (H2S) is an important endogenous modulator of diverse physiological and pathophysiological processes. Its role in post-ischemic ventricular remodeling and the associated neurohormonal responses has not been defined. Here, we aimed at evaluating whether the slow-releasing water-soluble H2S donor GYY4137 (GYY) exerts cardioprotective effects and modulates the neurohormonal response to cardiac ischemic injury. Methods and resultsTreatment for 2 or 7days with GYY (100mg/Kg/48h, IP) after acute myocardial infarction (MI) in rats preserved LV dimensions and function in vivo, compared to untreated infarcted (MI), placebo- and dl-propargylglycine- (PAG, an inhibitor of endogenous H2S synthesis) treated animals (n=9/group/time-point). LV dimensions and function in GYY-treated animals were comparable to healthy sham-operated rats. GYY-treated hearts had significantly less LV fibrosis than MI, placebo and PAG hearts. A higher density of blood vessels was found in the LV scar area of GYY-treated animals compared to all other infarcted groups. Despite preserved LV structure and function, treatment with GYY increased the levels of the natriuretic peptides ANP and BNP in association with enhanced cyclic GMP levels, paralleled by higher cGMP-dependent protein kinase type I (cGKI) protein levels. ConclusionsOur data suggest that the slow-releasing H2S donor, GYY4137, preserves cardiac function, attenuates adverse remodeling and may exert post-ischemic cardioprotective (pro-angiogenic, anti-apoptotic, anti-hypertrophic and anti-fibrotic) effects in part through enhanced early post-ischemic endogenous natriuretic peptide activation.

Role of hydrogen sulfide in paramyxovirus infections.

Hydrogen sulfide (H2S) is an endogenous gaseous mediator that has gained increasing recognition as an important player in modulating acute and chronic inflammatory diseases. However, its role in virus-induced lung inflammation is currently unknown. Respiratory syncytial virus (RSV) is a major cause of upper and lower respiratory tract infections in children for which no vaccine or effective treatment is available. Using the slow-releasing H2S donor GYY4137 and propargylglycin (PAG), an inhibitor of cystathionine-γ-lyase (CSE), a key enzyme that produces intracellular H2S, we found that RSV infection led to a reduced ability to generate and maintain intracellular H2S levels in airway epithelial cells (AECs). Inhibition of CSE with PAG resulted in increased viral replication and chemokine secretion. On the other hand, treatment of AECs with the H2S donor GYY4137 reduced proinflammatory mediator production and significantly reduced viral replication, even when administered several hours after viral absorption. GYY4137 also significantly reduced replication and inflammatory chemokine production induced by human metapneumovirus (hMPV) and Nipah virus (NiV), suggesting a broad inhibitory effect of H2S on paramyxovirus infections. GYY4137 treatment had no effect on RSV genome replication or viral mRNA and protein synthesis, but it inhibited syncytium formation and virus assembly/release. GYY4137 inhibition of proinflammatory gene expression occurred by modulation of the activation of the key transcription factors nuclear factor κB (NF-κB) and interferon regulatory factor 3 (IRF-3) at a step subsequent to their nuclear translocation. H2S antiviral and immunoregulatory properties could represent a novel treatment strategy for paramyxovirus infections. RSV is a global health concern, causing significant morbidity and economic losses as well as mortality in developing countries. After decades of intensive research, no vaccine or effective treatment, with the exception of immunoprophylaxis, is available for this infection as well as for other important respiratory mucosal viruses. This study identifies hydrogen sulfide as a novel cellular mediator that can modulate viral replication and proinflammatory gene expression, both important determinants of lung injury in respiratory viral infections, with potential for rapid translation of such findings into novel therapeutic approaches for viral bronchiolitis and pneumonia.

GYY4137 protects against myocardial ischemia and reperfusion injury by attenuating oxidative stress and apoptosis in rats.

Hydrogen sulfide (H2S) is a gasotransmitter that regulates cardiovascular functions. The present study aimed to determine the protective effect of slow-releasing H2S donor GYY4137 on myocardial ischemia and reperfusion (I/R) injury and to investigate the possible signaling mechanisms involved. Male Sprague-Dawley rats were treated with GYY4137 at 12.5 mg/(kg·day), 25 mg/(kg·day) or 50 mg/(kg·day) intraperitoneally for 7 days. Then, rats were subjected to 30 minutes of left anterior descending coronary artery occlusion followed by reperfusion for 24 hours. We found that GYY4137 increased the cardiac ejection fraction and fractional shortening, reduced the ischemia area, alleviated histological injury and decreased plasma creatine kinase after myocardial I/R. Both H2S concentration in plasma and cystathionine-γ-lyase (CSE) activity in the myocardium were enhanced in the GYY4137 treated groups. GYY4137 also decreased malondialdehyde and myeloperoxidase levels in serum, attenuated superoxide anion level and suppressed phosphorylation of mitogen activated protein kinases in the myocardium after I/R. Meanwhile, GYY4137 increased the expression of Bcl-2 but decreased the expression of Bax, caspase-3 activity and apoptosis in the myocardium. The data suggest that GYY4137 protects against myocardial ischemia and reperfusion injury by attenuating oxidative stress and apoptosis.

Abstract 106: GYY4137 Attenuates Post-Ischemic Adverse Remodeling and Modulates ANP-Mediated cGMP Signal Transduction

Myocardial infarction is the most frequent proximate cause of heart failure, which remains a leading cause of death in the developed world. Hydrogen sulfide (H 2 S) is emerging as an important endogenous modulator in diverse physiological and pathophysiological events. We hypothesized that the slow-releasing water-soluble H 2 S donor GYY4137 (GYY) may exert cardioprotective effects through modulation of neurohormonal response to cardiac injury. We have found that treatment for 1 week with GYY (100mg/Kg/48hr, IP) after acute myocardial infarction in rats, provides powerful sustained preservation of left ventricular (LV) dimensions and function in vivo, compared to untreated infarcted (MI), placebo- and D-propargylglycine- (PAG, an inhibitor of endogenous H 2 S synthesis) treated animals 2 and 7 days after infarct (n=6/group/time-point). LV dimensions and function in GYY-treated animals were comparable to healthy sham-operated rats. GYY-treated hearts displayed a significantly lower percentage of LV fibrosis than MI, placebo and PAG hearts, whereas PAG treated animals had significantly bigger scar size relative to GYY, at days 2 and 7 after MI. A higher density of blood vessels was found in the scar area of GYY-treated animals compared to all other infarcted groups at days 2 (P&lt;0.0002) and 7 (P&lt;0.01) post-MI. Furthermore, treatment with GYY resulted in increased levels of plasma atrial natriuretic peptide (ANP) compared to all groups at days 2 and 7 after MI, while cardiac mRNA expression of ANP was also significantly increased in GYY-treated rats compared to Sham (P&lt;0.001), and all the infarcted groups (P&lt;0.05) at day 2 after MI. Concordantly, ANP second messenger cGMP was increased in plasma at day 7 in GYY rats compared to sham and all infarcted groups (p&lt;0.05), paralleled by higher cGMP-dependent protein kinase type I (cGKI) protein levels than sham (P&lt;0.01), and vasodilator-stimulated phosphoprotein phosphorylation (pVASP) at the cGKI preferred site (Ser239) compared to sham and PAG (P&lt;0.05) at day 2 post-MI. In conclusion, our data suggest that H2S attenuates adverse remodeling and may exert post-ischemic cardioprotective (pro-angiogenic, anti-fibrotic) effects in part through modulation of ANP-mediated cGMP signal transduction.