Slow-releasing H2S Donor Research Articles

Lifespan and healthspan benefits of exogenous H2S in C. elegans are independent from effects downstream of eat-2 mutation

Caloric restriction (CR) is one of the most effective interventions to prolong lifespan and promote health. Recently, it has been suggested that hydrogen sulfide (H2S) may play a pivotal role in mediating some of these CR-associated benefits. While toxic at high concentrations, H2S at lower concentrations can be biologically advantageous. H2S levels can be artificially elevated via H2S-releasing donor drugs. In this study, we explored the function of a novel, slow-releasing H2S donor drug (FW1256) and used it as a tool to investigate H2S in the context of CR and as a potential CR mimetic. We show that exposure to FW1256 extends lifespan and promotes health in Caenorhabditis elegans (C. elegans) more robustly than some previous H2S-releasing compounds, including GYY4137. We looked at the extent to which FW1256 reproduces CR-associated physiological effects in normal-feeding C. elegans. We found that FW1256 promoted healthy longevity to a similar degree as CR but with fewer fitness costs. In contrast to CR, FW1256 actually enhanced overall reproductive capacity and did not reduce adult body length. FW1256 further extended the lifespan of already long-lived eat-2 mutants without further detriments in developmental timing or fertility, but these lifespan and healthspan benefits required H2S exposure to begin early in development. Taken together, these observations suggest that FW1256 delivers exogenous H2S efficiently and supports a role for H2S in mediating longevity benefits of CR. Delivery of H2S via FW1256, however, does not mimic CR perfectly, suggesting that the role of H2S in CR-associated longevity is likely more complex than previously described.

H2S as a potential defense against COVID-19?

The outbreak of COVID-19 pneumonia caused by a new coronavirus (severe acute respiratory syndrome coronavirus 2, SARS-CoV-2) is posing a global health emergency and has led to more than 380,000 deaths worldwide. The cell entry of SARS-CoV-2 depends on two host proteins angiotensin-converting enzyme 2 (ACE2) and transmembrane protease serine 2 (TMPRSS2). There is currently no vaccine available and also no effective drug for the treatment of COVID-19. Hydrogen sulfide (H2S) as a novel gasotransmitter has been shown to protect against lung damage via its anti-inflammation, antioxidative stress, antiviral, prosurvival, and antiaging effects. In light of the research advances on H2S signaling in biology and medicine, this review proposed H2S as a potential defense against COVID-19. It is suggested that H2S may block SARS-CoV-2 entry into host cells by interfering with ACE2 and TMPRSS2, inhibit SARS-CoV-2 replication by attenuating virus assembly/release, and protect SARS-CoV-2-induced lung damage by suppressing immune response and inflammation development. Preclinical studies and clinical trials with slow-releasing H2S donor(s) or the activators of endogenous H2S-generating enzymes should be considered as a preventative treatment or therapy for COVID-19.

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.

Treatment with slow-releasing hydrogen sulfide donors inhibits the nociceptive and depressive-like behaviours accompanying chronic neuropathic pain: Endogenous antioxidant system activation.

Therapies to treat chronic neuropathic pain and its associated comorbidities are limited. Recent studies demonstrated that the administration of slow-releasing hydrogen sulfide (H2S) donors inhibited chemotherapy-induced neuropathic pain. However, the antidepressant or anxiolytic effects of these compounds and their mechanisms of action during chronic neuropathic pain have not been evaluated. To determine whether the administration of two slow-releasing H2S donors, allyl isothiocyanate (A-ITC) and phenyl isothiocyanate (P-ITC), inhibits the nociceptive and emotional disorders associated with chronic neuropathic pain. In C57BL/6 male mice with neuropathic pain caused by the chronic constriction of the sciatic nerve, we assessed the effects of intraperitoneal administration of A-ITC and P-ITC in (a) the mechanical allodynia, thermal hyperalgesia and thermal allodynia induced by nerve ligation; (b) the anxiety- and depressive-like behaviours linked with neuropathic pain; (c) glial activation and mitogen-activated protein kinases phosphorylation, and (d) expression of the antioxidant enzymes, heme oxygenase 1 (HO-1), NADPH quinone oxidoreductase1, and glutathione S-transferase mu-1 (GSTM1), and alpha-1 (GSTA1), in hippocampus and prefrontal cortex (PFC). Both treatments inhibited the allodynia and hyperalgesia, depressive-like behaviours, astroglial activation, and the extracellular signal-regulated kinase 1/2 phosphorylation but were unable to abolish the anxiety-like behaviours accompanying neuropathic pain. A-ITC and P-ITC also augmented the expression of HO-1, GSTM1, and GSTA1 in the hippocampus and/or PFC. The administration of slow-releasing H2S donors might be a promising treatment for the management of chronic neuropathic pain and some associated comorbidities via inhibiting the inflammatory and plasticity changes, and activating the endogenous antioxidant responses.

Divergent effect of fast- and slow-releasing H2S donors on boar spermatozoa under oxidative stress

Hydrogen sulphide (H2S) is involved in the physiology and pathophysiology of different cell types, but little is known about its role in sperm cells. Because of its reducing properties, we hypothesise that H2S protects spermatozoa against the deleterious effects of oxidative stress, a condition that is common to several male fertility disorders. This study aimed i) to determine the total antioxidant capacities of Na2S and GYY4137, which are fast- and slow-releasing H2S donors, respectively, and ii) to test whether H2S donors are able to protect spermatozoa against oxidative stress. We found that Na2S and GYY4137 show different antioxidant properties, with the total antioxidant capacity of Na2S being mostly unstable and even undetectable at 150 µM. Moreover, both H2S donors preserve sperm motility and reduce acrosome loss, although the effects were both dose and donor dependent. Within the range of concentrations tested (3–300 µM), GYY4137 showed positive effects on sperm motility, whereas Na2S was beneficial at the lowest concentration but detrimental at the highest. Our findings show that Na2S and GYY4137 have different antioxidant properties and suggest that both H2S donors might be used as in vitro therapeutic agents against oxidative stress in sperm cells, although the optimal therapeutic range differs between the compounds.

Glutathione-Allylsulfur Conjugates as Mesenchymal Stem Cells Stimulating Agents for Potential Applications in Tissue Repair.

The endogenous gasotransmitter H2S plays an important role in the central nervous, respiratory and cardiovascular systems. Accordingly, slow-releasing H2S donors are powerful tools for basic studies and innovative pharmaco-therapeutic agents for cardiovascular and neurodegenerative diseases. Nonetheless, the effects of H2S-releasing agents on the growth of stem cells have not been fully investigated. H2S preconditioning can enhance mesenchymal stem cell survival after post-ischaemic myocardial implantation; therefore, stem cell therapy combined with H2S may be relevant in cell-based therapy for regenerative medicine. Here, we studied the effects of slow-releasing H2S agents on the cell growth and differentiation of cardiac Lin− Sca1+ human mesenchymal stem cells (cMSC) and on normal human dermal fibroblasts (NHDF). In particular, we investigated the effects of water-soluble GSH–garlic conjugates (GSGa) on cMSC compared to other H2S-releasing agents, such as Na2S and GYY4137. GSGa treatment of cMSC and NHDF increased their cell proliferation and migration in a concentration dependent manner with respect to the control. GSGa treatment promoted an upregulation of the expression of proteins involved in oxidative stress protection, cell–cell adhesion and commitment to differentiation. These results highlight the effects of H2S-natural donors as biochemical factors that promote MSC homing, increasing their safety profile and efficacy after transplantation, and the value of these donors in developing functional 3D-stem cell delivery systems for cardiac muscle tissue repair and regeneration.

ADT-OH, a hydrogen sulfide-releasing donor, induces apoptosis and inhibits the development of melanoma in vivo by upregulating FADD

Hydrogen sulfide (H2S) is now widely considered the third endogenous gasotransmitter and plays critical roles in cancer biological processes. In this study, we demonstrate that 5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione (ADT-OH), the most widely used moiety for synthesising slow-releasing H2S donors, induces melanoma cell death in vitro and in vivo. Consistent with previous reports, ADT-OH inhibited IκBɑ degradation, resulting in reduced NF-κB activation and subsequent downregulation of the NF-κB-targeted anti-apoptotic proteins XIAP and Bcl-2. More importantly, we found that ADT-OH suppressed the ubiquitin-induced degradation of FADD by downregulating the expression of MKRN1, an E3 ubiquitin ligase of FADD. In addition, ADT-OH had no significant therapeutic effect on FADD-knockout B16F0 cells or FADD-knockdown A375 cells. Based on these findings, we evaluated the combined effects of ADT-OH treatment and FADD overexpression on melanoma cell death in vivo using a mouse xenograft model. As expected, tumour-specific delivery of FADD through a recombinant Salmonella strain, VNP-FADD, combined with low-dose ADT-OH treatment significantly inhibited tumour growth and induced cancer cell apoptosis. Taken together, our data suggest that ADT-OH is a promising cancer therapeutic drug that warrants further investigation into its potential clinical applications.

The Inhibitory Effects of Slow-Releasing Hydrogen Sulfide Donors in the Mechanical Allodynia, Grip Strength Deficits, and Depressive-Like Behaviors Associated with Chronic Osteoarthritis Pain.

Osteoarthritis and its associated comorbidities are important clinical problems that have a negative impact on the quality of life, and its treatment remains unresolved. We investigated whether the systemic administration of slow-releasing hydrogen sulfide (H2S) donors, allyl isothiocyanate (A-ITC) and phenyl isothiocyanate (P-ITC), alleviates chronic osteoarthritis pain and the associated emotional disorders. In C57BL/6 female mice with osteoarthritis pain induced by the intra-articular injection of monosodium iodoacetate, we evaluated the effects of repeated administration of A-ITC and P-ITC on the (i) mechanical allodynia and grip strength deficits; (ii) emotional conducts; and (iii) glial activity and expression of inducible nitric oxide synthase (NOS2), phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt), and antioxidant enzymes (heme oxygenase 1, NAD(P)H:quinone oxidoreductase-1, glutathione S-transferase mu 1 and alpha 1) in the hippocampus. The administration of A-ITC and P-ITC inhibited the mechanical allodynia, the grip strength deficits, and the depressive-like behaviors accompanying osteoarthritis. Both treatments inhibited microglial activation, normalized the upregulation of NOS2 and PI3K/p-Akt, and maintained high levels of antioxidant/detoxificant enzymes in the hippocampus. Data suggest that treatment with low doses of slow-releasing H2S donors might be an interesting strategy for the treatment of nociception, functional disability, and emotional disorders associated with osteoarthritis pain.

Altered dynamics in the circadian oscillation of clock genes in serum-shocked NIH-3T3 cells by the treatment of GYY4137 or AOAA

Background and purposeSeveral members of the core clock mechanism are equipped with a Per-Arnt-Sim (PAS) domain through which they can bind haem [Fe(II)]. Haem is a ligand for the orphan receptors REV-ERBα/β (NR1D1/2), which regulate circadian rhythm and metabolism. The ability to bind haem sensitises these clock components to the action of small molecule gases, including NO, CO and H2S. Studies conducted with European hamsters revealed that during winter sleep, key clock genes stop oscillating. At the same time, H2S, when administered at subtoxic concentrations, can induce a hypometabolic state in the cell. We suppose that core clock components, including the nuclear receptors REV-ERBs, neuronal PAS domain protein 2 (nPAS2) and PER2, can be H2S targets. The general objective of this study was to investigate the effect of the H2S system on the expression profile of the core clock genes in cells in vitro. Experimental approachWe analysed the expression of Per1, Per2, Per3, Bmal1, Cry1, Cry2, Nr1d1, Nfil-3 and Dbp messenger RNA (mRNA) in serum-shocked NIH-3T3 cells treated with a slow-releasing H2S donor (GYY4137) or the cystathionine beta-synthase (CBS) inhibitor (AOAA) cultured under constant darkness and collected during 3 days in 3 h interval. Key results and conclusions and implicationsWe found that pharmacological CBS inhibition increased the general expression and dynamics of several clock genes. On the other hand, increased H2S decreased Per2 expression. These data suggest that CBS can affect circadian clock and effect on clock-controlled transcription output.

Biological Effects of Morpholin-4-Ium 4 Methoxyphenyl (Morpholino) Phosphinodithioate and Other Phosphorothioate-Based Hydrogen Sulfide Donors.

Significance: Hydrogen sulfide (H2S) is regarded as the third gasotransmitter along with nitric oxide and carbon monoxide. Extensive studies have demonstrated a variety of biological roles for H2S in neurophysiology, cardiovascular disease, endocrine regulation, and other physiological and pathological processes. Recent Advances: Novel H2S donors have proved useful in understanding the biological functions of H2S, with morpholin-4-ium 4 methoxyphenyl (morpholino) phosphinodithioate (GYY4137) being one of the most common pharmacological tools used. One advantage of GYY4137 over sulfide salts is its ability to release H2S in a slow and sustained manner akin to endogenous H2S production, rather than the delivery of H2S as a single concentrated burst. Critical Issues: Here, we summarize recent progress made in the characterization of the biological activities and pharmacological effects of GYY4137 in a range of in vitro and in vivo systems. Recent developments in the structural modification of GYY4137 to generate new compounds and their biological effects are also discussed. Future Directions: Slow-releasing H2S donor, GYY4137, and other phosphorothioate-based H2S donors are potent tools to study the biological functions of H2S. Despite recent progress, more work needs to be performed on these new compounds to unravel the mechanisms behind H2S release and pace of its discharge, as well as to define the effects of by-products of donors after H2S liberation. This will not only lead to better in-depth understanding of the biological effects of H2S but will also shed light on the future development of a new class of therapeutic agents with potential to treat a wide range of human diseases.

Abstract 433: Hydrogen Sulfide Protects the Heart Against Homocysteine-Induced Remodeling by Regulating Autophagy and Pyroptosis

Hydrogen sulfide (H 2 S), a byproduct of homocysteine (Hcy) transsulfuration, protects against adverse cardiac remodeling in diabetic cardiomyopathy, hyperhomocysteinemia (HHcy), and other cardiomyopathies. However, the underlying molecular mechanisms are unclear. Pyroptosis is a caspase 1-mediated cell death mechanism that promotes remodeling. It is inhibited by autophagy, a lysosomal degradation process that maintains cellular homeostasis. We hypothesized that H 2 S supplementation inhibits Hcy-induced pyroptosis by increasing autophagy in the heart. We treated CBS +/- mice (a model of HHcy) and WT mice with SG1002 (slow-releasing H 2 S donor) or control diet for 14 weeks and performed deep sequencing of their hearts (LV tissue). Ingenuity Pathway Analysis revealed SG1002 downregulated cardiac hypertrophy genes, plausibly by altering upstream regulators involved in pyroptosis and autophagy (IL-1β and mTOR). Western blot results showed that SG1002 suppresses mTOR in CBS +/- hearts (WT: 2.1±0.83, CBS: 10±2.4, CBS+SG: 3.8±2.3, WT+SG: 0.96±0.39) to induce autophagy. SG1002 decreases IL-1β (WT: 0.61±0.08, CBS: 0.88±0.07, CBS+SG: 0.32±0.01, WT+SG: 0.38±0.03) and inhibits caspase-1 activity to suppress Hcy-induced pyroptosis. NF-kB p65, which activates pyroptosis, is also downregulated by SG1002. We conclude that H 2 S could alleviate cardiac remodeling by inducing autophagy and inhibiting NF-kB-mediated pyroptosis. This is the first report that H 2 S controls cross-talk of cardiac pyroptosis and autophagy.

An in situ slow-releasing H2S donor depot with long-term therapeutic effects for treating ischemic diseases.

Therapeutic angiogenesis is essential for rescuing necrotic tissues in cases of ischemic disease. The exogenous hydrogen sulfide (H2S) donor, diallyl trisulfide (DATS), has been investigated as a therapeutic agent that promotes angiogenesis. However, the short half-life of generated H2S limits its therapeutic efficacy. In an attempt to overcome this difficulty, a poly(D,L-lactic-co-glycolic acid) microparticle system that contains DATS (DATS@MPs) is prepared as an in situ depot for the controlled release of H2S, providing slow release and long-term effectiveness. The results of in vitro investigations indicate that the slow-released DATS from the DATS@MPs depot yields a longer intracellular production of H2S than that from a free DATS depot. The intracellular generation of H2S favors the translocation of the transcription factor, Nrf2, from the cytosol to nuclei, potentially upregulating the gene expressions of antioxidant enzymes, ultimately increasing cellular resistance to oxidative stress. Intramuscular injection of the slow-releasing H2S donor depot DATS@MPs in an ischemic limb that is experimentally generated in a mouse model promotes therapeutic angiogenesis and protects cells from apoptosis and tissues from necrosis, ultimately salvaging the limb. These analytical results reveal that DATS@MPs is potentially useful in H2S-based therapy for treating ischemic diseases.

Abstract 2641: Transactivation of EGFR and mTOR pathway by H2Sin cancer cells

Abstract Hydrogen sulfide regulates a variety of physiological and pathological processes. Previous studies have suggested that H2S plays a significant role as a secondary messenger in cellular signaling. Interestingly, it has been found that this unique gaseous molecule, or gasotransmitter, is capable of producing both pro- and anti- apoptotic activity in cultured cells. It has been speculated that these gasotransmitters are endogenously generated in mammalian systems through enzymatic catalysis. Our pursuits focus on comparing the effects of H2S on various lines of cancer cells (CaOV3-ovarian cancer cells, A375-melanoma cells, HeLa-cervical cancer cells, and WM 266-4-melanoma cells). Using a water-soluble, slow-releasing H2S donor, GYY4137 (200 μM), we studied how both concentration and time affect cell viability and investigated further whether H2S causes pro- or anti- apoptotic activity. Existing data have indicated that GYY4137 may activate molecular targets, including EGFR, ERK and JNK. We hypothesize that GYY4137 activates mTOR (mammalian target of rapamycin) with result of phosphorylation of S6, leading to either cell death or cell proliferation. By monitoring various proteins through Western blot analysis and confocal microscopy we studied the activation of EGFR/mTOR pathway in response to GYY4137 treatment. Our data showed that the longer the treatment the more prevalent phosphor S6 and phosphor-EGFR are. Noticeably, we observed a significant difference between 15 minutes and 30 minutes, suggesting that GYY4137 has the largest effect after 30 minutes of treatment. We further studied cell growth and viability using MTT assay to investigate the effects of various concentrations of GYY4137 on the cells. Our data indicated that the stronger the concentration is, the lower the cell viability. Similarly, we used Mitotracker ® Red dye to stain for mitochondrial activity in CaOV3 and WM 266-4 cells treated at various time points. Overall, mitochondrial activity increased when cells were exposed to GYY4137, suggesting that H2S may cause anti-apoptotic activity, and instead favor cell growth at the concentration we used. Collectively, our data suggests that the H2S donor, GYY4137 has a significant effect on cell proliferation and survival in several cancerous cells likely mediated by transactivation of EGFR and mTOR pathways. Note: This abstract was not presented at the meeting. Citation Format: Jillian L. Higgins, Aileen K. Kraus, Dominic T. Arruda, Hao Guo, Mattew Lautato, Christopher Lautato, Eliana DaCunha, Ashley Chen, Ricky Yao, Dongqin Yang, Yinsheng Wan. Transactivation of EGFR and mTOR pathway by H2Sin cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2641.

Hydrogen Sulfide Ameliorates Homocysteine-Induced Cardiac Remodeling and Dysfunction.

Patients with diabetes, a methionine-rich meat diet, or certain genetic polymorphisms show elevated levels of homocysteine (Hcy), which is strongly associated with the development of cardiovascular disease including diabetic cardiomyopathy. However, reducing Hcy levels with folate shows no beneficial cardiac effects. We have previously shown that a hydrogen sulfide (H2S), a by-product of Hcy through transsulfuration by cystathionine beta synthase (CBS), donor mitigates Hcy-induced hypertrophy in cardiomyocytes. However, the in vivo cardiac effects of H2S in the context of hyperhomocysteinemia (HHcy) have not been studied. We tested the hypothesis that HHcy causes cardiac remodeling and dysfunction in vivo, which is ameliorated by H2S. Twelve-week-old male CBS+/− (a model of HHcy) and sibling CBS+/+ (WT) mice were treated with SG1002 (a slow release H2S donor) diet for 4 months. The left ventricle of CBS+/− mice showed increased expression of early remodeling signals c-Jun and c-Fos, increased interstitial collagen deposition, and increased cellular hypertrophy. Notably, SG1002 treatment slightly reduced c-Jun and c-Fos expression, decreased interstitial fibrosis, and reduced cellular hypertrophy. Pressure volume loop analyses in CBS+/− mice revealed increased end systolic pressure with no change in stroke volume (SV) suggesting increased afterload, which was abolished by SG1002 treatment. Additionally, SG1002 treatment increased end-diastolic volume and SV in CBS+/− mice, suggesting increased ventricular filling. These results demonstrate SG1002 treatment alleviates cardiac remodeling and afterload in HHcy mice. H2S may be cardioprotective in conditions where H2S is reduced and Hcy is elevated.

Hydrogen sulfide intervention in cystathionine-β-synthase mutant mouse helps restore ocular homeostasis.

To investigate the applications of hydrogen sulfide (H2S) in eye-specific ailments in mice. Heterozygous cystathionine-β-synthase (CBS+/-) and wild-type C57BL/6J (WT) mice fed with or without high methionine diet (HMD) were administered either phosphate buffered saline (PBS) or the slow-release H2S donor: GYY4137. Several analyses were performed to study GYY4137 effects by examining retinal lysates for key protein expressions along with plasma glutamate and glutathione estimations. Intraocular pressure (IOP) was monitored during GYY4137 treatment; barium sulfate and bovine serum albumin conjugated fluorescein isothiocyanate (BSA-FITC) angiographies were performed for examining vasculature and its permeability post-treatment. Vision-guided behavior was also tested employing novel object recognition test (NORT) and light-dark box test (LDBT) recordings. CBS deficiency (CBS+/-) coupled with HMD led disruption of methionine/homocysteine (Hcy) metabolism leading to hyperhomocysteinemia (HHcy) in CBS+/- mice as reflected by increased Hcy, and s-adenosylhomocysteine hydrolase (SAHH) levels. Unlike CBS, cystathionine-γ lyase (CSE), methylenetetrahydrofolate reductase (MTHFR) levels which were reduced but compensated by GYY4137 intervention. Heightened oxidative and endoplasmic reticulum (ER) stress responses were mitigated by GYY4137 effects along with enhanced glutathione (GSH) levels. Increased glutamate levels in CBS+/- strain were prominent than WT mice and these mice also exhibited higher IOP that was lowered by GYY4137 treatment. CBS deficiency also resulted in vision-guided behavioral impairment as revealed by NORT and LDBT findings. Interestingly, GYY4137 was able to improve CBS+/- mice behavior together with lowering their glutamate levels. Blood-retinal barrier (BRB) appeared compromised in CBS+/- with vessels' leakage that was mitigated in GYY4137 treated group. This corroborated the results for occludin (an integral plasma membrane protein of the cellular tight junctions) stabilization. Findings reveal that HHcy-induced glutamate excitotoxicity, oxidative damage, ER-stress and vascular permeability alone or together can compromise ocular health and that GYY4137 could serve as a potential therapeutic agent for treating HHcy induced ocular disorders.

GYY4137 Modulates Renal Remodeling in Hyperhomocysteinemia

IntroductionElevated plasma levels of homocysteine, also known as hyperhomocysteinemia (HHcy), is associated with chronic kidney disease and end‐stage renal failure. HHcy is also a vascular risk factor that causes glomerular remodeling by inducing and accumulating extracellular matrix (ECM) in the glomerular and peri‐glomerular spaces. This remodeling is, in part, due to Hcy‐mediated imbalance between matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) and disruption of cell‐to‐cell gap junction protein, connexins (Cxs). HHcy also contribute to renal microvascular impairment and vasoconstriction due to decreased endogenous hydrogen sulfide (H2S) production and homocysteinylation of endothelial nitric‐oxide synthase (eNOS). However, the molecular events targeting Caveolin, eNOS, MMPs/TIMPs, Cxs and the role of H2S in HHcy associated ECM turnover and renovascular remodeling remain elusive.HypothesisWe investigated whether GYY4137, slow releasing H2S donor ameliorates HHcy effect and renal remodeling.MethodsCaveolin protein 1 knockout B6.Cg‐Cav1 (Cav1−/ −), C57BL6/J (WT) and hyperhomocysteinemic CBS+/− mice were supplemented without or with Hcy (2% in diet) and treated without or with GYY4137 (0.5 mg/Kg/d, IP) for a period of 8 weeks.ResultsHHcy decreased plasma H2S levels, upregulated Cav1 and decreased eNOS activity in the kidney. HHcy also resulted in the upregulation of mRNA and protein levels of MMPs −1, 2, −9, −8, −13 as well as connexins – 40 and −43 and decreased the activities of (TIMPs) −1, −2, −3, −4. Immunohistochemistry results indicated increased collagen IV deposition and expression of connexins in the glomerular and interstitial areas. GYY4137 treatment mitigated these changes in HHcy. In conclusion, our data suggests that H2S alleviates renovascular remodeling by modulating Cav‐1, eNOS, MMPs/TIMPs and Cxs in HHcy.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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.

Mitochondria-targeted hydrogen sulfide attenuates endothelial senescence by selective induction of splicing factors HNRNPD and SRSF2.

Cellular senescence is a key driver of ageing, influenced by age-related changes to the regulation of alternative splicing. Hydrogen sulfide (H2S) has similarly been described to influence senescence, but the pathways by which it accomplishes this are unclear.We assessed the effects of the slow release H2S donor Na-GYY4137 (100 µg/ml), and three novel mitochondria-targeted H2S donors AP39, AP123 and RT01 (10 ng/ml) on splicing factor expression, cell proliferation, apoptosis, DNA replication, DNA damage, telomere length and senescence-related secretory complex (SASP) expression in senescent primary human endothelial cells.All H2S donors produced up to a 50% drop in senescent cell load assessed at the biochemical and molecular level. Some changes were noted in the composition of senescence-related secretory complex (SASP); IL8 levels increased by 24% but proliferation was not re-established in the culture as a whole. Telomere length, apoptotic index and the extent of DNA damage were unaffected. Differential effects on splicing factor expression were observed depending on the intracellular targeting of the H2S donors. Na-GYY4137 produced a general 1.9 – 3.2-fold upregulation of splicing factor expression, whereas the mitochondria-targeted donors produced a specific 2.5 and 3.1-fold upregulation of SRSF2 and HNRNPD splicing factors only. Knockdown of SRSF2 or HNRNPD genes in treated cells rendered the cells non-responsive to H2S, and increased levels of senescence by up to 25% in untreated cells.Our data suggest that SRSF2 and HNRNPD may be implicated in endothelial cell senescence, and can be targeted by exogenous H2S. These molecules may have potential as moderators of splicing factor expression and senescence phenotypes.

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.