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•  Introduction •  Reactive nitrogen species (RNS) in the heart and vasculature •  (H2S) biosynthesis •  H2S measurement, catabolism and removal •  H2S in the heart and vasculature •  Evidence for ‘cross‐talk’ between nitric oxide and H2S •  Evidence for the formation of a novel intermediate between nitric oxide and H2S at physiological pH •  Concluding remarks Hydrogen sulfide (H2S) is a well known and pungent toxic gas that has recently been shown to be synthesised in man from the amino acids cystathionine, homocysteine and cysteine by at least two distinct enzymes; cystathionine‐γ‐lyase and cystathionine‐β‐synthase. In the past few years, H2S has emerged as a novel and increasingly important mediator in the cardiovascular system but delineating the precise physiology and pathophysiology of H2S is proving to be complex and difficult to unravel with disparate findings reported with cell types, tissue types and animal species reported. Therefore, in this review we summarize the mechanisms by which H2S has been proposed to regulate blood pressure and cardiac function, discuss the mechanistic discrepancies reported in the literature as well as the therapeutic potential of H2S. We also examine the methods of H2S detection in biological fluids, processes for H2S removal and discuss the reported blood levels of H2S in man and animal models of cardiovascular pathology. We also highlight the complex interaction of H2S with nitric oxide in regulating cardiovascular function in health and disease.

Alliums and allied plant species are rich sources of sulfur compounds that have effects on vascular homeostasis and the control of metabolic systems linked to nutrient metabolism in mammals. In view of the multiple biological effects ascribed to these sulfur molecules, researchers are now using these compounds as inspiration for the synthesis and development of novel sulfur-based therapeutics. This research has led to the chemical synthesis and biological assessment of a diverse array of sulfur compounds representative of derivatives of S-alkenyl-l-cysteine sulfoxides, thiosulfinates, ajoene molecules, sulfides, and S-allylcysteine. Many of these synthetic derivatives have potent antimicrobial and anticancer properties when tested in preclinical models of disease. Therefore, the current review provides an overview of advances in the development and biological assessment of synthetic analogs of allium-derived sulfur compounds.

The role of hydrogen sulfide (H2S) in inflammation remains unclear with both pro‐ and anti‐inflammatory actions of this gas described. We have now assessed the effect of GYY4137 (a slow‐releasing H2S donor) on lipopolysaccharide (LPS)‐evoked release of inflammatory mediators from human synoviocytes (HFLS) and articular chondrocytes (HAC) in vitro. We have also examined the effect of GYY4137 in a complete Freund's adjuvant (CFA) model of acute joint inflammation in the mouse. GYY4137 (0.1–0.5 mM) decreased LPS‐induced production of nitrite (NO2−), PGE2, TNF‐α and IL‐6 from HFLS and HAC, reduced the levels and catalytic activity of inducible nitric oxide synthase (iNOS) and cyclooxygenase‐2 (COX‐2) and reduced LPS‐induced NF‐κB activation in vitro. Using recombinant human enzymes, GYY4137 inhibited the activity of COX‐2, iNOS and TNF‐α converting enzyme (TACE). In the CFA‐treated mouse, GYY4137 (50 mg/kg, i.p.) injected 1 hr prior to CFA increased knee joint swelling while an anti‐inflammatory effect, as demonstrated by reduced synovial fluid myeloperoxidase (MPO) and N‐acetyl‐β‐D‐glucosaminidase (NAG) activity and decreased TNF‐α, IL‐1β, IL‐6 and IL‐8 concentration, was apparent when GYY4137 was injected 6 hrs after CFA. GYY4137 was also anti‐inflammatory when given 18 hrs after CFA. Thus, although GYY4137 consistently reduced the generation of pro‐inflammatory mediators from human joint cells in vitro, its effect on acute joint inflammation in vivo depended on the timing of administration.

Adipose tissue hormone leptin induces endothelium-dependent vasorelaxation mediated by nitric oxide (NO) and endothelium-derived hyperpolarizing factors (EDHF). Previously it has been demonstrated that in short-term obesity the NO-dependent and the EDHF-dependent components of vascular effect of leptin are impaired and up-regulated, respectively. Herein we examined the mechanism of the EDHF-dependent vasodilatory effect of leptin and tested the hypothesis that alterations of acute vascular effects of leptin in obesity are accounted for by chronic hyperleptinemia. The study was performed in 5 groups of rats: (1) control, (2) treated with exogenous leptin for 1 week to induce hyperleptinemia, (3) obese, fed highly-palatable diet for 4 weeks, (4) obese treated with pegylated superactive rat leptin receptor antagonist (PEG-SRLA) for 1 week, (5) fed standard chow and treated with PEG-SRLA. Acute effect of leptin on isometric tension of mesenteric artery segments was measured ex vivo. Leptin relaxed phenylephrine-preconstricted vascular segments in NO- and EDHF-dependent manner. The NO-dependent component was impaired and the EDHF-dependent component was increased in the leptin-treated and obese groups and in the latter group both these effects were abolished by PEG-SRLA. The EDHF-dependent vasodilatory effect of leptin was blocked by either the inhibitor of cystathionine γ-lyase, propargylglycine, or a hydrogen sulfide (H2S) scavenger, bismuth (III) subsalicylate. The results indicate that NO deficiency is compensated by the up-regulation of EDHF in obese rats and both effects are accounted for by chronic hyperleptinemia. The EDHF-dependent component of leptin-induced vasorelaxation is mediated, at least partially, by H2S.

Hydrogen sulfide (H2S) has recently been proposed as an endogenous mediator of inflammation and is present in human synovial fluid. This study determined whether primary human articular chondrocytes (HACs) and mesenchymal progenitor cells (MPCs) could synthesize H2S in response to pro‐inflammatory cytokines relevant to human arthropathies, and to determine the cellular responses to endogenous and pharmacological H2S. HACs and MPCs were exposed to IL‐1β, IL‐6, TNF‐α and lipopolysaccharide (LPS). The expression and enzymatic activity of the H2S synthesizing enzymes cystathionine‐β‐synthase (CBS) and cystathionine‐γ‐lyase (CSE) were determined by Western blot and zinc‐trap spectrophotometry, respectively. Cellular oxidative stress was induced by H2O2, the peroxynitrite donor SIN‐1 and 4‐hydroxynonenal (4‐HNE). Cell death was assessed by 3‐(4,5‐dimethyl‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays. Mitochondrial membrane potential (DCm) was determined in situ by flow cytometry. Endogenous H2S synthesis was inhibited by siRNA‐mediated knockdown of CSE and CBS and pharmacological inhibitors D,L‐propargylglycine and aminoxyacetate, respectively. Exogenous H2S was generated using GYY4137. Under basal conditions HACs and MPCs expressed CBS and CSE and synthesized H2S in a CBS‐dependent manner, whereas CSE expression and activity was induced by treatment of cells with IL‐1β, TNF‐α, IL‐6 or LPS. Oxidative stress‐induced cell death was significantly inhibited by GYY4137 treatment but increased by pharmacological inhibition of H2S synthesis or by CBS/CSE‐siRNA treatment. These data suggest CSE is an inducible source of H2S in cultured HACs and MPCs. H2S may represent a novel endogenous mechanism of cytoprotection in the inflamed joint, suggesting a potential opportunity for therapeutic intervention.

Background: We have evidence that hydrogen sulfide (H2S)-releasing compounds can reduce intraocular pressure in normotensive and glaucomatous rabbits by increasing the aqueous humor (AH) outflow through the trabecular meshwork. Since H2S has been reported to possess neuroprotective actions, the prevention of retinal ganglion cell loss is an important strategy in the pharmacotherapy of glaucoma. Consequently, the present study aimed to investigate the neuroprotective actions of H2S-releasing compounds against hydrogen peroxide (H2O2)-induced oxidative stress in an isolated bovine retina. Materials and Methods: The isolated neural retinae were pretreated with a substrate for H2S biosynthesis called L-cysteine, with the fast H2S-releasing compound sodium hydrosulfide, and with a mitochondrial-targeting H2S-releasing compound, AP123, for thirty minutes before a 30-min oxidative insult with H2O2 (100 µM). Lipid peroxidation was assessed via an enzyme immunoassay by measuring the stable oxidative stress marker, 8-epi PGF2α (8-isoprostane), levels in the retinal tissues. To determine the role of endogenous H2S, studies were performed using the following biosynthesis enzyme inhibitors: aminooxyacetic acid (AOAA, 30 µM); a cystathione-β-synthase/cystathionine-γ-lyase (CBS/CSE) inhibitor, α–ketobutyric acid (KBA, 1 mM); and a 3-mercaptopyruvate-s-sulfurtransferase (3-MST) inhibitor, in the absence and presence of H2S-releasing compounds. Results: Exposure of the isolated retinas to H2O2 produced a time-dependent (10–40 min) and concentration-dependent (30–300 µM) increase in the 8-isoprostane levels when compared to the untreated tissues. L-cysteine (10 nM–1 µM) and NaHS (30 –100 µM) significantly (p < 0.001; n = 12) prevented H2O2-induced oxidative damage in a concentration-dependent manner. Furthermore, AP123 (100 nM–1 µM) attenuated oxidative H2O2 damage resulted in an approximated 60% reduction in 8-isoprostane levels compared to the tissues treated with H2O2 alone. While AOAA (30 µM) and KBA (1 mM) did not affect the L-cysteine evoked attenuation of H2O2-induced oxidative stress, KBA reversed the antioxidant responses caused by AP123. Conclusions: In conclusion, various forms of H2S-releasing compounds and the substrate, L-cysteine, can prevent H2O2-induced lipid peroxidation in an isolated bovine retina.

Donors of H 2 S may be beneficial in treating cardiovascular diseases where the plasma levels of H 2 S are decreased. Therefore, we investigated the mechanisms involved in relaxation of small arteries induced by GYY4137 [(4-methoxyphenyl)-morpholin-4-yl-sulfanylidene-sulfido-λ5-phosphane;morpholin-4-ium], which is considered a slow-releasing H 2 S donor. Sulfides were measured by use of 5,5′-dithiobis-(2-nitro benzoic acid), and small rat mesenteric arteries with internal diameters of 200–250 µm were mounted in microvascular myographs for isometric tension recordings. GYY4137 produced similar low levels of sulfides in the absence and the presence of arteries. In U46619-contracted small mesenteric arteries, GYY4137 (10 −6 –10 –3  M) induced concentration-dependent relaxations, while a synthetic, sulfur-free, GYY4137 did not change the vascular tone. L-cysteine (10 −6 –10 –3  M) induced only small relaxations reaching 24 ± 6% at 10 –3  M. Premixing L-cysteine (10 –3  M) with Na 2 S and GYY4137 decreased Na 2 S relaxation and abolished GYY4137 relaxation, an effect prevented by an nitric oxide (NO) synthase inhibitor, L-NAME (N ω -nitro-L-arginine methyl ester). In arteries without endothelium or in the presence of L-NAME, relaxation curves for GYY4137 were rightward shifted. High extracellular K + concentrations decreased Na 2 S and abolished GYY4137 relaxation suggesting potassium channel-independent mechanisms are also involved Na 2 S relaxation while potassium channel activation is pivotal for GYY4137 relaxation in small arteries. Blockers of large-conductance calcium-activated (BK Ca ) and voltage-gated type 7 (K V 7) potassium channels also inhibited GYY4137 relaxations. The present findings suggest that L-cysteine by reaction with Na 2 S and GYY4137 and formation of sulfides, inhibits relaxations by these compounds. The low rate of release of H 2 S species from GYY4137 is reflected by the different sensitivity of these relaxations towards high K + concentration and potassium channel blockers compared with Na 2 S. The perspective is that the rate of release of sulfides plays an important for the effects of H 2 S salt vs. donors in small arteries, and hence for a beneficial effect of GYY4137 for treatment of cardiovascular disease.

Hydrogen sulfide (H2S) has been shown in previous studies to cause hypothermia and hypometabolism in mice, and its thermoregulatory effects were subsequently investigated. However, the molecular target through which H2S triggers its effects on deep body temperature has remained unknown. We investigated the thermoregulatory response to fast-(Na2S) and slow-releasing (GYY4137) H2S donors in C57BL/6 mice, and then tested whether their effects depend on the transient receptor potential ankyrin-1 (TRPA1) channel in Trpa1 knockout (Trpa1−/−) and wild-type (Trpa1+/+) mice. Intracerebroventricular administration of Na2S (0.5–1 mg/kg) caused hypothermia in C57BL/6 mice, which was mediated by cutaneous vasodilation and decreased thermogenesis. In contrast, intraperitoneal administration of Na2S (5 mg/kg) did not cause any thermoregulatory effect. Central administration of GYY4137 (3 mg/kg) also caused hypothermia and hypometabolism. The hypothermic response to both H2S donors was significantly (p < 0.001) attenuated in Trpa1−/− mice compared to their Trpa1+/+ littermates. Trpa1 mRNA transcripts could be detected with RNAscope in hypothalamic and other brain neurons within the autonomic thermoeffector pathways. In conclusion, slow- and fast-releasing H2S donors induce hypothermia through hypometabolism and cutaneous vasodilation in mice that is mediated by TRPA1 channels located in the brain, presumably in hypothalamic neurons within the autonomic thermoeffector pathways.

Mutations in the dystrophin gene cause Duchenne muscular dystrophy (DMD), a common muscle disease that manifests with muscle weakness, wasting, and degeneration. An emerging theme in DMD pathophysiology is an intramuscular deficit in the gasotransmitter hydrogen sulfide (H 2 S). Here we show that the C. elegans DMD model displays reduced levels of H 2 S and expression of genes required for sulfur metabolism. These reductions can be offset by increasing bioavailability of sulfur containing amino acids (L-methionine, L-homocysteine, L-cysteine, L-glutathione, and L-taurine), augmenting healthspan primarily via improved calcium regulation, mitochondrial structure and delayed muscle cell death. Additionally, we show distinct differences in preservation mechanisms between sulfur amino acid vs H 2 S administration, despite similarities in required health-preserving pathways. Our results suggest that the H 2 S deficit in DMD is likely caused by altered sulfur metabolism and that modulation of this pathway may improve DMD muscle health via multiple evolutionarily conserved mechanisms. A C. elegans model of Duchenne muscular dystrophy reveals a potential role for disrupted sulfur metabolism in the disease and thus the therapeutic potential of sulfur amino acid supplementation.

Cold preservation is the standard of care for renal grafts. However, research on alternatives like perfusion at higher temperatures and supplementing preservation solutions with hydrogen sulfide (H2S) has gained momentum. In this study, we investigated whether adding H2S donor AP39 to porcine blood during subnormothermic perfusion at 21 °C improves renal graft outcomes. Porcine kidneys were nephrectomized after 30 min of clamping the renal pedicles and treated to 4 h of static cold storage (SCS) on ice or ex vivo subnormothermic perfusion at 21 °C with autologous blood alone (SNT) or with AP39 (SNTAP). All kidneys were reperfused ex vivo with autologous blood at 37 °C for 4 h. Urine output, histopathology and RNAseq were used to evaluate the renal graft function, injury and gene expression profiles, respectively. The SNTAP group exhibited significantly higher urine output than other groups during preservation and reperfusion, along with significantly lower apoptotic injury compared to the SCS group. The SNTAP group also exhibited differential pro-survival gene expression patterns compared to the SCS (downregulation of pro-apoptotic genes) and SNT (downregulation of hypoxia response genes) groups. Subnormothermic perfusion at 21 °C with H2S-supplemented blood improves renal graft outcomes. Further research is needed to facilitate the clinical translation of this approach.