Peter Rose

Hydrogen Sulfide and Cell Signaling

Hydrogen sulfide (H₂S) is a gaseous mediator synthesized from cysteine by cystathionine γ lyase (CSE) and other naturally occurring enzymes. Pharmacological experiments using H₂S donors and genetic experiments using CSE knockout mice suggest important roles for this vasodilator gas in the regulation of blood vessel caliber, cardiac response to ischemia/reperfusion injury, and inflammation. That H₂S inhibits cytochrome c oxidase and reduces cell energy production has been known for many decades, but more recently, a number of additional pharmacological targets for this gas have been identified. H₂S activates K(ATP) and transient receptor potential (TRP) channels but usually inhibits big conductance Ca²(+)-sensitive K(+) (BK(Ca)) channels, T-type calcium channels, and M-type calcium channels. H₂S may inhibit or activate NF-κB nuclear translocation while affecting the activity of numerous kinases including p38 mitogen-activated protein kinase (p38 MAPK), extracellular signal-regulated kinase (ERK), and Akt. These disparate effects may be secondary to the well-known reducing activity of H₂S and/or its ability to promote sulfhydration of protein cysteine moieties within the cell.

The effect of hydrogen sulfide donors on lipopolysaccharide-induced formation of inflammatory mediators in macrophages

The role of hydrogen sulfide (H(2)S) in inflammation is controversial, with both pro- and antiinflammatory effects documented. Many studies have used simple sulfide salts as the source of H(2)S, which give a rapid bolus of H(2)S in aqueous solutions and thus do not accurately reflect the enzymatic generation of H(2)S. We therefore compared the effects of sodium hydrosulfide and a novel slow-releasing H(2)S donor (GYY4137) on the release of pro- and antiinflammatory mediators in lipopolysaccharide (LPS)-treated murine RAW264.7 macrophages. For the first time, we show that GYY4137 significantly and concentration-dependently inhibits LPS-induced release of proinflammatory mediators such as IL-1beta, IL-6, TNF-alpha, nitric oxide (*NO), and PGE(2) but increased the synthesis of the antiinflammatory chemokine IL-10 through NF-kappaB/ATF-2/HSP-27-dependent pathways. In contrast, NaHS elicited a biphasic effect on proinflammatory mediators and, at high concentrations, increased the synthesis of IL-1beta, IL-6, NO, PGE(2) and TNF-alpha. This study clearly shows that the effects of H(2)S on the inflammatory process are complex and dependent not only on H(2)S concentration but also on the rate of H(2)S generation. This study may also explain some of the apparent discrepancies in the literature regarding the pro- versus antiinflammatory role of H(2)S.

Peroxynitrite mediates calcium-dependent mitochondrial dysfunction and cell death via activation of calpains

Chondrocyte cell death is a hallmark of inflammatory and degenerative joint diseases such as rheumatoid arthritis (RA) and osteoarthritis (OA), but the molecular and cellular mechanisms involved have yet to be elucidated. Because 3‐nitrotyrosine, a marker for reactive nitrogen species such as peroxynitrite, has been observed in OA and RA cartilage and has been associated with chondrocyte cell death, we investigated the mechanisms by which peroxynitrite induces cell death in human articular chondrocytes. The earliest biochemical event observed, subsequent to treatment with either peroxynitrite or the peroxynitrite generator SIN‐1, was a rapid rise in intracellular calcium that lead to mitochondrial dysfunction and cell death. Although, chondrocyte death exhibited several classical hallmarks of apoptosis, including annexin V labeling, increased fraction of cells with subG1 DNA content and DNA condensation, we did not find evidence for caspase involvement either by Western blotting, fluorimetric assays, or caspase inhibition. Additionally, peroxynitrite did not inhibit cellular caspase activity. Furthermore, using other established assays of cell viability, including the MTT assay and release of lactate dehydrogenase, we found that the predominant mode of cell death involved calcium‐dependent cysteine proteases, otherwise known as calpains. Our data show, for the first time, that peroxynitrite induces mitochondrial dysfunction in cells via a calcium‐dependent process that leads to caspase‐independent apoptosis mediated by calpains.

Protective Effects of Cysteine Analogues on Acute Myocardial Ischemia: Novel Modulators of Endogenous H2S Production

The current study was designed to evaluate the pharmacologic effects of three novel cysteine-containing compounds: S-propyl-l-cysteine (SPC), S-allyl-l-cysteine (SAC), and S-propargyl-l-cysteine (SPRC) on H(2)S production and antioxidant defenses in an acute myocardial infarction (MI) rat model. The enzymatic activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), as well as glutathione redox status and malonaldehyde (MDA) content, also were determined. All three compounds were found to preserve SOD and GPx activities and also tissue GSH levels while reducing the formation of the lipid peroxidation product MDA in ventricular tissues. With immunfluorescence assays, we observed the expression of CSE and Mn-SOD. The morphologic changes of the cardiac cells are seen with both light and electron microscopy. The corresponding pathologic alterations were characterized mainly as loss of adherence between cardiac myocytes and swollen or ruptured mitochondria at the ultrastructural level. Propargylglycine, a selective inhibitor of CSE, abolished the protective effects of each compound used in the current model. Our study provides novel evidence that SPC, SAC, and SPRC have cardioprotective effects in MI by reducing the deleterious effects of oxidative stress by modulating the endogenous levels of H(2)S and preserving the activities of antioxidant defensive enzymes like SOD.

S-propargyl-cysteine protects both adult rat hearts and neonatal cardiomyocytes from ischemia/hypoxia injury: the contribution of the hydrogen sulfide-mediated pathway.

In this study, we determined the cardioprotective effects of S-propargyl-cysteine (SPRC), a structural analog of S-allylcysteine (SAC), using in vivo models of acute myocardial infarction (MI) and in vitro hypoxic cardiomyocytes models. MI was created in rats by ligating the left anterior descending coronary artery. Plasma enzymes levels and cystathionine-γ-lyase (CSE) activities were determined. Primary cultures of newborn rats cardiomyocytes were injured by hypoxia for 6 h. Cell viabilities were measured with the thiazolyl blue assay. RT-PCR and western blot analysis revealed the expression of CSE in both models. The protective effects of SPRC were associated with an observed reduction in infarct size (20.8 ± 2.4% vs. 36.0 ± 1.3%), decreased plasma enzymes levels and reduced malondialdehyde levels when compared to the MI vehicle group (P < 0.05); cardiac function was also improved. SPRC increased CSE activity and plasma H2S concentration by 1.6-fold and 1.3-fold, respectively, in MI rats. Decreased cell viability (64.5 ± 5.4%) in hypoxic cardiomyocytes could be rescued with use of SPRC (81.0 ± 3.1%). Similarly, mRNA and protein expression of CSE were upregulated in the SPRC group. Treatment with the CSE inhibitor propargylglycine abolished the protective effects of SPRC. Our study provides novel evidence that SPRC is protective in myocardial infarctions via a H2S-related pathway.

Malignant transformation of acoustic neuroma/vestibular schwannoma 10 years after gamma knife stereotactic radiosurgery.

Only a handful of cases of de-novo malignancies of the vestibulocochlear nerve have been reported. Even rarer is the malignant transformation of a previously histologically diagnosed benign vestibular schwannoma. We present the case of a young adult who had combined operative/Gamma knife treatment for a benign vestibular schwannoma, followed by further surgery 2 years later. He represented 10 years after original diagnosis with facial numbness and ataxia, MRI showing gross tumor recurrence. After radical resection, histology showed malignant transformation to a malignant peripheral nerve sheath tumor. Within 3 months there was rapid, aggressive recurrence with brainstem compression, requiring further surgery for brainstem decompression. Histology confirmed further de-differentiation to an anaplastic sarcoma. While awaiting radiotherapy the tumor recurred again, the patient succumbing. The patient had no features of neurofibromatosis type 2. In the literature there are 13 other cases of malignant vestibular schwannomata. Only six had radiotherapy and of these only two had histological confirmation of a benign lesion preradiotherapy. Neither of these had neurofibromatosis. Three other cases had histological proof of malignancy postradiosurgery, but with no preradiotherapy histology; of these, two were positive for neurofibromatosis. The tumor biology of vestibular schwannomata as well as the radiobiology in the context of malignant transformation is discussed.

Hydrogen Sulfide Is an Endogenous Regulator of Aging in Caenorhabditis elegans

AIMSnTo investigate the role of endogenous hydrogen sulfide (H2S) in the control of aging and healthspan of Caenorhabditis elegans.nnnRESULTSnWe show that the model organism, C. elegans, synthesizes H2S. Three H2S-synthesizing enzymes are present in C. elegans, namely cystathionine γ lyase (CSE), cystathionine β synthetase, and 3-mercaptopyruvate transferase (MPST or 3-MST). Genetic deficiency of mpst-1 (3-MST orthologue 1), but not cth-2 (CSE orthologue), reduced the lifespan of C. elegans. This effect was reversed by a pharmacological H2S donor (GYY4137). GYY4137 also reduced detrimental age-dependent changes in a range of physiological indices, including pharyngeal contraction and defecation. Treatment of C. elegans with GYY4137 increased the expression of several age-related, stress response, and antioxidant genes, whereas MitoSOX Red fluorescence, indicative of reactive oxygen species generation, was increased in mpst-1 knockouts and decreased by GYY4137 treatment. GYY4137 additionally increased the lifespan in short-lived mev-1 mutants with elevated oxidative stress and protected wild-type C. elegans against paraquat poisoning. The lifespan-prolonging and health-promoting effects of H2S in C. elegans are likely due to the antioxidant action of this highly cell-permeable gas.nnnINNOVATIONnThe possibility that novel pharmacological agents based on the principle of H2S donation may be able to retard the onset of age-related disease by slowing the aging process warrants further study.nnnCONCLUSIONnOur results show that H2S is an endogenous regulator of oxidative damage, metabolism, and aging in C. elegans and provide new insight into the mechanisms, which control aging in this model organism.

H2S biosynthesis and catabolism: new insights from molecular studies

Hydrogen sulfide (H2S) has profound biological effects within living organisms and is now increasingly being considered alongside other gaseous signalling molecules, such as nitric oxide (NO) and carbon monoxide (CO). Conventional use of pharmacological and molecular approaches has spawned a rapidly growing research field that has identified H2S as playing a functional role in cell-signalling and post-translational modifications. Recently, a number of laboratories have reported the use of siRNA methodologies and genetic mouse models to mimic the loss of function of genes involved in the biosynthesis and degradation of H2S within tissues. Studies utilising these systems are revealing new insights into the biology of H2S within the cardiovascular system, inflammatory disease, and in cell signalling. In light of this work, the current review will describe recent advances in H2S research made possible by the use of molecular approaches and genetic mouse models with perturbed capacities to generate or detoxify physiological levels of H2S gas within tissues.

GYY4137, a novel water-soluble, H2S-releasing molecule.

Hydrogen sulfide (H2S) is now recognized as the so called third gasotransmitter taking its place alongside nitric oxide and carbon monoxide. In recent years, H2S has been reported to exhibit a diverse range of pharmacological effects in biological systems. Much of this evidence is derived from a combination of conventional pharmacological and genetic approaches coupled with the use of chemical compounds such as sodium hydrosulfide, a rapid H2S releasing donor. Developments in the design of new drug entities which attempt to take into account physicochemical properties, targeting to specific cellular organelles, triggering of H2S release upon specific chemical reactions in the cell, and controlling the release of H2S over extended periods of time have been described. For most of these molecules, little or no work has been conducted to determine their biological activity or possible therapeutic effects. It is therefore not clear whether such molecules have therapeutic potential which highlights the need for further in vivo studies. One exception to the general rule is GYY4137 (morpholin-4-ium 4-methoxyphenyl(morpholino) phosphinodithioate), a slow releasing H2S donor, which has been evaluated for activity in a range of pharmacological models both in vitro and in vivo. GYY4137 was first reported to release H2S and exhibit vasodilator activity over 5 years ago and, to date, GYY4137 is becoming increasingly employed as a pharmacological tool to explore the biological functions of H2S.

ZYZ-168 alleviates cardiac fibrosis after myocardial infarction through inhibition of ERK1/2-dependent ROCK1 activation

Selective treatments for myocardial infarction (MI) induced cardiac fibrosis are lacking. In this study, we focus on the therapeutic potential of a synthetic cardio-protective agent named ZYZ-168 towards MI-induced cardiac fibrosis and try to reveal the underlying mechanism. ZYZ-168 was administered to rats with coronary artery ligation over a period of six weeks. Ecocardiography and Masson staining showed that ZYZ-168 substantially improved cardiac function and reduced interstitial fibrosis. The expression of α–smooth muscle actin (α-SMA) and Collagen I were reduced as was the activity of matrix metalloproteinase 9 (MMP-9). These were related with decreased phosphorylation of ERK1/2 and expression of Rho-associated coiled-coil containing protein kinase 1 (ROCK1). In cardiac fibroblasts stimulated with TGF-β1, phenotypic switches of cardiac fibroblasts to myofibroblasts were observed. Inhibition of ERK1/2 phosphorylation or knockdown of ROCK1 expectedly reduced TGF-β1 induced fibrotic responses. ZYZ-168 appeared to inhibit the fibrotic responses in a concentration dependent manner, in part via a decrease in ROCK 1 expression through inhibition of the phosphorylation status of ERK1/2. For inhibition of ERK1/2 phosphorylation with a specific inhibitor reduced the activation of ROCK1. Considering its anti-apoptosis activity in MI, ZYZ-168 may be a potential drug candidate for treatment of MI-induced cardiac fibrosis.