A novel slow-releasing hydrogen sulfide donor, FW1256, exerts anti-inflammatory effects in mouse macrophages and in vivo.

Exogenous hydrogen sulfide (H2S) is known to exert anti-inflammatory effects both in macrophages and in animal models. In this study, we first showed that NaHS caused a concentration dependent reduction in TNFα and IL-6 secretion in LPS-stimulated RAW264.7 macrophages in the absence of cell death. Thereafter, we screened a series of novel slow H2S donors for similar activity. One such compound, FW1256, concentration dependently decreased TNFα, IL-6, PGE2 and NO generation in LPS-stimulated RAW264.7 macrophages and BMDMs. FW1256 also significantly reduced IL-1ß, COX-2 and iNOS mRNA and protein in LPS-stimulated RAW264.7 macrophages. Mechanistically, FW1256 decreased NFκB activation as evidenced by reduced cytosolic phospho-IκBα levels and reduced nuclear p65 levels in LPS-stimulated RAW264.7 macrophages treated with FW1256. Using a H2S fluorescent probe in FW1256-treated RAW264.7 macrophages, H2S release from FW1256 was apparent over a period of 24h in these cells. Moreover, the effect of FW1256 on TNFα and IL-6 by FW1256 in LPS-stimulated RAW264.7 macrophages was reversed by treatment with the H2S scavenger, vitamin B12a. FW1256 had no cytotoxic effect on LPS-stimulated RAW264.7 macrophages or BMDMs. In vivo, FW1256 administration also reduced IL-1ß, TNFα, nitrate/nitrite and PGE2 levels in LPS-treated mice. We show here a novel slow H2S-releasing compound that exerts anti-inflammatory effects in macrophages and in vivo. FW1256 may be a useful tool to study the biological effects of exogenous H2S and could also have future therapeutic value in inflammatory conditions.

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

AIMS: Myocardial 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 RESULTS: Treatment for 2 or 7 days with GYY (100 mg/Kg/48 h, 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. CONCLUSIONS: Our 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.

Hydrogen sulfide promotes adipogenesis in 3T3L1 cells.

The effect of hydrogen sulfide (H2S) on differentiation of 3T3L1-derived adipocytes was examined. Endogenous H2S was increased after 3T3L1 differentiation. The expression of the H2S-synthesising enzymes, cystathionine γ-lyase (CSE), cystathionine ß-synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (3-MST), was increased in a time-dependent manner during 3T3L1 differentiation. Expression of genes associated with adipogenesis related genes including fatty acid binding protein 4 (FABP4/aP2), a key regulator of this process, was increased by GYY4137 (a slow-releasing H2S donor compound) and sodium hydrosulfide (NaHS, a classical H2S donor) but not by ZYJ1122 or time-expired NaHS. Furthermore expression of these genes were reduced by aminooxyacetic acid (AOAA, CBS inhibitor), DL-propargylglycine (PAG, CSE inhibitor) as well as by CSE small interference RNA (siCSE) and siCBS. The size and number of lipid droplets in mature adipocytes was significantly increased by both GYY4137 and NaHS, which also impaired the ability of CL316,243 (ß3-agonist) to promote lipolysis in these cells. In contrast, AOAA and PAG had the opposite effect. Taken together, we show that the H2S-synthesising enzymes CBS, CSE and 3-MST are endogenously expressed during adipogenesis and that both endogenous and exogenous H2S modulate adipogenesis and adipocyte maturation.

Effect of feeding a high fat diet on hydrogen sulfide (H2S) metabolism in the mouse.

Hydrogen sulfide (H2S) has complex effects in inflammation with both pro- and anti-inflammatory actions of this gas reported. Recent work suggests that a deficiency of H2S occurs in, and may contribute to, the chronic inflammation which underpins ongoing atherosclerotic disease. However, whether a high fat diet, predisposing to atherosclerosis, affects H2S metabolism is not known. In this study we assessed H2S metabolism in different tissues of mice fed a high fat diet for up to 16 weeks. Ex vivo biosynthesis of H2S was reduced in liver, kidney and lung of high fat fed mice. Western blotting revealed deficiency of cystathionine γ lyase (CSE) in liver and lung with increased expression of cystathionine ß synthetase (CBS) in liver and kidney. Expression of 3-mercaptopyruvate sulfurtransferase (3-MST) was reduced in liver but not other tissues. Aortic endothelial cell CSE was also reduced in high fat fed animals as determined immunohistochemically. Plasma H2S concentration was not changed in these animals. No evidence of lipid deposition was apparent in aortae from high fat fed animals and plasma serum amyloid A (SAA) and C-reactive protein (CRP) were also unchanged suggesting lack of frank atherosclerotic disease. Plasma IL-6, IL12p40 and G-CSF levels were increased by high fat feeding whilst other cytokines including IL-1α, IL-1b and TNF-α were not altered. These results suggest that deficiency of tissue CSE and H2S occurs in mice fed a high fat diet and that this change takes place prior to development of frank atherosclerotic disease.

Hydrogen sulfide is an endogenous regulator of aging in Caenorhabditis elegans.

AIMS: To investigate the role of endogenous hydrogen sulfide (H2S) in the control of aging and healthspan of Caenorhabditis elegans. RESULTS: We show that the model organism, C. elegans, synthesizes H2S. Three H2S-synthesizing enzymes are present in C. elegans, namely cystathionine γ lyase (CSE), cystathionine ß 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. INNOVATION: The 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. CONCLUSION: Our 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.

The hydrogen sulfide donor, GYY4137, exhibits anti-atherosclerotic activity in high fat fed apolipoprotein E(-/-) mice.

BACKGROUND AND PURPOSE: Atherosclerosis is associated with reduced vascular hydrogen sulfide (H2 S) biosynthesis. GYY4137 is a novel slow-releasing H2 S compound that may effectively mimic the time course of H2 S release in vivo. However, it is not known whether GYY4137 affects atherosclerosis. EXPERIMENTAL APPROACH: RAW 264.7 cells and human blood monocyte-derived macrophages were incubated with oxidized low density lipoprotein (ox-LDL) with/without GYY4137. ApoE(-/-) mice were fed a high-fat diet for 4 weeks and administered GYY4137 for 30 days. Lipid and atherosclerotic lesions were measured by oil red O staining. Endothelium-dependent relaxation was assessed in response to acetylcholine. Superoxide production was detected by dihydroethidium staining. Expression of mRNA and protein were evaluated by quantitative real-time PCR and Western blot. KEY RESULTS: GYY4137 inhibited ox-LDL-induced foam cell formation and cholesterol esterification in cultured cells. GYY4137 decreased the expression of lectin-like ox-LDL receptor-1, iNOS, phosphorylated IκBα, NF-κB, ICAM-1, VCAM-1 and chemokines, including CXCL2, CXCR4, CXCL10 and CCL17, but increased the scavenger protein CD36, in ox-LDL-treated RAW 264.7 cells. In vivo, GYY4137 decreased aortic atherosclerotic plaque formation and partially restored aortic endothelium-dependent relaxation in apoE(-/-) mice. GYY4137 decreased ICAM-1, TNF-α and IL-6 mRNA expression as well as superoxide (O2 (-) ) generation in aorta. In addition, GYY4137 increased aortic eNOS phosphorylation and expression of PI3K, enhanced Akt Ser(473) phosphorylation and down-regulated the expression of LOX-1. CONCLUSION AND IMPLICATIONS: GYY4137 inhibits lipid accumulation induced by ox-LDL in RAW 264.7 cells. In vivo, GYY4137 decreased vascular inflammation and oxidative stress, improved endothelial function and reduced atherosclerotic plaque formation in apoE(-/-) mice.

Aspirin regulates SNARE protein expression and phagocytosis in dendritic cells.

Since being introduced globally as Aspirin in 1899, acetylsalicylic acid (ASA) has been widely used as an analgesic, immune-regulatory, anti-pyretic and anti-thrombotic drug. ASA and its metabolite, salicylate, were also reported to be able to modulate antigen presenting functions of dendritic cells (DC). However, the intracellular targets of ASA in DC are still poorly understood. Since phagocytosis is the initial step taken by antigen-presenting cells in the uptake of antigens for processing and presentation, ASA might exerts its immune-regulatory effects by regulating phagocytosis. Here we show that ASA inhibits phagocytosis and modulates expression of endosomal SNAREs, such as Vti1a, Vti1b, VAMP-3, VAMP-8 and Syn-8 (but not syn-6 and syn-16) in DC. We further show that the phagocytic inhibitory effect of ASA is dependent on the expression of Vti1a and Vti1b. Consistently, Vti1a and Vti1b localize to the phagosomes and up-regulation of Vti1a and Vti1b inhibits phagocytosis in DC. Our results suggest that ASA modulates phagocytosis in part through the control of endosomal SNARE protein expression and localization in DC. All experiments were performed using either a murine DC line (DC2.4) or primary DC derived from murine bone marrow cells.