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For many years it has been recognized that inhibition of cyclooxygenase enzymes is effective in reducing the incidence of many types of cancer, but the adverse effects of these drug, particularly in the gastrointestinal and cardiovascular systems, limits their utility. Recently developed hydrogen sulfide-releasing anti-inflammatory drugs may be a promising option for cancer chemoprevention. In this paper we review evidence suggesting that these novel compounds are effective in a range of animal models of various types of cancer, while exhibiting greatly reduced toxicity relative to currently marketed non-steroidal anti-inflammatory drugs. Some of the possible mechanisms of action of hydrogen sulfide-releasing anti-inflammatory drugs are also discussed. [ABSTRACT FROM AUTHOR]

Graphical abstract Abstract The incidence and severity of asthma preponderate in women versus men. Leukotrienes (LTs) are lipid mediators involved in asthma pathogenesis, and sex disparities in LT biosynthesis and anti-LT pharmacology in inflammation have recently emerged. Here, we report on sex dimorphism in LT production during allergen sensitization and its correlation to lung function. While high plasma levels of IgE, as sensitization index, were elevated in both sexes, LT levels increased only in lungs of female ovalbumin-sensitized BALB/c mice. Sex-dependent elevated LT levels strictly correlated to an enhanced airway hyperreactivity, pulmonary inflammation and mast cell infiltration/activation in female mice. Importantly, this sex bias was coupled to superior therapeutic efficacy of different types of clinically used LT modifiers like zileuton, MK886 and montelukast in female animals. Our findings reveal sex-dependent LT production as a basic mechanism of sex dimorphism in allergic asthma, and suggest that women might benefit more from anti-LT asthma therapy. [ABSTRACT FROM AUTHOR]

Hydrogen sulfide (H 2 S) is produced by the action of cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE) or 3-mercaptopyruvate sulfurtransferase (3-MST). 3-MST converts 3-mercaptopyruvate (MPT) to H 2 S and pyruvate. H 2 S is recognized as an endogenous gaseous mediator with multiple regulatory roles in mammalian cells and organisms. In the present study we demonstrate that MPT, the endogenous substrate of 3-MST, acts also as endogenous H 2 S donor. Colorimetric, amperometric and fluorescence based assays demonstrated that MPT releases H 2 S in vitro in an enzyme-independent manner. A functional study was performed on aortic rings harvested from C57BL/6 (WT) or 3-MST-knockout (3-MST −/− ) mice with and without endothelium. MPT relaxed mouse aortic rings in endothelium-independent manner and at the same extent in both WT and 3-MST −/− mice. N5-(1-Iminoethyl)- l -ornithine dihydrochloride (L-NIO, an inhibitor of endothelial nitric oxide synthase) as well as 1H-[1,2,4]oxadiazolo [4,3-a]quinoxalin-1-one (ODQ, a soluble guanylyl cyclase inhibitor) did not affect MPT relaxant action. Conversely, hemoglobin (as H 2 S scavenger), as well as glybenclamide (an ATP-dependent potassium channel blocker) markedly reduced MPT-induced relaxation. The functional data clearly confirmed a non enzymatic vascular effect of MPT. In conclusion, MPT acts also as an endogenous H 2 S donor and not only as 3-MST substrate. MPT could, thus, be further investigated as a means to increase H 2 S in conditions where H 2 S bioavailability is reduced such as hypertension, coronary artery disease, diabetes or urogenital tract disease. [ABSTRACT FROM AUTHOR]

The corpus cavernosum (CC) is a highly vascularized tissue and represents an excellent example of microcirculation. Indeed, erectile dysfunction is considered an early index of cardiovascular disease. Hydrogen sulfide (H 2 S) at the vascular level is endogenously produced from L -cysteine mainly by the action of cystathionine-γ-lyase (CSE) and plays a role in CC vascular homeostasis. Here we have evaluated the involvement of the endogenous H 2 S in the regulation of the soluble guanylate cyclase (sCG) redox state. The lack of CSE-derived endogenous H 2 S, in CSE-/- mice, disrupted the eNOS/NO/sGC/PDE pathway. Indeed, the absence of CSE-derived endogenous H 2 S caused a significant reduction of the relaxant response to riociguat, an sGC redox-dependent stimulator. Conversely, the response to cinaciguat, an sGC redox-independent activator, was not modified. The relevance of the role played at the redox level of the endogenous H 2 S was confirmed by the findings that in CC harvested from CSE-/- mice there was a significant reduction of GC β1 expression coupled with a decrease in CYP5R3, a reductase involved in the regulation of the redox state of sGC. These molecular changes driven by the lack of endogenous H 2 S translate into a significant reduction in cGMP levels. The replenishment of the lack of H 2 S with an H 2 S donor rescued the relaxant response to riociguat in CC of CSE-/- mice. In conclusion, the endogenous CSE-derived H 2 S plays a physiological key role in the regulation of the redox state of sGC in CC microcirculation. [Display omitted] • CSE-/- mice showed an impairment in eNOS/NO/sGC/PDE signaling in isolated strips of the corpus cavernosum. • The relaxation of riociguat, an sGC redox-dependent stimulator, was reduced in the corpus cavernosum of CSE−/− mice. • The supplement of H 2 S donor recovered the relaxant response to riociguat in the corpus cavernosum of CSE−/− mice. [ABSTRACT FROM AUTHOR]

Erectile function is a widely accepted indicator of systemic endothelial activity since from a clinical standpoint erectile dysfunction (ED) often precedes cardiovascular events. Recently it has been described a potential role for β 3 adrenoceptor in cardiovascular diseases emphasizing a possible development of new drugs. β 3 adrenoceptor stimulation relaxes human corpus cavernosum (HCC) strips in cyclic guanosine monophosphate (cGMP)-dependent and endothelium/nitric oxide (NO)-independent manner. Hydrogen sulfide (H 2 S), along with NO, is another gaseous molecule involved in cardiovascular system and as a consequence also in penile erection. Cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE), the enzymes mainly responsible for H 2 S biosynthesis, are constitutively expressed in HCC. CSE rather than CBS is more abundant in human penile tissue. Herein we investigated the involvement of H 2 S pathway in β 3 adrenoceptor-induced relaxation in HCC and penile artery. Penile artery expresses both CSE and β 3 adrenoceptor. BRL37344, a β 3 selective agonist, relaxed HCC strips and penile artery rings and this effect was significantly reduced by CSE inhibition. Incubation of HCC and penile artery homogenate with BRL37344 significantly increased H 2 S production. This effect was significantly reduced by the inhibition of either CSE or β 3 adrenoceptor. Finally, the BRL37344-induced increase in cGMP was reduced by CSE inhibition in both tissues. Thus, BRL37344-induced relaxation in HCC and penile artery occurs in a H 2 S/cGMP-dependent manner. In conclusion, β 3 /H 2 S/cGMP pathway can act as an alternative to NO. Since about 15% of patients do not respond to phosphodiesterase-5 inhibitors, β 3 agonists could represent a therapeutic alternative or a useful adjuvant therapy to treat these patients. [ABSTRACT FROM AUTHOR]

Inflammation plays a key role in tumor promotion and development. Indeed, cyclooxygenase-2 (COX-2) expression is strongly associated with different types of cancer. An emerging class of compounds with significant anti-inflammatory properties is the hydrogen sulfide-releasing non-steroidal anti-inflammatory drugs (H 2 S-NSAIDs). They consist of a traditional NSAID to which an H 2 S-releasing moiety is covalently attached. We have recently demonstrated that H 2 S donors inhibit melanoma cell proliferation. In the current study, we evaluated the potential beneficial effects of a new H 2 S-releasing derivative of naproxen, ATB-346 [2-(6-methoxynapthalen-2-yl)-propionic acid 4-thiocarbamoyl phenyl ester] which inhibits COX activity but also releases H 2 S. We used cell culture and a mouse melanoma model to evaluate the effect of ATB-346 on: i) in vitro growth of human melanoma cells; ii) in vivo melanoma development in mice. Cell culture studies demonstrated that ATB-346 reduced the in vitro proliferation of human melanoma cells and this effect was associated to induction of apoptosis and inhibition of NF-κB activation. Moreover, ATB-346 had novel Akt signaling inhibitory properties. Daily oral dosing of ATB-346 (43 μmol/kg) significantly reduced melanoma development in vivo. This study shows that ATB-346, a novel H 2 S-NSAID, inhibits human melanoma cell proliferation by inhibiting pro-survival pathways associated with NF-κB and Akt activation. Furthermore, oral treatment with ATB-346 inhibits melanoma growth in mice. In conclusion, the combination of inhibition of cyclooxygenase and delivery of H 2 S by ATB-346 may offer a promising alternative to existing therapies for melanoma. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is one of the most prevalent forms of neurodegenerative disorders. Previously, we have shown that in vivo administration of an IL-17 neutralizing antibody (IL-17Ab) rescues amyloid‐β‐induced neuro-inflammation and memory impairment, demonstrating the pivotal role of IL-17 in AD-derived cognitive deficit. Recently, AD has been recognized as a more intriguing pathology affecting vascular networks and platelet function. However, not much is known about peripheral vascular inflammation and how pro-inflammatory circulating cells/mediators could affect peripheral vessels' function. This study aimed to evaluate whether IL‐17Ab treatment could also impact peripheral AD features, such as systemic inflammation, peripheral vascular dysfunction, and related pro-thrombotic state in a non-genetic mouse model of AD. Mice were injected intracerebroventricularly with Aβ 1–42 peptide (3 μg/3 μl). To evaluate the systemic/peripheral protective profile of IL-17Ab, we used an intranasal administration of IL-17Ab (1 μg/10 μl) at 5, 12, and 19 days after Aβ 1–42 injection. Circulating Th17/Treg cells and related cyto-chemokines, haematological parameters, vascular/endothelial reactivity, platelets and coagulation function in mice were evaluated. IL-17Ab treatment ameliorates the systemic/peripheral inflammation, immunological perturbance, vascular/endothelial impairment and pro-thrombotic state, suggesting a key role for this cytokine in fostering inflammatory processes that characterize the multifaced aspects of AD. [Display omitted] • Alzheimer's disease onset and progression are sustained by IL-17 with an impact on systemic/peripheral inflammation, endothelial impairment, and pro-aggregative state. • Administration of a neutralizing-IL‐17A antibody rescues AD-related systemic/peripheral immunological and inflammatory-based disfunction of AD. • The non-genetic mouse model of Aβ 1–42 -induced Alzheimer's disease recapitulates the main features of pathology. [ABSTRACT FROM AUTHOR]

Adrenergic β2-agonists represent a mainstay in asthma management. Their chronic use has been associated with decreased bronchoprotection and rebound hyperresponsiveness. Here we investigate on the possible therapeutic advantage of a pharmacological association of β2-agonists with montelukast, a highly selective leukotriene receptor antagonist, in modulating bronchial reactivity and controlling asthma features. The study has been conducted in vitro and in vivo and also takes advantage of the synthesis of a salt that gave us the possibility to simultaneously administer in vivo formoterol and montelukast (MFS). In vitro studies demonstrate that montelukast (1) preserves β2-agonist response in isolated bronchi by preventing homologous β2-adrenoceptor desensitization; (2) reduces desensitization by modulating β2-receptor translocation in bronchial epithelial cells. In vivo studies demonstrate that sensitized mice receiving formoterol or montelukast display a significant reduction in airway hyperresponsiveness, but the β2-agonist relaxing response is still impaired. Allergen challenge causes β 2 heterologous desensitization that is further increased by treatment in vivo with formoterol. Conversely MFS not only inhibits airway hyperresponsiveness but it rescues the β2-agonist response. Histological analysis confirms the functional data, demonstrating an enhanced therapeutic efficiency of MSF in controlling also pulmonary metaplasia and lung inflammation. MFS is efficacious also when sensitized mice received the drug by local administration. In conclusion, the data obtained evidenced a therapeutic advantage in the association of β2-agonists with montelukast in the control of asthma-like features and a better rescue bronchodilation response to β2-agonists. [Display omitted] [ABSTRACT FROM AUTHOR]

The endogenous gasotransmitter hydrogen sulphide (H 2 S) is an important regulator of the cardiovascular system, particularly of myocardial function. Moreover, H 2 S exhibits cardioprotective activity against ischemia/reperfusion (I/R) or hypoxic injury, and is considered an important mediator of “ischemic preconditioning”, through activation of mitochondrial potassium channels, reduction of oxidative stress, activation of the endogenous “anti-oxidant machinery” and limitation of inflammatory responses. Accordingly, H 2 S-donors, i.e. pro-drugs able to generate exogenous H 2 S, are viewed as promising therapeutic agents for a number of cardiovascular diseases. The novel H 2 S-donor 4-carboxy phenyl-isothiocyanate (4CPI), whose vasorelaxing effects were recently reported, was tested here in different experimental models of myocardial I/R. In Langendorff-perfused rat hearts subjected to I/R, 4CPI significantly improved the post-ischemic recovery of myocardial functional parameters and limited tissue injury. These effects were antagonized by 5-hydroxydecanoic acid (a blocker of mitoK ATP channels). Moreover, 4CPI inhibited the formation of reactive oxygen species. We found the whole battery of H 2 S-producing enzymes to be present in myocardial tissue: cystathionine γ-lyase (CSE), cystathionine β-synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (MPST). Notably, 4CPI down-regulated the post-ischemic expression of CSE. In Langendorff-perfused mouse hearts, 4CPI reduced the post-ischemic release of norepinephrine and the incidence of ventricular arrhythmias. In both rat and mouse hearts, 4CPI did not affect the degranulation of resident mast cells. In isolated rat cardiac mitochondria, 4CPI partially depolarized the mitochondrial membrane potential; this effect was antagonized by ATP (i.e., the physiological inhibitor of K ATP channels). Moreover, 4CPI abrogated calcium uptake in the mitochondrial matrix. Finally, in an in vivo model of acute myocardial infarction in rats, 4CPI significantly decreased I/R-induced tissue injury. In conclusion, H 2 S-donors, and in particular isothiocyanate-based H2S-releasing drugs like 4CPI, can actually be considered a suitable pharmacological option in anti-ischemic therapy. [ABSTRACT FROM AUTHOR]

Compelling evidence suggests the involvement of sphingosine-1-phosphate (S1P) in the pathogenesis of asthma. The systemic administration of S1P causes asthma like features in the mouse involving mast cells. In this study we investigated whether disodium cromoglycate (DSCG), administered as a preventative treatment as in human therapy, could affect S1P effects on airways. BALB/c mice, treated with DSCG, received subcutaneous administration of S1P. Bronchi and pulmonary tissues were collected and functional, molecular and cellular studies were performed. DSCG inhibited S1P-induced airway hyper-reactivity as well as pulmonary inflammation. DSCG decreased the recruitment of solely mast cells and B cells in the lung. IgE serum levels, prostaglandin D 2 , mucus production and IL-13 were also reduced when mice were pretreated with DSCG. S1P induced pulmonary expression of CD23 on T and B cells, that was reversed by DSCG. Conversely, S1P failed to upregulate CD23 in mast cell-deficient Kit W−sh/W−sh mice. In conclusion we have shown that DSCG inhibits S1P-induced asthma like features in the mouse. This beneficial effect is due to a regulatory action on mast cell activity, and in turn to an inhibition of IgE-dependent T and B cells responses. [ABSTRACT FROM AUTHOR]

Sildenafil, a selective phosphodiesterase type 5 (PDE5) inhibitor, commonly used in the oral treatment for erectile dysfunction, relaxes smooth muscle of human bladder through the activation of hydrogen sulfide (H 2 S) signaling. H 2 S is an endogenous gaseous transmitter with myorelaxant properties predominantly formed from l -cysteine ( l -Cys) by cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE). Sildenafil also relaxes rat and human myometrium during preterm labor but the underlying mechanism is still unclear. In the present study we investigated the possible involvement of H 2 S as a mediator of sildenafil-induced effect in uterine mouse contractility. We firstly demonstrated that both enzymes, CBS and CSE were expressed, and able to convert l -Cys into H 2 S in mouse uterus. Thereafter, sildenafil significantly increased H 2 S production in mouse uterus and this effect was abrogated by CBS or CSE inhibition. In parallel, l -Cys, sodium hydrogen sulfide or sildenafil but not d -Cys reduced spontaneous uterus contractility in a functional study. The blockage of CBS and CSE reduced this latter effect even if a major role for CSE than CBS was observed. This data was strongly confirmed by using CSE −/− mice. Indeed, the increase in H 2 S production mediated by l -Cys or by sildenafil was not found in CSE −/− mice. Besides, the effect of H 2 S or sildenafil on spontaneous contractility was reduced in CSE −/− mice. A decisive proof for the involvement of H 2 S signaling in sildenafil effect in mice uterus was given by the measurement of cGMP. Sildenafil increased cGMP level that was significantly reduced by CSE inhibition. In conclusion, l -Cys/CSE/H 2 S signaling modulates the mouse uterus motility and the sildenafil effect. Therefore the study may open different therapeutical approaches for the management of the uterus abnormal contractility disorders. [ABSTRACT FROM AUTHOR]

Soluble guanylate cyclase (sGC) is a receptor for nitric oxide (NO). Binding of NO to ferrous (Fe 2+ ) heme increases its catalytic activity, leading to the production of cGMP from GTP. Hydrogen sulfide (H 2 S) is a signaling molecule that exerts both direct and indirect anti-oxidant effects. In the present, study we aimed to determine whether H 2 S could regulate sGC redox state and affect its responsiveness to NO-releasing agents and sGC activators. Using cultured rat aortic smooth muscle cells, we observed that treatment with H 2 S augmented the response to the NO donor DEA/NO, while attenuating the response to the heme-independent activator BAY58-2667 that targets oxidized sGC. Similarly, overexpression of H 2 S-synthesizing enzyme cystathionine-γ lyase reduced the ability of BAY58-2667 to promote cGMP accumulation. In experiments with phenylephrine-constricted mouse aortic rings, treatment with rotenone (a compound that increases ROS production), caused a rightward shift of the DEA/NO concentration-response curve, an effect partially restored by H 2 S. When rings were pre-treated with H 2 S, the concentration-response curve to BAY 58-2667 shifted to the right. Using purified recombinant human sGC, we observed that treatment with H 2 S converted ferric to ferrous sGC enhancing NO-donor-stimulated sGC activity and reducing BAY 58-2667-triggered cGMP formation. The present study identified an additional mechanism of cross-talk between the NO and H 2 S pathways at the level of redox regulation of sGC. Our results provide evidence that H 2 S reduces sGC heme Fe, thus, facilitating NO-mediated cellular signaling events. [ABSTRACT FROM AUTHOR]

Compelling evidence suggests that hydrogen sulfide represents an important gaseous transmitter in the mammalian respiratory system. In the present study, we have evaluated the role of mast cells in hydrogen sulfide-induced effects on airways in a mouse model of asthma. Mice were sensitized to ovalbumin and received aerosol of a hydrogen sulfide donor (NaHS; 100 ppm) starting at day 7 after ovalbumin challenge. Exposure to hydrogen sulfide abrogated ovalbumin-induced bronchial hypereactivity as well as the increase in lung resistance. Concomitantly, hydrogen sulfide prevented mast cell activity as well as FGF-2 and IL-13 upregulation. Conversely, pulmonary inflammation and the increase in plasmatic IgE levels were not affected by hydrogen sulfide. A lack of hydrogen sulfide effects in mast cell deficient mice occurred. Primary fibroblasts harvested from ovalbumin-sensitized mice showed an increased proliferation rate that was inhibited by hydrogen sulfide aerosol. Furthermore, ovalbumin-induced transdifferentiation of pulmonary fibroblasts into myofibroblasts was reversed. Finally, hydrogen sulfide did abrogate in vitro the degranulation of the mast cell-like RBL-2H3 cell line. Similarly to the in vivo experiments the inhibitory effect was present only when the cells were activated by antigen exposure. In conclusion, inhaled hydrogen sulfide improves lung function and inhibits bronchial hyper-reactivity by modulating mast cells and in turn fibroblast activation. [ABSTRACT FROM AUTHOR]

There are numerous gaseous substances that can act as signaling molecules, but the best characterized of these are nitric oxide, hydrogen sulfide and carbon monoxide. Each has been shown to play important roles in many physiological and pathophysiological processes. This article is focused on the effects of these gasotransmitters in the context of inflammation. There is considerable overlap in the actions of nitric oxide, hydrogen sulfide and carbon monoxide with respect to inflammation, and these mediators appear to act primarily as anti-inflammatory substances, promoting resolution of inflammatory processes. They also have protective and pro-healing effects in some tissues, such as the gastrointestinal tract and lung. Over the past two decades, significant progress has been made in the development of novel anti-inflammatory and cytoprotective drugs that release of one or more of these gaseous mediators. [ABSTRACT FROM AUTHOR]

Glucocorticoid (GC)-induced hypertension is a common clinical problem still poorly understood. The presence of GC receptor (GR) in vascular smooth muscle and endothelial cells suggests a direct role for GC in vasculature. In response to hemodynamic shear stress, endothelium tonically releases nitric oxide (NO), endothelial-derived hyperpolarizing factor (EDHF) and prostacyclin contributing to vascular homeostasis. Recently, hydrogen sulfide (H 2 S) has been proposed as a candidate for EDHF. H 2 S is endogenously mainly formed from L-cysteine by the action of cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE). It plays many physiological roles and contributes to cardiovascular function. Here we have evaluated the role played by H 2 S in mesenteric arterial bed and in carotid artery harvested from rats treated with vehicle or dexamethasone (DEX; 1.5 mg/kg/day) for 8 days. During treatments systolic blood pressure was significantly increased in conscious rats. EDHF contribution was evaluated in ex-vivo by performing a concentration–response curve induced by acetylcholine (Ach) in presence of a combination of indomethacin and L-NG-Nitroarginine methyl ester in both vascular districts. EDHF-mediated relaxation was significantly reduced in DEX-treated group in both mesenteric bed and carotid artery. EDHF-mediated relaxation was abolished by pre-treatment with both apamin and charybdotoxin, inhibitors of small and big calcium-dependent potassium channels respectively, or with propargylglycine, inhibitor of CSE. Western blot analysis revealed a marked reduction in CBS and CSE expression as well as H 2 S production in homogenates of mesenteric arterial bed and carotid artery from DEX-treated rats. In parallel, H 2 S plasma levels were significantly reduced in DEX group compared with vehicle. In conclusion, an impairment in EDHF/H 2 S signaling occurs in earlier state of GC-induced hypertension in rats suggesting that counteracting this dysfunction may be beneficial to manage DEX-associated increase in blood pressure. [ABSTRACT FROM AUTHOR]

Benefits for vitamin E intake in diseases with inflammatory components have been described and related in part, to endogenously formed metabolites (long-chain metabolites, LCM). Here, we have evaluated the role of LCM in relieving asthma features. To this aim, the endogenous vitamin E metabolite α-13′-carboxychromanol (α-T-13′-COOH) that acts as potent 5-lipoxygenase inhibitor has been administered either intraperitoneally or by oral gavage to BALB/c mice sensitized by subcutaneous injection of ovalbumin (OVA). We also have taken advantage of the metabolically stable α-T-13′-COOH derivative α-amplexichromanol (α-AC). Intraperitoneal treatment with α-T-13'-COOH reduced OVA-induced airway hyperreactivity (AHR) as well as peri-bronchial inflammatory cell infiltration. α-AC was more efficacious than α-T-13'-COOH, as demonstrated by better control of AHR and in reducing subepithelial. Both compounds exerted their protective function by reducing pulmonary leukotriene C 4 levels. Beneficial effects of α-AC were coupled to inhibition of the sensitization process, as indicated by a reduction of IgE plasma levels, lung mast cell infiltration and Th2 immune response. Metabololipidomics analysis revealed that α-AC raises the pulmonary levels of prostanoids, their degradation products, and 12/15-lipoxygenase metabolites. Following oral administration, the pharmacodynamically different profile in α-T-13′-COOH and α-AC was abrogated as demonstrated by a similar and improved efficacy in controlling asthma features as well as by metabololipidomics analysis. In conclusion, this study highlights a role for LCM and of vitamin E derivatives as pharmacologically active compounds that ameliorate asthmatic features and defines an important role for endogenous vitamin E metabolites in regulating immune response underlying the sensitization process. [Display omitted] • Vitamin E long-chain metabolites relieve allergen-induced bronchial hyperreactivity. • Vitamin E long-chain metabolites inhibit Th2 immune response induced by allergen. • Vitamin E long-chain metabolites modulate anti- and pro-inflammatory lipid mediators [ABSTRACT FROM AUTHOR]

Hydrogen sulfide (H 2 S), a gaseous mediator synthesized in several mammalian tissues by two main enzymes CBS and CSE, increases under inflammatory conditions or sepsis [1,2] . Inflammation per se does not represent an unwanted event, since it also provides a physiological response to an injury, keeping inflammation under control [3] . Annexin A1 (AnxA1) represent the front line of innate immunity as it is externalized on leukocytes membrane, preventing their adhesion to vascular endothelium and their transmigration into inflamed tissues [4] . Since H 2 S affords potent inhibitory properties on the process of leukocyte trafficking [5] , we tested whether endogenous AnxA1 could display intermediary functions. In order to achieve our aim we used different approaches: isolated human polymorphonuclear cells (PMNs), murine bone marrow-derived macrophages (BMDM) and intravital microscopy on mesenteric microcirculation in AnxA1 −/− mice. Treatment of human PMNs with the H 2 S donor NaHS (10–100 μM) provoked prompted and intense mobilization (>50%) of AnxA1 from the cytosolic pool to the cell surface. Such in vitro actions could be translated in the analysis of the inflamed microcirculation, where NaHS (100 μmol/kg s.c., −30 min) afforded marked inhibition of IL1 β -induced cell adhesion and emigration in the mesenteric vessels of wild type, but not AnxA1 −/− , mice. Next, we investigated whether endogenous AnxA1 could link to H 2 S pathway, indicating existence of a positive circuit. To this end, a marked increase in CBS and/or CSE expression in a variety of tissues (aorta, kidney, spleen) tested from AnxA1 −/− mice, as compared to wild type animals, was quantified by qPCR. Moreover, NaHS was able to counteract the increase in expression in iNOS and COX2 (4-fold and 7-fold reduction, respectively) upon LPS-stimulation of BMDM, though it was totally inactive in cells prepared from AnxA1 −/− mice. Taken together, these data strongly suggest – for the first time – the existence of a positive loop between AnxA1 and the H 2 S pathway, providing a novel mechanistic explanation for the exquisite properties of H 2 S in the control of experimental inflammation. These findings may be relevant to innovative discovery programmes aiming at harnessing the biological properties of H 2 S. [ABSTRACT FROM AUTHOR]

The gasotransmitter hydrogen sulfide (H 2 S) is involved in the regulation of the vascular tone and an impairment of its endogenous production may play a role in hypertension. Thus, the administration of exogenous H 2 S may be a possible novel and effective strategy to control blood pressure. Some natural and synthetic sulfur compounds are suitable H 2 S-donors, exhibiting long-lasting H 2 S release; however, novel H 2 S-releasing agents are needed to improve the pharmacological armamentarium for the treatment of cardiovascular diseases. For this purpose, N-phenylthiourea (PTU) and N,N'-diphenylthiourea (DPTU) compounds have been investigated as potential H 2 S-donors. The thioureas showed long-lasting H 2 S donation in cell free environment and in human aortic smooth muscle cells (HASMCs). In HASMCs, DPTU caused membrane hyperpolarization, mediated by activation of K ATP and Kv7 potassium channels. The thiourea derivatives promoted vasodilation in rat aortic rings, which was abolished by K ATP and Kv7 blockers. The vasorelaxing effects were also observed in angiotensin II-constricted coronary vessels. In conclusion, thiourea represents an original H 2 S-donor functional group, which releases H 2 S with slow and long lasting kinetic, and promotes typical H 2 S-mediated vascular effects. Such a moiety will be extremely useful for developing original cardiovascular drugs and new chemical tools for investigating the pharmacological roles of H 2 S. [ABSTRACT FROM AUTHOR]

• 5α-dihydrotestosterone is involved in sex bias in asthma disease. • 5α-dihydrotestosterone inhibits allergen-induced bronchial hyperreactivity in females. • 5α-dihydrotestosterone inhibits mast cell activation/degranulation induced by allergen. Androgen levels inversely correlate with the incidence, susceptibility and severity of asthma. However, whether male sex hormones such as 5α-dihydrotestosterone (DHT) have beneficial effects on asthma symptoms and/or could affect asthma susceptibility have not been investigated. DHT administration to female mice, during the sensitization phase, abrogates the sex bias in bronchial hyperreactivity. This effect correlates with inhibition of leukotriene biosynthesis in the lung. DHT significantly inhibits also other asthma-like features such as airway hyperplasia and mucus production in sensitized female mice. Conversely, DHT does not affect plasma IgE levels as well as CD3+CD4+ IL-4+ cell and IgE+c-Kit+ cell infiltration within the lung but prevents pulmonary mast cell activation. The in vitro study on RBL‐2H3 cells confirms that DHT inhibits mast cell degranulation. In conclusion, our data demonstrate that immunomodulatory effects of DHT on mast cell activation prevent the translation of allergen sensitization into clinical manifestation of asthma. [ABSTRACT FROM AUTHOR]

Hyperhomocysteinemia is a risk factor for neurovascular and cardiovascular disease associated to endothelial dysfunction and accelerated atherosclerosis [1–3] . Many clinical and epidemiological studies have demonstrated a positive correlation among homocysteine (Hcy) plasma levels and cardiovascular disorders [4] leading to the general conclusion that Hcy is a pro-thrombotic factor [4,5] . Hcy is metabolized to methionine by the action of 5,10 methylenetetrahydrofolate reductase (MTHFR) [6] . Alternatively, by the transulfuration pathway, homocysteine is transformed to hydrogen sulfide (H 2 S), through multiple steps involving cystathionine β -synthase (CBS) and cystathionine γ -lyase (CSE) [7] . To date, the influence of H 2 S on platelet function has been poorly explored. Here we have evaluated the involvement of H 2 S in platelet reactivity by using platelets harvested from healthy volunteers or patients firstly diagnosed with hyperhomocysteinemia (MTHFR++ carriers). Sodium hydrogen sulfide (NaHS) or l -cysteine were used as exogenous or endogenous source of H 2 S, respectively. NaHS (0.1–100 μM) or l -cysteine (0.1–100 μM) did not cause per se platelet aggregation. However both NaHS and l -cysteine significantly increased platelet aggregation induced by the thrombin receptor activator peptide-6 amide (TRAP-6, 2 μM) in a concentration-dependent manner in platelets harvested from healthy volunteers. When platelets harvested from MTHFR++ carriers were used platelet aggregation was further potentiated. The potentiating effect present in this latter group was reverted by the inhibition of either CBS or CSE. Therefore the l -cysteine/H 2 S pathway is involved in the increased responsivity in MTHFR++ carriers. The role played by this pathway is further supported by the finding that H 2 S levels were significantly higher in both platelets and plasma. To gain further insights into the downstream signaling we evaluated the involvement of thromboxaneA 2 (TXA 2 ). TXA 2 levels were markedly increased in response to both NaHS or l -cysteine in platelets of healthy volunteers. Inhibition of phospholipase A 2 , cyclooxygenase or blockade of thromboxane receptor markedly reduced TXA 2 production triggered by H 2 S. These results confirmed that H 2 S triggers the arachidonic acid cascade and that the main downstream signal is thromboxane. Since phospholipase A 2 is the rate limiting enzyme of the arachidonic cascade we evaluated if H 2 S can activate phospholipase A 2 by measuring its phosphorylation. Phospholipase A 2 phosphorylation was significantly higher in MTHFR++ carriers when compared to healthy volunteers. Our data suggest that in MTHFR++ carriers there is an increase in platelet activity dependent upon H 2 S generated within the platelet that boosts the arachidonic acid cascade. Therefore in MTHFR++ carriers the activation of the l -cysteine/H 2 S pathway within the platelets primes the cells making them more responsive to endogenous stimuli that normally do not activate healthy platelets. [ABSTRACT FROM AUTHOR]

It has been extensively demonstrated that hydrogen sulfyde (H 2 S) is implicated is several physiological and pathological conditions (J Mol Med, 2012 Mar;90(3):255–63). In particular, it has been shown that H 2 S causes relaxation in human penile tissues (Proc Natl Acad Sci USA, 2009 Mar 17;106(11):4513–8) and inhibits phosphodiesterase (PDE) activity in vessels (ATVB 2010, Oct;30(10):1998–2004). Beside sildenafil increases H 2 S generation in human bladder (Eur Urol. 2012 Dec;62(6):1174–80) and tadalafil in myocardial tissues (Circulation 2009, 120:S31-S36). Therefore, our aim was to demonstrate the link between H 2 S and PDE-5 in mice corpus cavernosum tissues. Thus, we investigated the effects of sildenafil (10 μM, 0.5 h); PDE-5 inhibitor, on H 2 S production as well as the H 2 S -induced relaxations in mice penile tissues. Penile tissues from CD1 mouse corpus cavernosum (MCC) were used. Functional studies were performed by myograph in Krebs solution. Western blot analysis was performed in order to evaluate CBS and CSE expression and methylene blue assay for measurement of H 2 S levels. In order to investigate functional significance of H 2 S on sildenafil-induced augmentation of endothelial relaxation in MCC the sildenafil effect was evaluated on acetylcholine (ACh; 10–9-10–4 M), L-cysteine (10–6-10–3 M) and NaHS-induced relaxations in presence or not of CSE enzyme inhibitor PPG (10 μM, 0.5 h). In order to achieve this issue the H 2 S production in MCC tissues was also evaluated by incubating the penile tissue with sildenafil in presence or absence of the CSE inhibitor PPG (10 μM, 0.5 h) Both CBS and CSE were expressed in MCC and the enzymes efficiently converted L-cysteine into H 2 S. Further we showed that sildenafil caused a significant increase in H 2 S production and this augmentation was reversed by CSE inhibition. We found that sildenafil induced an increase in both ACh and L-cysteine–induced relaxations and these augmentations reversed by CSE inhibitor PPG in MCC pre-contracted with phenylephrine (3.10–5 M). Beside sildenafil did not significantly increase the NaHS –induced relaxations. Therefore we suggest that both gaseous transmitters NO and H 2 S affect sildenafil action. In particular our results demonstrate that sildenafil effect is partially mediated by H 2 S pathway. Thus, H 2 S signaling may represent a new mechanism involved in the effect of sildenafil on erectile dysfunction. [ABSTRACT FROM AUTHOR]

Background: The need for new drugs in melanoma treatment is of great relevance. Indeed, current therapies for the treatment of metastatic melanoma offer a limited clinical benefit and only in recent years there has been an advancement due to the identification of new molecular targets . Hydrogen sulphide (H2S) is endogenously produced by the action of three enzymes CBS, CSE and the newly discovered 3-MST . While H2S is cytoprotective at physiological concentrations, it seems to have pro-apoptotic actions in cancer cells . However, to date there are not definitive reports on the role played by H2S in cancer development. Aim of this study was to determine the possible involvement of H2S in human melanoma. Methods: The study has been performed by using some relevant human melanoma cell lines such as A375, WM115 and SK-Mel-28. HaCat, a normal human fibroblast cell line, has been used as control. Cellular proliferation was evaluated by the MTT assay. Apoptosis was assayed by flow cytometry analysis by double staining with Annexin V and propidium iodide (PI). NF-kB/DNA-binding activity was evaluated by electrophoretic mobility shift assay. Expression of CBS, CSE, 3-MST was assayed by quantitative real time RT-PCR, expression of Bcl-2, XIAP, c-FLIP, caspase 3, PARP, IKBa and Akt/p-Akt was determined by western blotting. Levels of H2S in the supernatant and in total cellular extracts were assayed by colorimetric assay. Results: Diallyl trisulfide (DATS) is a garlic-derived polysulfide able to release H2S . Our results demonstrate that DATS greatly suppressed, in a time and concentration-dependent manner, proliferation of the three human melanoma cell lines used. The most striking effect was obtained on the A375 cell line whose proliferation was inhibited, following incubation with DATS (10–30–100μM, 72h) by 30%, 70%, and 78% respectively (p <0.001). This effect well correlated with the significant increase in H2S levels found in both supernatants and cellular lysates. Conversely, DATS up to 300 μM did not affect proliferation of HaCat. DATS-induced inhibition of A375 proliferation (10–100μM, 72h) was almost completely reversed by haemoglobin (10μM; p <0.001) a scavenger of H2S. Moreover, DATS-induced inhibition of A375 proliferation was due to the induction of apoptosis as demonstrated by FACS analysis with Annexin V/PI staining and further confirmed by the inhibition of Bcl-2, XIAP, FLIP, as well as by the cleavage and consequent activation of caspase-3 and inactivation of poly (ADP ribose) polymerase (PARP-1). Constitutive NF-kB and activated Akt expression have been described in melanoma. We also demonstrated that H2S released by DATS suppressed both constitutive NF-kB/DNA-binding activity and Akt phosphorylation suggesting that the apoptotic effect observed following exposure to H2S was consistent with the signal transduction pathways activated. Conclusion: In conclusion, we demonstrate that H2S triggers, in relevant human melanoma cell lines, an apoptotic effect. This effect, in turn, activates downstream a signal pattern that candidates H2S as a possible novel therapeutic/target or diagnostic tool. [Copyright &y& Elsevier]

Background: H2S is endogenously produced from l-cysteine (L-cys) by the action of three key enzymes cysthationine β-synthase (CBS), cysthationine-γ-lyase (CSE) and the newly discovered 3-mercaptopyruvate sulfurtransferase (3-MST) . It is present in human blood at micromolar concentrations and it is involved in the maintenance of cardiovascular homeostasis . To date, the influence of H2S on platelets, which play a central role in blood homeostasis, has been poorly explored. Therefore, we aimed to evaluate the effect of H2S signaling in human platelets. Methods: Human washed platelets were collected from 15 healthy volunteers. The expression of both CBS, CSE and 3-MST was evaluated by Western blot analysis. The enzymes activity was evaluated through H2S measurement by a colorimetric assay . Light transmission aggregometry technique was used to analyze platelet aggregation. H2S-induced effect was evaluated using both an exogenous source of H2S, sodium hydrogen sulphide (NaHS, 0.1μM–10mM) and the metabolic precursor, L-cys (0.1μM–10mM) on thrombin receptor activator peptide 6 amide (TRAP-6, 2μM) stimulus. We operated a pharmacological modulation by using specific inhibitors of arachidonic acid cascade, the main pathway involved in platelet function. Indomethacin (INDO, 10μM, 15min), a COX inhibitor, arachidonyl trifluoromethyl ketone (AACOCF3, 1μM, 15min), a phospholipase A2 (PLA2) inhibitor or SQ29548 (1μM), a thromboxane receptor antagonist were used. In addition, thromboxane (TXA2) and cAMP levels were evaluated. Results: Human washed platelets expressed CBS, CSE and 3-MST and generated detectable amounts of H2S. Incubation with L-cys significantly increased H2S release (p <0.001). Neither L-cys nor NaHS (0.1μM–10mM) affected human washed platelets in resting conditions, but both significantly increased TRAP-6-induced aggregation (p <0.001 for L-cys 0.1mM and NaHS 0.1mM and 10μM; p <0.01 for NaHS 1μM and p <0.05 for L-cys 10μM). Besides, H2S did not modify platelet cAMP levels. Conversely, INDO, AACOCF3 and SQ29548 blocked the potentiating effect of H2S on platelet aggregation induced by TRAP-6. Interestingly, both NaHS and L-cys induced a significantly increase in TXA2production (p <0.01). Conclusions: Our data imply that the H2S endogenously produced within human platelets is involved in platelet aggregation through PLA2 activation. These findings may open a new pharmacological approache in platelet dependent disorders. [Copyright &y& Elsevier]

Background: Malignant hyperthermia (MH) is a pharmacogenetic disorder of skeletal muscle. The incidence of MH reactions ranges from 1:5000 to 1:50,000 anesthesias, however genetic abnormalities have been estimated as great as one in 3000 subjects . The symptoms of MH include hyperthermia, increase in carbon dioxide production and oxygen consumption, muscle rigidity, rhabdomyolisis, tachycardia and, if untreated, death. Over 90 mutations have been identified in the ryanodine receptor (RYR1) gene and at least 30 are causal for MH. Thegenetic test only detects about 30% of people at riskof MH. Therefore, the “gold standard” for MH diagnosis is the in vitro contracture test. These tests enclose a positive response to caffeine, which is a well-known non specific PDE inhibitor , and to halothane, whose mechanism of action involves KATP channels . Since these actions are among the most accredited molecular mechanisms triggered by H2S we aimed to evaluate the role of H2S/L-Cys pathway in MH. Methods: The procedure is performed accordingly to the “European Group protocol for investigation of malignant hyperthermia susceptibly” Briefly, the muscular biopsy of the vastus group of the quadriceps muscle is harvested under regional anaesthesia. Eight muscular bundles of 15–25mm length and 2–3mm thickness are placed in a tissue bath with Krebs solution at 37°C and connected to an isometric transducer. Electrical stimulus is than applied and the muscle is stretched slowly up to 2g. After20min of equilibration caffeine or halothane are added in the tissue bath at progressive concentrations. An increase in resting tension of at least 2mN allows a MH susceptible (MHS) diagnosis. After diagnosis has been made, muscle bundles from both MHS and MH negative (MHN) subjects have been used for functional studies. Western blot analysis, qRT-PCR for H2S molecular machinery and plasmatic and tissutal H2S content were also performed. Results: H2S assay performed on tissues homogenate revealed an significant increase of H2S content in MHS patients compared to MHN. Conversely, no difference was founded in plasmatic levels H2S of both groups of patients. Western blot analysis showed a significant and selective increase of CBS expression in MHS patients compared to MHN, confirmed by qRT-PCR analysis. Functional studies performed on MHN showed that pre-incubation of the tissue with NaHS was able to switch an MHS typical response following challenge with either caffeine or halothane. Conclusion: Our data show that in MHS patients there is an increase in CBS expression that accounts for the enhanced concentration of H2S within the skeletal muscle. The involvement of H2S in MH is further confirmed by the finding that incubation of MHN tissues with NaHS prior to the addition of either caffeine or halothane generates a response similar to MHS tissues. In conclusion we demonstrate that L-Cys/CBS/H2S pathway is involved in malygnant hyperthermia. This finding may allows a different diagnostic and/or therapeutic approach. [Copyright &y& Elsevier]

Background: Hydrogen sulphide (H2S) is a novel gaseous mediator enzymatically produced by cystathionine-beta-synthase (CBS) and cystathionine-gamma-lyase (CSE). It is involved in physiological homeostatic processes and several pathological conditions; however, more importantly, H2S has a prominent role in cardiovascular system, where CSE deletion results in hypertension .ACE inhibitors are widely used in controlling blood pressure in hypertensive patients and they represent first line treatment in different cardiovascular diseases, since they also show additional beneficial effects unrelated to ACE inhibition .In particular, therapeutic use of zofenopril, a sulfhydrylated ACE inhibitor, has raised hypothesis over a potential role of thiol group in such beneficial effects, other than antioxidant activity.Here, we aimed to investigate on vascular effect of zofenoprilat, water-soluble metabolite of zofenopril, with respect to H2S pathway activation. Methods: In order to pursue our goal, we performed isolated organ bath experiments by using aorta and carotid arteries harvested from Wistar Kyoto rats, where we tested vascular effect of zofenoprilat, compared to other ACE inhibitors, in presence of CSE inhibitor PAG or after endothelium removal.In addition, we also performed in vivo experiment, where we tested vascular response after two weeks treatment with zofenopril compared to enalapril in spontaneously hypertensive rats.We also determined vascular response to l -cys, precursor for H 2 S production, and quantified H 2 S levels in plasma and tissues and CSE and CBS protein levels upon different treatments. Results: In vitro data showed that zofenoprilat, but not enalaprilat, was able to relax both aorta and carotid arteries in a concentration dependent fashion (100nM–1mM), reaching ∼60% vasodilation in both cases.Endothelium removal significantly reduced zofenoprilat induced vasorelaxation in aorta (EC50:8.5μM vs 381μM), but this effect was not observed in carotid.Next, CSE inhibition by PAG nearly abrogated zofenoprilat vasodilation in both aorta and carotid, indicating a role for H2S in this effect.In vivo data, first highlighted a significantly stronger effect of zofenopril vs enalapril treatment in lowering blood pressure (p <0.05).In addition, vascular reactivity, determined as response to Ach or phenylephrine, was better restored by zofenopril than enalapril administration.Furthermore, vasorelaxant response to l-cys was significantly improved in zofenopril vs enalapril treated animals (p <0.05) and levels of circulating H2S were restored by zofenopril only.At the same extent, only zofenopril treatment restored CSE, but not CBS, expression in aorta after two weeks treatment, while a slight and not significant increase was observed in carotid artery. Conclusion: In conclusion, we show that sulfhydrylated ACE inhibitor zofenopril triggers H2S pathway and regulates different signal transduction cascades, other than inhibition of ACE. We suggest that this action could be, at least partly, explicative of the additional effects reported in the clinical literature about sulfhydrylated ACE inhibitors. Our data also imply the chance to understand, by using a drug already adopted in clinic, the role played by the l-Cys/H2S pathway in human hypertension. [Copyright &y& Elsevier]