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Penile erection is a perfect example of microcirculation modulated by psychological factors and hormonal status. It is the result of a complex neurovascular process that involves the integrative synchronized action of vascular endothelium; smooth muscle; and psychological, neuronal, and hormonal systems. Therefore, the fine coordination of these events is essential to maintain penile flaccidity or allow erection; an alteration of these events leads to erectile dysfunction (ED). ED is defined as the consistent or recurrent inability of a man to attain and/or maintain a penile erection sufficient for sexual activity. A great boost to this research field was given by commercialization of phosphodiesterase-5 (PDE5) inhibitors. Indeed, following the discovery of sildenafil, research on the mechanisms underlying penile erection has had an enormous boost, and many preclinical and clinical papers have been published in the last 10 years. This review is structured to provide an overview of the mediators and peripheral mechanism(s) involved in penile function in men, the drugs used in therapy, and the future prospective in the management of ED. Indeed, 30% of patients affected by ED are classified as “nonresponders,” and there is still an unmet need for therapeutic alternatives. A flowchart suggesting the guidelines for ED evaluation and the ED pharmacological treatment is also provided.

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 trans-differentiation 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. © 2015 Elsevier Ltd. All rights reserved.

Introduction: Nitric oxide (NO) acts a pleiotropic biomodulator in several systems, including the cardiovascular, nervous and immune systems. The intracellular levels of cyclic guanylate monophosphate (cGMP) can be increased by NO or by inhibiting the breakdown to 5'cGMP operated by the cyclic nucleotide phosphodiesterases (PDEs). Platelets are anuclear circulating cells that are rich in both soluble guanylyl cyclase and PDEs. The purpose of this study was to standardize cGMP determination in human platelets. Methods: Fresh blood samples were obtained from a group of healthy volunteers in order to obtain washed platelets. Platelet (3 x 10(4)/mu l 5 x 10(5)/mu l) cGMP levels were measured in basal and stimulated conditions. To stimulate platelets two different NO-donors were used: sodium nitroprusside (SNP; 10, 100, 1000 mu M) or diethylamine NONOate (DEA-NONOate; 1, 10, 100 mu M). Different times of incubation were also studied (5, 15 and 30 min). As positive control has been used ODQ a well known inhibitor of guanylyl cyclase. Platelet cGMP accumulation was measured by using a standard ELISA kit using different sample dilutions. Results: The optimal stimulus was DEA-NONOate, the optimal washed platelet concentration was 5 x 10(5)/mu l, incubation time was 30 mm and dilution to be used was 1:2. Discussion: Platelets represent a valuable marker to investigate the effect of drugs interfering with the cGMP cascade. This standardized assay allows to measure ex vivo the inhibitory (PDE inhibitors) or stimulatory effect (NO donors) of drugs given in vivo to humans. (C) 2011 Elsevier Inc. All rights reserved.

Background: Phosphodiesterase 5 inhibitors (PDE5-Is) are a mainstay in the therapy of erectile dysfunction (ED). The primary end point of clinical efficacy, both in clinical studies and normal practice, is represented by the International Index of Erectile Function (IIEF). Objective: To evaluate if platelet cyclic guanosine monophosphate (cGMP) could represent a valuable marker for PDE5-I activity in ED. Design, setting, and participants: The study enrolled 46 patients with psychogenic, organic, and mixed ED (20–71 yr of age; IIEF score

Hydrogen sulphide is endogenously synthesized mainly by two pyridoxal-5′-phosphate-dependent enzymes involved in L-cysteine metabolism: cystathionine-ß-synthase (CBS) and cystathionine-γ-lyase (CSE) [1], [2] and [3]. The H2S pathway has been proposed to be involved in the pathophysiology of several diseases. At the present stage the research in this field is impaired by the lack of pharmacological tools such as selective enzymatic inhibitors [2], [3] and [4]. The goal of our study is the development of compounds that selectively regulate enzymatic activity. Here we present the synthesis and the activity of a selective CSE inhibitor. We preliminarily selected and tested commercially available cysteine surrogates because of the unavailability of a pharmacophoric model as a lead for rational design of targeted enzyme inhibitors. The catalytic profiles of recombinant CBS and CSE were assessed in the presence of the selected compounds. On the basis of the results obtained were designed new compounds aiming to obtain novel molecular entities embodying the structural features of both cysteine and the well known CSE inhibitor, DL-propargylglycine. The synthetically modified compounds, obtained in our laboratory, were tested in vitro by using rat aortic rings. The compound showing maximal inhibitory effects in this test was an oxothiazolidine derivative, dubbed ‘compound VII’. The effects of this compound on the enzyme kinetics were further tested on the purified enzymes using a metabolomic approach based on nuclear magnetic resonance techniques. These studies clearly showed that ‘compound VII’ is a potent enzyme inhibitor of CSE, without affecting the CBS kinetics. The identification of this highly selective CSE inhibitor may help to better define the role of CSE vs CBS in the pathophysiology of the diseases where a role for the H2S pathway has been proposed.

Protease-activated receptor-1 (PAR-1) is a G-coupled receptor activated by α-thrombin and other proteases. In this paper we describe the synthesis and the pharmacological evaluation of novel peptide-mimetic antagonists (compounds 1 – 16 ) characterized by the presence of new heterocyclic nuclei such as 2-methyl-indole (5- and 6-substituted) and 1,4-benzodiazepine moiety. The new derivatives, tested in order to evaluate their antagonist potency by using human platelet aggregation induced by PAR-1AP, resulted in some cases (compounds 1 and 4 ) more potent than the reference. The compounds, tested on aortic rings, confirmed the results obtained in the aggregation assay.

Peyronie's disease (PD) is characterized by an inflammatory response beneath the tunica albuginea with fibroblast proliferation forming a thickened fibrous plaque that may cause pain, penile curvature and erectile dysfunction. The progression of the PD plaque may eventually lead to calcification or ossification. Current therapeutic success is often unsatisfactory because of limited insight into disease mechanisms. Research has been hampered by the lack of a universally accepted animal model. We describe an animal model of spontaneous PD in tight skin (Tsk) mice, a C57Bl/6J subline that reproduces with age important features of the human disease (fibrous plaque formation, penile bending and areas of chondroid metaplasia with heterotopic ossification). Histological analysis demonstrated an evident structural disorganization of the tunica albuginea with excessive accumulation of type I collagen. At 12 months of age, fibrous plaques with areas of chondroid metaplasia and heterotopic ossification characterized Tsk penises. The up-regulation of hypoxia-inducible factor-1 (HIF-1) leads to an increased downstream expression of HIF-1 target genes, such as TGFbeta and iNOS. These factors, together with some PDGF family members, can cause collagen deposition in Tsk penises. They can also influence chondrocyte differentiation and heterotopic bone formation. In conclusion, hypoxia, HIF-1 and HIF-1 target genes appear to play an important role in the pathogenesis of PD in Tsk mice. This mouse model that is the first example of naturally occurring model of PD in laboratory animals may aid in the identification of signalling pathways crucial for PD and should facilitate the designing and testing of new therapeutic interventions.

Hydrogen sulfide, like nitric oxide, was best known as a toxic pollutant before becoming recognized as a key regulator of several physiologic processes. In recent years, evidence has accumulated to suggest important roles for hydrogen sulfide as a mediator of several aspects of gastrointestinal and liver function. Moreover, alterations in hydrogen sulfide production could contribute to disorders of the gastrointestinal tract and liver. For example, nonsteroidal anti-inflammatory drugs can reduce production of hydrogen sulfide in the stomach, and this has been shown to contribute to the generation of mucosal injury. Hydrogen sulfide has also been shown to play a key role in modulation of visceral hyperalgesia. Inhibitors of hydrogen sulfide synthesis and drugs that can generate safe levels of hydrogen sulfide in vivo have been developed and are permitting interventional studies in experimental models and, in the near future, humans.

Vascular system is constituted by a complex and articulate network, e.g. arteries, arterioles, venules and veins, that requires a high degree of coordination between different elemental cell types. Proteinase-activated receptors (PARs) constitute a recent described family of 7-transmembrane G protein-coupled receptors that are activated by proteolysis. In recent years several evidence have been accumulated for an involvement of this receptor in the response to endothelial injury in vitro and in vivo experimental settings suggesting a role for PAR 2 in the pathophysiology of cardiovascular system. This review will deal with the role of PAR 2 receptor in the cardiovascular system analyzing both in vivo and in vitro published data. In particular this review will deal with the role of this receptor in vascular reactivity, ischemia/reperfusion injury, coronary atherosclerotic lesions and angiogenesis.

NO biology has had an enormous boost and several aspects of its role in physiology and pathology has been extensively studied. NO acts as a double edge sword mediator that has beneficial physiological effects as well as detrimental pathological effects making very difficult to develop drugs. Studies on nitric oxide therapeutic approach can be divided into two simple approaches: one directed to increase the NO release and another to inhibit NO release. Gene therapy approach have been also developed and pre-clinical data on iNOS and eNOS have shown promising results in post-angioplasty restenosis. The major limitation to the use of NSAID is represented by their ability to cause ulceration and bleeding in the gastrointestinal tract. NO plays an important role in GI integrity. NO releasing NSAID have higher GI tolerability and retain their anti-inflammatory activity as well as the ability to inhibit platelet aggregation. NO-NSAIDs not only represents a new class of drugs but they represent the first “proof of concept” on the key role of NO in the gastrointestinal homeostasis.

A novel and convenient microwave-assisted dimerization of an active peptide compound using the DKPs as scaffold is described. The key reaction giving rise to the diketopiperazine scaffold is the intermolecular coupling. No epimerization was detected in the reactions used. Conventional and microwave heating of the reactions are compared. Synthesis by microwave irradiation gave the desired compounds in higher yields and in shorter reaction times than those obtained by conventional heating. © 2003 Elsevier Science Ltd. All rights reserved.

A hallmark of inflammation is increased vascular permeability. Increases in vascular permeability and the migration of inflammatory cells are linked to complex interactions of inflammatory mediators with the vascular endothelium. Normally, endothelial nitric oxide synthase (eNOS) produces a tonic amount of nitric oxide (NO), which is responsible for the homeostasis between the endothelium and surrounding tissues. However, most agonists that act on endothelial cells cause a series of post-translational modifications that influence eNOS activity. Furthermore, stimulation by shear stress, autacoids or growth factors either induces eNOS or shifts it to a more active state, which produces a burst of NO. Here, we highlight recent findings about eNOS and propose how new pharmacological tools can be used to dissect the involvement and contribution of eNOS to inflammatory responses.

Inflammation occurs in the context of integrin-mediated adhesive interactions of cells with their extracellular matrix environment. We investigated the role of the collagen binding integrin alpha1beta1 in a model of colitis. alpha1beta1 was expressed on lamina propria T cells and monocytes during disease. Both alpha1 deficiency and anti-alpha1 mAb treatment (prophylactic and therapeutic) protected against colitis. In vivo alpha1beta1 blockade improved macroscopic and histologic scores, decreased inflammatory cytokine production, and profoundly affected the ability of lamina propria mononuclear cells to proliferate and produce IFN-gamma in vitro. Development and alpha1-mediated inhibition of colitis can be lymphocyte independent, suggesting that activated monocytes also represent a key alpha1beta1-expressing cell type involved in colitis. These results underscore the importance of innate immunity and, specifically, of leukocyte/matrix interactions in regulating local inflammatory responses.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are a mainstay in the treatment of inflammatory disease and are among the most widely used drugs worldwide. They are anti-inflammatory, antipyretic, and analgesic and are prescribed as first choice for the treatment of rheumatic disorders and, in general, inflammation. The main limitation in using NSAIDs consists in their side-effects, including gastrointestinal ulcerogenic activity and bronchospasm. The mechanism of action of these drugs is attributed to the inhibition of cyclooxygenase (COX), and, consequently, the conversion of arachidonic acid into prostaglandins. It is hypothesized that the undesirable side-effects of NSAIDs are due to the inhibition of COX-1 (constitutive isoform), whereas the beneficial effects are related to the inhibition of COX-2 (inducible isoform). Arachidonic acid can also be converted to leukotrienes (LTs) by the action of 5-lipoxygenase (5-LOX). LTC4, LTD4, and LTE4 are potent bronchoconstrictors, whereas LTB4 is chemotactic for leukocytes and plays an important role in the development of gastrointestinal ulcers by contributing to the inflammatory process. Thus, developing dual inhibitor compounds that will simultaneously inhibit COX and 5-LOX could enhance their individual anti-inflammatory effects and reduce the undesirable side-effects associated with NSAIDs, especially of the gastrointestinal tract. The most promising COX/5-LOX inhibitor is ML3000 ([2,2-dimethyl-6-(4-chlorophenyl)-7-phenyl-2,3-dihydro-1H-pyrrolizine-5-yl]-acetic acid), now in Phase III clinical trials. This new approach will certainly help to unravel the mechanisms at the root of the undesirable effects of NSAIDs and to develop safer NSAIDs.

Inflammation and coagulation cannot be considered as two separate processes, since there are several connecting points making them part of unique, defensive host response. The endothelium can be considered as the first link between inflammation and coagulation, since damaged endothelium during inflammation represents a surface where proteins involved in both coagulation and the development of inflammation are expressed. During inflammation, cytokines modulate the coagulation system by downregulating the expression of thrombomodulin and the activation of protein C pattern but, at the same time, they induce the expression of tissue factor, modifying, in this way, the balance between procoagulant and anticoagulant activities. At the same time, at the site of tissue injury, platelets become activated and release several mediators that modify tissue integrity. Thrombin, formed following activation of the coagulation cascade, is essential to promote haemostasis but also stimulates several cell functions, including chemotaxis and mitogenesis, which are responsible for the spreading of the lesion and the tissue repair process. Therefore, in the study of inflammation the involvement of the coagulation pathway has to be taken into account, since the interaction between coagulation and inflammation pathways is a critical issue.

Inflammation occurs as a defensive response to invasion of the host by foreign material, often of microbial nature. This response is normally a localized protective response that at the microscopic level involves a complex series of events including dilatation of arterioles, venules, and capillaries with increased vascular permeability, exudation of fluids including plasma proteins, and leukocyte migration into the inflammatory area. Since disease characterized by inflammation is an important cause of morbidity and mortality in humans, the processes involved in the host defense in inflammation have been and continue to be the object of several experimental studies. The role of several mediators such as histamine, serotonin, bradykinin, prostaglandins, and, more recently, cytokines and nitric oxide has been evaluated, and a contribution for each one of these mediators has been proposed. With the development of powerful molecular biology tools, it has become possible to study enzymes involved in this complex phenomenon by measuring the expression or evaluating the signaling pathways following a specific stimulus. These techniques have generated a proliferation of studies on the role of several enzymes and cytokines in inflammation. Most of these studies have been conducted in vitro on cell lines, and not many of the results have been confirmed by in vivo studies. This commentary does not pretend to analyze all of the studies and their possible in congruences, but endeavors to provoke in the reader a critical review of dogmas and current beliefs that most of the time are built on unilateral interpretation of the data.

Hirulog(TM) is a thrombin catalytic site inhibitor which exhibits specificity for the anionic binding exosite of alpha thrombin. Here, we have evaluated the effect of Hirulog(TM) (1, 5 and 10 mg/kg, 30 min pretreatment) in a rat model of endotoxemia. Intravenous injection of lipopolysaccharide from E. coli (25 mg/kg) serotype 0127:B8) caused decreases in blood pressure which were significantly reduced (about 60%) in animals pretreated with Hirulog(TM). Rat survival to endotoxin was significantly increased in Hirulog(TM) pretreated group (5 and 10 mg/kg) up to 24 hours. Hirulog(TM) at the dose of 10 mg/kg inhibited both endotoxin-induced leukopenia at 30 and 60 minute points and thrombocytopenia at 30 minute point but not at 90 and 120 minute points. Fibrinogen levels were significantly reduced after 2 hours following endotoxin administration. Pretreatment with Hirulog(TM) (5-10 mg/kg i.v.) 30 min prior to administration of endotoxin prevented changes in fibrinogen plasma levels. These results demonstrate that Hirulog(TM)-induced inhibition of thrombin is effective in reducing toxic and lethal effects of endotoxin.

In this study, we assessed the effects of addition of a nitroxybutyl moiety to diclofenac on its ulcerogenic properties. The diclofenac derivative, ‘nitrofenac’, was examined in terms of its ability to induce acute gastric erosions and chronic-type gastric ulcers in rats and rabbits, respectively. The effects of these compounds on prostaglandin synthesis in the stomach and at a site of peripheral inflammation were also assessed, as were their anti-inflammatory properties in a model of acute inflammation. Diclofenac dose-dependently caused acute gastric mucosal injury in the rat at all doses tested (10–40 mg/kg), that was significantly greater in severity than that observed with the same doses of nitrofenac. In rabbits, twice-daily administration of diclofenac induced penetrating antral ulcers and small intestinal damage. No damage was observed in the stomach or small intestine of rabbits receiving nitrofenac. Diclofenac and nitrofenac exerted similar inhibitory effects on prostaglandin E 2 synthesis in the stomach and in a carrageenan-sponge model of peripheral inflammation. These compounds exerted similar inhibitory effects on carrageenan-induced paw edema. Nitrofenac, but not diclofenac, caused a significant increase in plasma levels of nitrate/nitrite. These results suggest that the addition of a nitroxybutyl moiety to diclofenac markedly reduces the ulcerogenic properties of this compound without interfering with its ability exhibit cyclo-oxygenase activity or to reduce acute inflammation.

Platelets contain a phospholipase A2 in their granules which can be released in response to various activating stimuli in vitro as well as in vivo. Human recombinant phospholipase A2 (1-10 micrograms/ml) had no direct effect on platelet aggregation in vitro using rabbit platelet rich plasma. In contrast human recombinant phospholipase A2 (1-20 micrograms/ml) was able to inhibit aggregation of washed rabbit platelet in vitro induced by collagen (0.250-2.0 micrograms/ml). When rabbit platelet rich plasma was recalcified with CaCl2 1 M in the presence of the thrombin inhibitor hirulog (10 micrograms/ml), human recombinant phospholipase A2 (10-40 micrograms/ml) was able to inhibit platelet aggregation. The anti-aggregatory effect was removed by incubation of platelet rich plasma with a monoclonal anti-human recombinant phospholipase A2 antibody. Human recombinant phospholipase A2 (1-10 micrograms) inhibited 111In-labelled platelet accumulation within the thoracic region of rats and guinea pigs induced by i.v. administration of submaximal doses of collagen or adenosine diphosphate. Phospholipase A2 (1-20 micrograms/ml) from Naja mocambique mocambique snake venom had no direct effect on platelet aggregation in vitro. However, Naja phospholipase A2 administered i.v. to rats or guinea pigs was able to induce a dose related accumulation of 111In-labelled platelet within the thoracic region. The inhibitory effect of exogenously added human recombinant phospholipase A2 on platelet aggregation in vivo suggests a possible pathophysiological role for the extracellular form of phospholipase A2 but this property is not a feature of all phospholipase A2 preparations.

Addition of a nitroxybutyl moiety to diclofenac greatly reduces its damaging effects on the gastric mucosa without altering its ability to suppress prostaglandin synthesis and exert anti-inflammatory actions. The present study was performed in order to determine if this derivative of diclofenac, called nitrofenac, would also have less toxicity in the small and large intestine when administered repeatedly over a 1-2 week period. Healthy rats were given equimolar doses of diclofenac (10 mg/kg) or nitrofenac (15 mg/kg) twice daily for up to two weeks. All 10 rats receiving diclofenac died prior to completion of the study, exhibiting massive small intestinal ulceration and perforation. No deaths were observed in the rats treated with nitrofenac, and the only small intestinal abnormality observed was diffuse hyperemia. As nonsteroidal anti-inflammatory drugs have been shown to exacerbate colitis, we compared the effects of twice daily treatment with diclofenac (1-10 mg/kg) or nitrofenac (1.5-15 mg/kg) for 1 week in rats in which colitis had been induced with trinitrobenzene sulfonic acid. Diclofenac administration resulted in mortality which increased dose-dependently (e.g. 86% at 5 mg/kg) and was associated with perforation of the colon. Mortality was not observed with nitrofenac at doses of 1.5 or 7.5 mg/kg, while at 15 mg/kg the mortality rate was 33%. None of the doses of nitrofenac significantly augmented colonic injury or granulocyte infiltration (measured by myeloperoxidase activity). Suppression of colonic prostaglandin E2 synthesis was comparable with equimolar doses of diclofenac and nitrofenace. These studies demonstrate that nitrofenac has markedly reduced intestinal toxicity in healthy and colitic rats when compared to diclofenac.

Extracellular phospholipase A2 is secreted from the platelets upon activation by a stimulus such as thrombin. The secreted enzyme has been recently cloned and the recombinant protein produced. Snake venom PLA2 effect on platelet and coagulation has been extensively studied (for review see 3,4) and it has been proposed that the anticoagulant phospholipases may inhibit coagulation by competing with clotting proteins for the lipid surface. Structure function relationship for PLA2s with anticoagulant activity indicates that the activity is conferred by positively charged aminoacids between residues 54 and 77. The corresponding segment of human recombinant secreted platelet PLA2 (r-hnps-PLA2) possesses five positively charged aminoacids in this region being at the identical positions to those of PLA2s with known anticoagulant activities. Here using human and rat plasma we have demonstrated for the first time that the recombinant human extracellular secreted platelet PLA2 increases activated partial thromboplastin time but not prothrombin time.

1. The rat paw oedema produced by a local injection of phospholipase A2 from Naja mocambique mocambique has been shown to be mainly driven by the liberation of serotonin and kinins. 2. Using specific bradykinin receptor antagonists we have shown that kinins are acting through B2 receptors. 3. Using endothelium-derived relaxing factor (EDRF) synthesis inhibitors NG-monomethyl-L-arginine and L-NG-nitro arginine we have tested the possible envolvement of EDRF as mediator. 4. Our work supports the view that extracellular phospholipases A2 are involved in inflammation, and suggests a role for EDRF as mediator of extravasation in this model of inflammation.

Hydrogen sulfide (H2S), 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 H2S 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 H2S 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 IL1β-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 H2S 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 H2S pathway, providing a novel mechanistic explanation for the exquisite properties of H2S in the control of experimental inflammation. These findings may be relevant to innovative discovery programmes aiming at harnessing the biological properties of H2S.

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) [1] , [2] . It is present in human blood at micromolar concentrations [3] and it is involved in the maintenance of cardiovascular homeostasis [4] . 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 [5] . 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, 0.1 μM–10 mM) and the metabolic precursor, L-cys (0.1 μM–10 mM) on thrombin receptor activator peptide 6 amide (TRAP-6, 2 μM) stimulus. We operated a pharmacological modulation by using specific inhibitors of arachidonic acid cascade, the main pathway involved in platelet function. Indomethacin (INDO, 10 μM, 15 min), a COX inhibitor, arachidonyl trifluoromethyl ketone (AACOCF3, 1 μM, 15 min), a phospholipase A2 (PLA2) inhibitor or SQ29548 (1 μM), 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 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.

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 [1] . 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 [2] , and to halothane, whose mechanism of action involves KATP channels [3] . Since these actions are among the most accredited molecular mechanisms triggered by H2S [4] , [5] 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 15–25 mm length and 2–3 mm 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 2 g. After20 min of equilibration caffeine or halothane are added in the tissue bath at progressive concentrations. An increase in resting tension of at least 2 mN 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.

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 [1] . Hydrogen sulphide (H 2 S) is endogenously produced by the action of three enzymes CBS, CSE and the newly discovered 3-MST [2] . While H 2 S is cytoprotective at physiological concentrations, it seems to have pro-apoptotic actions in cancer cells [3] . However, to date there are not definitive reports on the role played by H 2 S in cancer development. Aim of this study was to determine the possible involvement of H 2 S 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 H 2 S 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 H 2 S [4] . 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 (10–30–100 μM, 72 h) by 30%, 70%, and 78% respectively ( p 2 S 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 (10–100 μM, 72 h) was almost completely reversed by haemoglobin (10 μM; p 2 S. 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 [5] . We also demonstrated that H 2 S released by DATS suppressed both constitutive NF-kB/DNA-binding activity and Akt phosphorylation suggesting that the apoptotic effect observed following exposure to H 2 S was consistent with the signal transduction pathways activated. Conclusion In conclusion, we demonstrate that H 2 S triggers, in relevant human melanoma cell lines, an apoptotic effect. This effect, in turn, activates downstream a signal pattern that candidates H 2 S as a possible novel therapeutic/target or diagnostic tool.

The therapeutic use of sulfhydrylated inhibitor zofenopril has raised different hypotheses regarding the role played by its thiol group in the beneficial clinical effects. In order to evaluate the involvement of the H 2 S pathway in the extra-beneficial effects in vascular function we performed an in vitro and an ex vivo study by using SHR and WKY rats. Both rat strains were treated with either zofenopril or enalapril in vivo. Aorta and carotid were harvested and ex vivo vascular reactivity to Ach and l -cysteine assessed. CBS, CSE and 3MST expression, as well as H 2 S levels, were evaluated in both vascular tissues. The vascular response to Ach in both carotid and aorta was impaired in SHR (∼30%, P l -cysteine-induced relaxation was enhanced. CSE expression in vessels, and tissue/plasma H 2 S levels were restored to WKY values in SHR rats receiving zofenopril. In contrast, CBS and 3MST expression were not modified by treatments. Zofenoprilat, the active metabolite of zofenopril, releases H 2 S in a cell-free assay and it directly relaxed vessels in vitro in a concentration-dependent manner ( P In vivo administration of R-zofenoprilat diasteroisomer, which does not inhibit ACE, did not modify blood pressure; nonetheless, it retained the beneficial effect on SHR vascular function as well as restored plasma/tissue H 2 S levels. In conclusion our findings establish that zofenopril improves vascular function by potentiating the H 2 S pathway in a model of spontaneous hypertension explaining the mechanism underlying the beneficial effects of sulfhydrylated ACE inhibitors beyond ACE inhibition reported in clinical literature. Moreover zofenopril can represent a proof of concept of the role played by H 2 S in hypertension.

It has been extensively demonstrated that hydrogen sulfyde (H2S) is implicated is several physiological and pathological conditions (J Mol Med, 2012 Mar;90(3):255–63). In particular, it has been shown that H2S 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 H2S 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 H2S and PDE-5 in mice corpus cavernosum tissues. Thus, we investigated the effects of sildenafil (10 μM, 0.5 h); PDE-5 inhibitor, on H2S production as well as the H2S -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 H2S levels. In order to investigate functional significance of H2S 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 H2S 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 H2S. Further we showed that sildenafil caused a significant increase in H2S 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 H2S affect sildenafil action. In particular our results demonstrate that sildenafil effect is partially mediated by H2S pathway. Thus, H2S signaling may represent a new mechanism involved in the effect of sildenafil on erectile dysfunction.

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 [1] .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 [2] , [3] .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 H2S production, and quantified H2S 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 (100 nM–1 mM), reaching ∼60% vasodilation in both cases.Endothelium removal significantly reduced zofenoprilat induced vasorelaxation in aorta (EC50:8.5 μM vs 381 μM), 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 l -cys was significantly improved in zofenopril vs enalapril treated animals (p 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.