A reverse translational approach reveals the protective roles of Mangifera indica in inflammatory bowel disease.

Inflammatory bowel diseases (IBDs) are chronic intestinal disorders often characterized by a dysregulation of T cells, specifically T helper (Th) 1, 17 and T regulatory (Treg) repertoire. Increasing evidence demonstrates that dietary polyphenols from Mangifera indica L. extract (MIE, commonly known as mango) mitigate intestinal inflammation and splenic Th17/Treg ratio. In this study, we aimed to dissect the immunomodulatory and anti-inflammatory properties of MIE using a reverse translational approach, by initially using blood from an adult IBD inception cohort and then investigating the mechanism of action in a preclinical model of T cell-driven colitis. Of clinical relevance, MIE modulates TNF-α and IL-17 levels in LPS spiked sera from IBD patients as an ex vivo model of intestinal barrier breakdown. Preclinically, therapeutic administration of MIE significantly reduced colitis severity, pathogenic T-cell intestinal infiltrate and intestinal pro-inflammatory mediators (IL-6, IL-17A, TNF-α, IL-2, IL-22). Moreover, MIE reversed colitis-induced gut permeability and restored tight junction functionality and intestinal metabolites. Mechanistic insights revealed MIE had direct effects on blood vascular endothelial cells, blocking TNF-α/IFN-γ-induced up-regulation of COX-2 and the DP2 receptors. Collectively, we demonstrate the therapeutic potential of MIE to reverse the immunological perturbance during the onset of colitis and dampen the systemic inflammatory response, paving the way for its clinical use as nutraceutical and/or functional food.

Searching for Novel Sources of Hydrogen Sulfide Donors: Chemical Profiling of Polycarpa aurata Extract and Evaluation of the Anti-Inflammatory Effects.

Hydrogen sulfide (H2S) is a signaling molecule endogenously produced within mammals' cells that plays an important role in inflammation, exerting anti-inflammatory effects. In this view, the research has shown a growing interest in identifying natural H2S donors. Herein, for the first time, the potential of marine extract as a source of H2S-releasing agents has been explored. Different fractions obtained by the Indonesian ascidian Polycarpa aurata were evaluated for their ability to release H2S in solution. The main components of the most active fraction were then characterized by liquid chromatography-high-resolution mass spectrometry (LC-HRMS) and NMR spectroscopy. The ability of this fraction to release H2S was evaluated in a cell-free assay and J774 macrophages by a fluorimetric method, and its anti-inflammatory activity was evaluated in vitro and in vivo by using carrageenan-induced mouse paw edema. The anti-inflammatory effects were assessed by inhibiting the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX2), and interleukin-6 (IL-6), coupled with a reduction in nitric oxide (NO) and IL-6 levels. Thus, this study defines the first example of a marine source able to inhibit inflammatory responses in vivo through the release of H2S.

Biphasic inflammatory response induced by intra-plantar injection of L-cysteine: Role of CBS-derived H2S and S1P/NO signaling.

This study investigates the inflammatory response to intra-plantar injection of L-cysteine in a murine model. L-cysteine induces a two-phase response: an early phase lasting 6 h and a late phase peaking at 24 h and declining by 192 h. The early phase shows increased neutrophil accumulation at 2 h up to 24 h, followed by a reduction at 48 h. On the other hand, the late phase exhibits increased macrophage infiltration peaking at 96 h. Inhibition of cystathionine ß-synthase (CBS), the first enzyme in the transsulfuration pathway, significantly reduces L-cysteine-induced edema, suggesting its dependence on CBS-derived hydrogen sulfide (H2S). Sequential formation of sphingosine-1-phosphate (S1P) preceding nitric oxide (NO) generation suggests the involvement of a CBS/S1P/NO axis in the inflammatory response. Inhibition of de novo sphingolipid biosynthesis, S1P1 receptor, and endothelial NO synthase (eNOS) attenuates L-cysteine-induced paw edema. These findings indicate a critical role of the CBS/H2S/S1P/NO signaling pathway in the development and maintenance of L-cysteine-induced inflammation. The co-presence of H2S and NO is necessary for inducing and sustaining the inflammatory response, as NaHS or L-arginine alone do not replicate the marked and prolonged inflammatory effect observed with L-cysteine. This study enhances our understanding of the complex molecular mechanisms of the interplay between NO and H2S pathways in inflammation and identifies potential therapeutic targets for inflammatory disorders.

Hydrogen sulfide regulates the redox state of soluble guanylate cyclase in CSE-/- mice corpus cavernosum microcirculation.

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 (H2S) 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 H2S in the regulation of the soluble guanylate cyclase (sCG) redox state. The lack of CSE-derived endogenous H2S, in CSE-/- mice, disrupted the eNOS/NO/sGC/PDE pathway. Indeed, the absence of CSE-derived endogenous H2S 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 H2S was confirmed by the findings that in CC harvested from CSE-/- mice there was a significant reduction of GCß1 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 H2S translate into a significant reduction in cGMP levels. The replenishment of the lack of H2S with an H2S donor rescued the relaxant response to riociguat in CC of CSE-/- mice. In conclusion, the endogenous CSE-derived H2S plays a physiological key role in the regulation of the redox state of sGC in CC microcirculation.

Hydrogen sulfide donor AP123 restores endothelial nitric oxide-dependent vascular function in hyperglycemia via a CREB-dependent pathway.

Diabetes is associated with severe vascular complications involving the impairment of endothelial nitric oxide synthase (eNOS) as well as cystathionine γ-lyase (CSE) activity. eNOS function is suppressed in hyperglycaemic conditions, resulting in reduced NO bioavailability, which is paralleled by reduced levels of hydrogen sulfide (H2S). Here we have addressed the molecular basis of the interplay between the eNOS and CSE pathways. We tested the impact of H2S replacement by using the mitochondrial-targeted H2S donor AP123 in isolated vessels and cultured endothelial cells in high glucose (HG) environment, at concentrations not causing any vasoactive effect per se. Aorta exposed to HG displayed a marked reduction of acetylcholine (Ach)-induced vasorelaxation that was restored by the addition of AP123 (10 nM). In HG condition, bovine aortic endothelial cells (BAEC) showed reduced NO levels, downregulation of eNOS expression, and suppression of CREB activation (p-CREB). Similar results were obtained by treating BAEC with propargylglycine (PAG), an inhibitor of CSE. AP123 treatment rescued eNOS expression, as well as NO levels, and restored p-CREB expression in both the HG environment and the presence of PAG. This effect was mediated by a PI3K-dependent activity since wortmannin (PI3K inhibitor) blunted the rescuing effects operated by the H2S donor. Experiments performed in the aorta of CSE-/- mice confirmed that reduced levels of H2S not only negatively affect the CREB pathway but also impair Ach-induced vasodilation, significantly ameliorated by AP123. We have demonstrated that the endothelial dysfunction due to HG involves H2S/PI3K/CREB/eNOS route, thus highlighting a novel aspect of the H2S/NO interplay in the vasoactive response.

Interleukin-17 (IL-17) triggers systemic inflammation, peripheral vascular dysfunction, and related prothrombotic state in a mouse model of Alzheimer's disease.

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-ß-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 Aß1-42 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 Aß1-42 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.

Effects of Subtoxic Concentrations of Atrazine, Cypermethrin, and Vinclozolin on microRNA-Mediated PI3K/Akt/mTOR Signaling in SH-SY5Y Cells.

Endocrine-disrupting chemicals (EDCs) are different natural and synthetic chemicals that may interfere with several mechanisms of the endocrine system producing adverse developmental, metabolic, reproductive, and neurological effects in both human beings and wildlife. Among pesticides, numerous chemicals have been identified as EDCs. MicroRNAs (miRNAs) can regulate gene expression, making fine adjustments in mRNA abundance and regulating proteostasis. We hypothesized that exposure to low doses of atrazine, cypermethrin, and vinclozolin may lead to effects on miRNA expression in SH-SY5Y cells. In particular, the exposure of SH-SY5Y cells to subtoxic concentrations of vinclozolin is able to downregulate miR-29b-3p expression leading to the increase in the related gene expression of ADAM12 and CDK6, which may promote a pro-oncogenic response through the activation of the PI3K/Akt/mTOR pathway and counteracting p53 activity. A better understanding of the molecular mechanisms of EDCs could provide important insight into their role in human disease.

Boosting lung accumulation of gallium with inhalable nano-embedded microparticles for the treatment of bacterial pneumonia.

The potential of intra-venous gallium nitrate (GaN) administration against Pseudomonas aeruginosa pneumonia was recently demonstrated in mice and in cystic fibrosis (CF) patients. Likewise, the added value of direct lung delivery of Ga(III) has been shown in rats. Therefore, the design of a drug delivery system specifically engineered for Ga(III) inhalation is imperative to improve its accumulation in lungs. To this purpose, Ga(III) was efficiently encapsulated into hyaluronic acid/chitosan nanoparticles (Ga_HA/CS NPs), whose features were tuned to facilitate access to the target by overcoming mucus and biofilm surrounding bacteria. Then, to improve in vivo lung deposition, Ga_HA/CS NPs were engineered into mannitol-based NEM (Ga_Man NEM). The powders showed optimal in vitro aerosol performance, and sustained release kinetics in lung lining fluids. Moreover, good tolerability and antimicrobial properties were shown in vitro. Intratracheal insufflation of Ga_Man NEM in rats resulted in a significant improvement of Ga(III) persistence in the lungs coupled to a lower Ga(III) concentration in plasma and urine, compared to GaN solution. Noteworthy, the developed formulation significantly modifies the unfavorable Ga(III) kinetic increasing the Ga(III) to the lung and preventing Ga(III) accumulation in the kidney, key responsible for adverse effects, conclusively demonstrating the benefit of Ga_Man NEM to exploit the therapeutic effect of Ga(III) via inhalation route.

ß3 Relaxant Effect in Human Bladder Involves Cystathionine γ-Lyase-Derived Urothelial Hydrogen Sulfide.

It is now well established that the urothelium does not act as a passive barrier but contributes to bladder homeostasis by releasing several signaling molecules in response to physiological and chemical stimuli. Here, we investigated the potential contribution of the hydrogen sulfide (H2S) pathway in regulating human urothelium function in ß3 adrenoceptor-mediated relaxation. The relaxant effect of BRL 37344 (0.1-300 µM), a selective ß3 adrenoceptor agonist, was evaluated in isolated human bladder strips in the presence or absence of the urothelium. The relaxant effect of BRL 37344 was significantly reduced by urothelium removal. The inhibition of cystathionine-γ-lyase (CSE), but not cystathionine-ß-synthase (CBS), significantly reduced the BRL 37344 relaxing effect to the same extent as that given by urothelium removal, suggesting a role for CSE-derived H2S. ß3 adrenoceptor stimulation in the human urothelium or in T24 urothelial cells markedly increased H2S and cAMP levels that were reverted by a blockade of CSE and ß3 adrenoceptor antagonism. These findings demonstrate a key role for urothelium CSE-derived H2S in the ß3 effect on the human bladder through the modulation of cAMP levels. Therefore, the study establishes the relevance of urothelial ß3 adrenoceptors in the regulation of bladder tone, supporting the use of ß3 agonists in patients affected by an overactive bladder.

Beneficial Effect of H2S-Releasing Molecules in an In Vitro Model of Sarcopenia: Relevance of Glucoraphanin.

Sarcopenia is a gradual and generalized skeletal muscle (SKM) syndrome, characterized by the impairment of muscle components and functionality. Hydrogen sulfide (H2S), endogenously formed within the body from the activity of cystathionine-γ-lyase (CSE), cystathionine- ß-synthase (CBS), and mercaptopyruvate sulfurtransferase, is involved in SKM function. Here, in an in vitro model of sarcopenia based on damage induced by dexamethasone (DEX, 1 µM, 48 h treatment) in C2C12-derived myotubes, we investigated the protective potential of exogenous and endogenous sources of H2S, i.e., glucoraphanin (30 µM), L-cysteine (150 µM), and 3-mercaptopyruvate (150 µM). DEX impaired the H2S signalling in terms of a reduction in CBS and CSE expression and H2S biosynthesis. Glucoraphanin and 3-mercaptopyruvate but not L-cysteine prevented the apoptotic process induced by DEX. In parallel, the H2S-releasing molecules reduced the oxidative unbalance evoked by DEX, reducing catalase activity, O2- levels, and protein carbonylation. Glucoraphanin, 3-mercaptopyruvate, and L-cysteine avoided the changes in myotubes morphology and morphometrics after DEX treatment. In conclusion, in an in vitro model of sarcopenia, an impairment in CBS/CSE/H2S signalling occurs, whereas glucoraphanin, a natural H2S-releasing molecule, appears more effective for preventing the SKM damage. Therefore, glucoraphanin supplementation could be an innovative therapeutic approach in the management of sarcopenia.

Endogenous and exogenous hydrogen sulfide modulates urothelial bladder carcinoma development in human cell lines.

The role of H2S in urothelial carcinoma (UC) is still unclear. Here we have evaluated the expression of H2S producing enzymes as well as the effect of endogenous and exogenous H2S on human bladder UC cells. In human UC cells the expression of cystathionine ß-synthase (CBS), cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3-MST); is significantly lower as compared to healthy cells. A modulatory role for the H2S pathway is supported by the finding that, the overexpression of CSE or CBS, but not 3-MST, inhibits cell proliferation and promotes apoptosis. A similar effect is obtained by using exogenous H2S. Diallyl trisulfide (DATS), which is a fully characterized H2S donor, inhibits the proliferation of UC cells in a time and concentration-dependent manner as well as promotes apoptosis. Moreover, DATS also induces autophagy, as determined by transcriptomic and western blot analysis. Finally, DATS inhibits mRNA expression levels of canonical markers of epithelial-mesenchymal transition by limiting migration and clonogenic ability of human UC cells in vitro. In conclusion, in urothelial carcinoma, there is an impairment of H2S pathway that involves CSE and CBS- derived hydrogen sulfide. Thus, targeting H2S signaling pathway in urothelial carcinoma could represent a novel therapeutic strategy.

The Role of Perivascular Adipose Tissue-Derived Hydrogen Sulfide in the Control of Vascular Homeostasis.

Significance: Emerging evidence suggests that perivascular adipose tissue (PVAT) has a relevant role in the control of vascular tone in physiology and pathology. Healthy PVAT has anticontractile, anti-inflammatory, and antioxidative actions. Accumulating data from both human and experimental animal models indicate that PVAT dysfunction is conceivably coupled to cardiovascular diseases, and it is associated with vascular inflammation, oxidative stress, and arterial remodeling. Therefore, "healthy" PVAT may constitute a novel therapeutic target for the prevention and treatment of cardiovascular diseases. Recent Advances: Hydrogen sulfide (H2S) has been recognized as a vascular anti-contractile factor released from PVAT. The enzymes deputed to H2S biosynthesis are variously expressed in PVAT and strictly dependent on the vascular bed and species. Metabolic and cardiovascular diseases can alter the morphological and secretory characteristics of PVAT, influencing also the H2S signaling. Here, we discuss the role of PVAT-derived H2S in healthy conditions and its relevance in alterations occurring in vascular disorders. Critical Issues: We discuss how a better understanding may help in the prevention of vascular dysfunction related to alteration in PVAT-released H2S as well as the importance of the interplay between PVAT and H2S. Future Directions: We propose future directions to evaluate the contribution of each enzyme involved in H2S biosynthesis and their alteration/switch occurring in vascular disorders and the remaining challenges in investigating the role of H2S. Antioxid. Redox Signal. 37, 84-97.

A Highly Sensitive Luminescent Biosensor for the Microvolumetric Detection of the Pseudomonas aeruginosa Siderophore Pyochelin.

The pyochelin (PCH) siderophore produced by the pathogenic bacterium Pseudomonas aeruginosa is an important virulence factor, acting as a growth promoter during infection. While strong evidence exists for PCH production in vivo, PCH quantification in biological samples is problematic due to analytical complexity, requiring extraction from large volumes and time-consuming purification steps. Here, the construction of a bioluminescent whole cell-based biosensor, which allows rapid, sensitive, and single-step PCH quantification in biological samples, is reported. The biosensor was engineered by fusing the promoter of the PCH biosynthetic gene pchE to the luxCDABE operon, and the resulting construct was inserted into the chromosome of the ΔpvdAΔpchDΔfpvA siderophore-null P. aeruginosa mutant. A bioassay was setup in a 96-well microplate format, enabling the contemporary screening of several samples in a few hours. A linear response was observed for up to 40 nM PCH, with a lower detection limit of 1.64 ± 0.26 nM PCH. Different parameters were considered to calibrate the biosensor, and a detailed step-by-step operation protocol, including troubleshooting specific problems that can arise during sample preparation, was established to achieve rapid, sensitive, and specific PCH quantification in both P. aeruginosa culture supernatants and biological samples. The biosensor was implemented as a screening tool to detect PCH-producing P. aeruginosa strains on a solid medium.

A new original nutraceutical formulation ameliorates the effect of Tadalafil on clinical score and cGMP accumulation.

OBJECTIVE: To assess the efficacy of the combination of Tadalafil 5 mg and nutritional supplements composed by Panax ginseng, Moringa Oleifera and Rutin on erectile function in men with mild and moderate vasculogenic ED. METHODS: we prospectively enrolled 86 patients divided into two groups A (45), B (33) in this multicenter randomized, doubleblind, placebo-controlled trial . Drop out was 8 patients (3 patients in group A and 5 in Group B). At screening visit patients underwent clinical examination, blood test (hormonal and metabolic profile) and filled out the IIEF-5 questionnaire and the SEP-2, SEP-3. Patients were randomized by a computergenerated list to receive either Tadalafil 5 mg once daily plus nutritional supplement once daily (group A) or Tadalafil 5 mg plus placebo with the same administration schedule (group B) for 3 months. Blood samples, IIEF-5, SEP-2 and SEP-3 have been collected again after 3 months. cGMP was measured in platelets of 38 patients at baseline and after one months. RESULTS: Mean age was 59.98 ± 6.90 (range 38-69), mean IIEF-5 score at baseline was 13.59 ± 3.90. After three months of treatment, IIEF-5 score significantly improved in both groups compared to baseline (13.18 ± 3.75 vs 20.48 ± 2.24, p < 0.0001; 14.15 ± 4.09 vs 19.06 ± 4.36, p < 0.0001, in group A and group B respectively). Patients treated with Tadalafil plus nutritional supplement showed a significantly higher increase in IIEF-5 score compared to those who received placebo (7.27 ± 2.20 and 4.9 ± 2.79, respectively; p < 0.0001;). No hormonal differences and metabolic effects were found. According cGMP result, nutritional supplements ameliorates and extends the activity of the chronic treatment. CONCLUSIONS: IIEF-5 significant increase in group B, can be ascribed to the nutritional supplement properties and antioxidant effects of moringa oleifera, ginseng and rutin and this can enhance the endothelial NO and cGMP production.

Intra-tracheal administration increases gallium availability in lung: implications for antibacterial chemotherapy.

The emergence of pan-resistant strains in nosocomial settings underscores the urgent need of novel therapies targeting vital bacterial functions. Bacterial iron metabolism is a fascinating target for new antimicrobials. Iron mimetic metal Ga(III) has been repurposed as an antimicrobial drug, in pre-clinical studies and recent clinical studies have raised the possibility of using Ga(III) for the treatment of P. aeruginosa pulmonary infection. Ga(III) has been approved by FDA for the treatment of cancer, autoimmune and bone resorption disorders. However, some critical issues affect the therapeutic schedule of Ga(III), principally the intra-venous (i.v.) administration, and the nephrotoxicity caused by prolonged administration. Ga(III) aerosolization could represent a viable alternative for treatment of lung infections, since delivery of antimicrobial agents to the airways maximizes drug concentration at the site of infection, improves the therapeutic efficacy, and alleviates systemic toxic effects. We demonstrate the advantage of inhaled vs i.v. administered Ga(III), in terms of bio-distribution and lung acute toxicity, by using a rat model. In vivo results support the use of Ga(III) for inhalation since intra-tracheal Ga(III) delivery improved its persistence in the lung, while the i.v. administration caused rapid clearance and did not allow to attain a significant Ga(III) concentration in this organ. Moreover, local and systemic acute toxicity following intra-tracheal administration was not observed, since no significant signs of inflammation were found. At this stage of evidence, the direct administration of Ga(III) to the lung appears feasible and safe, boosting the development of Ga(III)-based drugs for inhalation therapy.

Involvement of 3',5'-cyclic inosine monophosphate in cystathionine γ-lyase-dependent regulation of the vascular tone.

BACKGROUND AND PURPOSE: l-cysteine or hydrogen sulfide (H2 S) donors induce a biphasic effect on precontracted isolated vessels. The contractile effect occurs within a concentration range of 10 nM to 3 µM followed by vasodilatation at 30-100 µM. Here, we have investigated the signalling involved in the H2 S-induced contraction. EXPERIMENTAL APPROACH: Vascular response to NaHS or l-cysteine is evaluated on isolated precontracted with phenylephrine vessel rings harvested from wild type, cystathionine γ-lyase (CSE-/- ), soluble guanylyl cyclase (sGCα1-/- ) and endothelial nitric oxide synthase (eNOS-/- ) knock-out mice. The cAMP, cGMP and inosine 3',5'-cyclic monophosphate (cIMP) levels are simultaneously quantified using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis. The involvement of sGC, phosphodiesterase (PDE) 4A and PDE5 are also evaluated. KEY RESULTS: CSE-derived H2 S-induced contraction requires an intact eNOS/NO/sGC pathway and involves cIMP as a second messenger. H2 S contractile effect involves a transient increase of cGMP and cAMP metabolism caused by PDE5 and PDE4A, thus unmasking cIMP contracting action. The stable cell-permeable analogue of cIMP elicits concentration-dependent contraction on a stable background tone induced by phenylephrine. The lack of cIMP, coupled to the hypocontractility displayed by vessels harvested from CSE-/- mice, confirms that H2 S-induced contraction involves cIMP. CONCLUSION AND IMPLICATIONS: The endothelium dynamically regulates vessel homeostasis by modulating contractile tone. This also involves CSE-derived H2 S that is mediated by cIMP.

Uterine Dysfunction in Diabetic Mice: The Role of Hydrogen Sulfide.

It is well-known that the physiological uterine peristalsis, related to several phases of reproductive functions, plays a pivotal role in fertility and female reproductive health. Here, we have addressed the role of hydrogen sulfide (H2S) signaling in changes of uterine contractions driven by diabetes in non-obese diabetic (NOD) mice, a murine model of type-1 diabetes mellitus. The isolated uterus of NOD mice showed a significant reduction in spontaneous motility coupled to a generalized hypo-contractility to uterotonic agents. The levels of cyclic nucleotides, cAMP and cGMP, notoriously involved in the regulation of uterus homeostasis, were significantly elevated in NOD mouse uteri. This increase was well-correlated with the higher levels of H2S, a non-specific endogenous inhibitor of phosphodiesterases. The exposure of isolated uterus to L-cysteine (L-Cys), but not to sodium hydrogen sulfide, the exogenous source of H2S, showed a weak tocolytic effect in the uterus of NOD mice. Western blot analysis revealed a reorganization of the enzymatic expression with an upregulation of 3-mercaptopyruvate-sulfurtransferase (3-MST) coupled to a reduction in both cystathionine-ß-synthase (CBS) and cystathionine-γ-lyase (CSE) expression. In conclusion, the increased levels of cyclic nucleotides dysregulate the uterus peristalsis and contractility in diabetic mice through an increase in basal H2S synthesis suggesting a role of 3-MST.

Pharmacology and perspectives in erectile dysfunction in man.

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.

L-cysteine/cystathionine-ß-synthase-induced relaxation in mouse aorta involves a L-serine/sphingosine-1-phosphate/NO pathway.

BACKGROUND AND PURPOSE: Among the three enzymes involved in the transsulfuration pathway, only cystathionine ß-synthase (CBS) converts L-cysteine into L-serine and H2 S. L-serine is also involved in the de novo sphingolipid biosynthesis through a condensation with palmitoyl-CoA by the action of serine palmitoyltransferase (SPT). Here, we have investigated if L-serine contributes to the vasorelaxant effect. EXPERIMENTAL APPROACH: The presence of CBS in mouse vascular endothelium was assessed by immunohistochemistry and immunofluorescence. The relaxant activity of L-serine (0.1-300 µM) and L-cysteine (0.1-300 µM) was estimated on mouse aorta rings, with or without endothelium. A pharmacological modulation study evaluated NO and sphingosine-1-phosphate (S1P) involvement. Levels of NO and S1P were also measured following incubation of aorta tissue with either L-serine (1, 10, and 100 µM) or L-cysteine (10, 100 µM, and 1 mM). KEY RESULTS: L-serine relaxed aorta rings in an endothelium-dependent manner. The vascular effect was reduced by L-NG-nitro-arginine methyl ester and wortmaninn. A similar pattern was obtained with L-cysteine. The S1P1 receptor antagonist (W146) or the SPT inhibitor (myriocin) reduced either L-serine or L-cysteine relaxant effect. L-serine or L-cysteine incubation increased NO and S1P levels in mouse aorta. CONCLUSIONS AND IMPLICATIONS: L-serine, a by-product formed within the transsulfuration pathway starting from L-cysteine via CBS, contributes to the vasodilator action of L-cysteine. The L-serine effect involves both NO and S1P. This mechanism could be involved in the marked dysregulation of vascular tone in hyperhomocysteinemic patients (CBS deficiency) and may represent a feasible therapeutic target. LINKED ARTICLES: This article is part of a themed section on Hydrogen Sulfide in Biology & Medicine. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v177.4/issuetoc.