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.

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.

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.

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.

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.

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.

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.

Mercaptopyruvate acts as endogenous vasodilator independently of 3-mercaptopyruvate sulfurtransferase activity.

Hydrogen sulfide (H2S) is produced by the action of cystathionine-ß-synthase (CBS), cystathionine-γ-lyase (CSE) or 3-mercaptopyruvate sulfurtransferase (3-MST). 3-MST converts 3-mercaptopyruvate (MPT) to H2S and pyruvate. H2S 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 H2S donor. Colorimetric, amperometric and fluorescence based assays demonstrated that MPT releases H2S 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 H2S 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 H2S donor and not only as 3-MST substrate. MPT could, thus, be further investigated as a means to increase H2S in conditions where H2S bioavailability is reduced such as hypertension, coronary artery disease, diabetes or urogenital tract disease.

ß3 adrenergic receptor activation relaxes human corpus cavernosum and penile artery through a hydrogen sulfide/cGMP-dependent mechanism.

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 ß3 adrenoceptor in cardiovascular diseases emphasizing a possible development of new drugs. ß3 adrenoceptor stimulation relaxes human corpus cavernosum (HCC) strips in cyclic guanosine monophosphate (cGMP)-dependent and endothelium/nitric oxide (NO)-independent manner. Hydrogen sulfide (H2S), along with NO, is another gaseous molecule involved in cardiovascular system and as a consequence also in penile erection. Cystathionine-ß-synthase (CBS) and cystathionine-γ-lyase (CSE), the enzymes mainly responsible for H2S biosynthesis, are constitutively expressed in HCC. CSE rather than CBS is more abundant in human penile tissue. Herein we investigated the involvement of H2S pathway in ß3 adrenoceptor-induced relaxation in HCC and penile artery. Penile artery expresses both CSE and ß3 adrenoceptor. BRL37344, a ß3 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 H2S production. This effect was significantly reduced by the inhibition of either CSE or ß3 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 H2S/cGMP-dependent manner. In conclusion, ß3/H2S/cGMP pathway can act as an alternative to NO. Since about 15% of patients do not respond to phosphodiesterase-5 inhibitors, ß3 agonists could represent a therapeutic alternative or a useful adjuvant therapy to treat these patients.

l-Cys/CSE/H2S pathway modulates mouse uterus motility and sildenafil effect.

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 (H2S) signaling. H2S is an endogenous gaseous transmitter with myorelaxant properties predominantly formed from l-cysteine (l-Cys) by cystathionine-ß-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 H2S 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 H2S in mouse uterus. Thereafter, sildenafil significantly increased H2S 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 H2S production mediated by l-Cys or by sildenafil was not found in CSE(-/-) mice. Besides, the effect of H2S or sildenafil on spontaneous contractility was reduced in CSE(-/-) mice. A decisive proof for the involvement of H2S 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/H2S 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.

Hydrogen sulfide compensates nitric oxide deficiency in murine corpus cavernosum.

Erectile dysfunction (ED) is considered as a marker for cardiovascular diseases. Nitric oxide (NO) deficiency is the major cause of erectile dysfunction (ED). The role of hydrogen sulfide (H2S) in erection has recently been recognized and is receiving attention as a pharmacological target. Several studies have focused on the effect of H2S on NO-dependent relaxation, but the role of NO on H2S in penile tissue has not been studied yet. Unlike NO, H2S is mainly synthesized from smooth muscle cells rather than endothelial cells. We hypothesized that H2S may compensate for the decreased NO bioavailability and may be beneficial in severe ED where endothelial dysfunction is present. Thus we studied the effect of NO deficiency on H2S formation and vasorelaxation induced by l-cysteine, which is the substrate of the H2S producing enzymes in mice corpus cavernosum (MCC). NO deficiency induced by Nω-Nitro-l-arginine (L-NNA) was confirmed by the inhibition of acetylcholine-induced relaxation. l-cysteine, the substrate for the endogenous H2S production, caused a concentration-dependent relaxation that was reduced by CBS/CSE inhibitor aminooxyacetic acid (AOAA) in MCC strips. L-NNA caused a significant increase in l-cysteine-induced relaxation, and this effect was reversed by AOAA. On the contrary, no change in relaxation to NaHS (exogenous H2S donor) in MCC was observed. L-NNA increased H2S formation stimulated by l-cysteine in wild type MCC but not in CSE-/- mice. In parallel, the expression of both cysthationine γ lyase (CSE) and 3-mercaptopyruvate sulphurtransferase (3-MST) was increased, whereas cysthationine-ß synthase (CBS) was decreased in eNOS-/- MCC. We conclude that H2S plays a compensatory role in the absence of NO by enhancing the relaxation induced by endogenous H2S through CSE and 3-MPST in MCC, without altering downstream mechanisms. We suggest that H2S-targeting drugs may provide the maintenance of compensatory treatment in ED patients. This may be more relevant in ED with severe endothelial dysfunction, as H2S is mainly derived from smooth muscle.

Vascular effects of linagliptin in non-obese diabetic mice are glucose-independent and involve positive modulation of the endothelial nitric oxide synthase (eNOS)/caveolin-1 (CAV-1) pathway.

AIM: To test the effect of linagliptin in non-obese diabetic (NOD) mice, a murine model of type 1 diabetes, to unveil a possible direct cardiovascular action of dipeptidyl peptidase 4 (DPP-4) inhibitors beyond glycaemia control. METHODS: NOD mice were grouped according to glycosuria levels as NODI: none; NODII: high; NODIII: severe. Linagliptin treatment was initiated once they reached NODII levels. Vascular reactivity was assessed ex vivo on aorta harvested from mice upon reaching NODIII level. In a separate set of experiments, the effect of linagliptin was tested directly in vitro on vessels harvested from untreated NODIII, glucagon-like peptide-1 (GLP-1) receptor knockout and soluble guanylyl cyclase-α1 knockout mice. Molecular and cellular studies were performed on endothelial and endothelial nitric oxide synthase (eNOS)-transfected cells. RESULTS: In this ex vivo vascular study, endothelium-dependent vasorelaxation was ameliorated and eNOS/nitric oxide (NO)/soluble guanylyl cyclase (sGC) signalling was enhanced. In the in vitro vascular study, linagliptin exerted a direct vasodilating activity on vessels harvested from both normo- or hyperglycaemic mice. The effect was independent from GLP-1/GLP-1 receptor (GLP-1R) interaction and required eNOS/NO/sGC pathway activation. Molecular studies performed on endothelial cells show that linagliptin rescues eNOS from caveolin-1 (CAV-1)-binding in a calcium-independent manner. CONCLUSION: Linagliptin, by interfering with the protein-protein interaction CAV-1/eNOS, led to an increased eNOS availability, thus enhancing NO production. This mechanism accounts for the vascular effect of linagliptin that is independent from glucose control and GLP-1/GLP-1R interaction.

Hydrogen sulphide pathway contributes to the enhanced human platelet aggregation in hyperhomocysteinemia.

Homocysteine is metabolized to methionine by the action of 5,10 methylenetetrahydrofolate reductase (MTHFR). Alternatively, by the transulfuration pathway, homocysteine is transformed to hydrogen sulphide (H2S), through multiple steps involving cystathionine ß-synthase and cystathionine γ-lyase. Here we have evaluated the involvement of H2S in the thrombotic events associated with hyperhomocysteinemia. To this purpose we have used platelets harvested from healthy volunteers or patients newly diagnosed with hyperhomocysteinemia with a C677T polymorphism of the MTHFR gene (MTHFR++). NaHS (0.1-100 µM) or l-cysteine (0.1-100 µM) significantly increased platelet aggregation harvested from healthy volunteers induced by thrombin receptor activator peptide-6 amide (2 µM) in a concentration-dependent manner. This increase was significantly potentiated in platelets harvested from MTHFR++ carriers, and it was reversed by the inhibition of either cystathionine ß-synthase or cystathionine γ-lyase. Similarly, in MTHFR++ carriers, the content of H2S was significantly higher in either platelets or plasma compared with healthy volunteers. Interestingly, thromboxane A2 production was markedly increased in response to both NaHS or l-cysteine in platelets of healthy volunteers. The inhibition of phospholipase A2, cyclooxygenase, or blockade of the thromboxane receptor markedly reduced the effects of H2S. Finally, phosphorylated-phospholipase A2 expression was significantly higher in MTHFR++ carriers compared with healthy volunteers. In conclusion, the H2S pathway is involved in the prothrombotic events occurring in hyperhomocysteinemic patients.

Toward Repositioning Niclosamide for Antivirulence Therapy of Pseudomonas aeruginosa Lung Infections: Development of Inhalable Formulations through Nanosuspension Technology.

Inhaled antivirulence drugs are currently considered a promising therapeutic option to treat Pseudomonas aeruginosa lung infections in cystic fibrosis (CF). We have recently shown that the anthelmintic drug niclosamide (NCL) has strong quorum sensing (QS) inhibiting activity against P. aeruginosa and could be repurposed as an antivirulence drug. In this work, we developed dry powders containing NCL nanoparticles that can be reconstituted in saline solution to produce inhalable nanosuspensions. NCL nanoparticles were produced by high-pressure homogenization (HPH) using polysorbate 20 or polysorbate 80 as stabilizers. After 20 cycles of HPH, all formulations showed similar properties in the form of needle-shape nanocrystals with a hydrodynamic diameter of approximately 450 nm and a zeta potential of -20 mV. Nanosuspensions stabilized with polysorbate 80 at 10% w/w to NCL (T80_10) showed an optimal solubility profile in simulated interstitial lung fluid. T80_10 was successfully dried into mannitol-based dry powder by spray drying. Dry powder (T80_10 DP) was reconstituted in saline solution and showed optimal in vitro aerosol performance. Both T80_10 and T80_10 DP were able to inhibit P. aeruginosa QS at NCL concentrations of 2.5-10 µM. NCL, and these formulations did not significantly affect the viability of CF bronchial epithelial cells in vitro at microbiologically active concentrations (i.e., ≤10 µM). In vivo acute toxicity studies in rats confirmed no observable toxicity of the NCL T80_10 DP formulation upon intratracheal administration at a concentration 100-fold higher than the anti-QS activity concentration. These preliminary results suggest that NCL repurposed in the form of inhalable nanosuspensions has great potential for the local treatment of P. aeruginosa lung infections as in the case of CF patients.

Hydrogen sulfide is involved in dexamethasone-induced hypertension in rat.

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 (H2S) has been proposed as a candidate for EDHF. H2S is endogenously mainly formed from L-cysteine by the action of cystathionine-ß-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 H2S 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 H2S production in homogenates of mesenteric arterial bed and carotid artery from DEX-treated rats. In parallel, H2S plasma levels were significantly reduced in DEX group compared with vehicle. In conclusion, an impairment in EDHF/H2S 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.

Annexin A1 mediates hydrogen sulfide properties in the control of inflammation.

Hydrogen sulfide (H2S) is a gaseous mediator synthesized in mammalian tissues by three main enzymes-cystathionine-ß-synthase (CBS), cystathionine-γ-lyase (CSE), and 3-mercaptopyruvate-sulfurtransferase-and its levels increase under inflammatory conditions or sepsis. Since H2S and H2S-releasing molecules afford inhibitory properties in leukocyte trafficking, we tested whether endogenous annexin A1 (AnxA1), a glucocorticoid-regulated inhibitor of inflammation acting through formylated-peptide receptor 2 (ALX), could display intermediary functions in the anti-inflammatory profile of H2S. We first investigated whether endogenous AnxA1 could modulate H2S biosynthesis. To this end, a marked increase in CBS and/or CSE gene products was quantified by quantitative real-time polymerase chain reaction in aortas, kidneys, and spleens collected from AnxA1(-/-) mice, as compared with wild-type animals. When lipopolysaccharide-stimulated bone marrow-derived macrophages were studied, H2S-donor sodium hydrosulfide (NaHS) counteracted the increased expression of inducible nitric oxide synthase and cyclooxygenase 2 mRNA evoked by the endotoxin, yet it was inactive in macrophages harvested from AnxA1(-/-) mice. Next we studied the effect of in vivo administration of NaHS in a model of interleukin-1ß (IL-1ß)-induced mesenteric inflammation. AnxA1(+/+) mice treated with NaHS (100 µmol/kg) displayed inhibition of IL-1ß-induced leukocyte adhesion/emigration in the inflamed microcirculation, not observed in AnxA1(-/-) animals. These results were translated by testing human neutrophils, where NaHS (10-100 µM) prompted an intense mobilization (>50%) of AnxA1 from cytosol to cell surface, an event associated with inhibition of cell/endothelium interaction under flow. Taken together, these data strongly indicate the existence of a positive interlink between AnxA1 and H2S pathway, with nonredundant functions in the control of experimental inflammation.

The endocrine disruptor vinclozolin causes endothelial injury via eNOS/Nox4/IRE1α signaling.

Vinclozolin (VCZ) is a common dicarboximide fungicide used to protect crops from diseases. It is also an endocrine disruptor, and its effects on various organs have been described but its influence on vasculature has not yet been addressed. This study focuses on the potential mechanism of VCZ-induced vascular injury. The effect of VCZ on vascular function in terms of relaxing and contracting response was evaluated in mice aorta. A short exposure to VCZ affected the endothelial but not the smooth muscle component. Specifically, it caused a disruption of the eNOS/NO signaling. In line, a short exposure to VCZ in bovine aortic endothelial cells promoted eNOS uncoupling resulting in a reduction of NO bioavailability and eNOS dimer/monomer ratio, and in turn an increase of nitro-tyrosine levels and ROS formation. Prolonging the exposure to VCZ (3 and 6h) an up-regulation of Nox4, enzyme-generating ROS constitutively expressed in endothelial cells, and an increase in ROS and malondialdehyde content coupled with a reduction in NO levels were found. These events were strictly linked to endoplasmic reticulum stress as demonstrated by the phosphorylation of inositol-requiring transmembrane kinase endoribonuclease 1α (IRE1α), a stress sensor and its reversion by using a selective inhibitor. Collectively, these results demonstrated that VCZ provokes endothelial dysfunction by oxidative stress involving eNOS/Nox4/IRE1α axis. The rapid exposure affected the endothelial function promoting eNOS uncoupling while a post-transcriptional modification, involving Nox4/IRE1α signaling, occurred following prolonged exposure. Thus, exposure to VCZ could contribute to the onset and/or progression of cardiovascular diseases associated with endothelial dysfunction.

N-Palmitoylethanolamide protects the kidney from hypertensive injury in spontaneously hypertensive rats via inhibition of oxidative stress.

Hypertension is an important risk factor for kidney failure and renal events in the general population. Palmitoylethanolamide (PEA) is a member of the fatty acid ethanolamine family with profound analgesic and anti-inflammatory effects, resulting from its ability to activate peroxisome proliferator activated receptor (PPAR)α. A role for this nuclear receptor has been addressed in cardiovascular system and PPARα ligands have been shown to protect against inflammatory damage especially resulting from angiotensin II hypertension. In this study, we demonstrated that PEA significantly reduced blood pressure in spontaneously hypertensive rats (SHR) and limited kidney damage secondary to high perfusion pressure. To investigate the mechanisms involved in PEA effect, we found that PEA reduced cytochrome P450 (CYP) hydroxylase CYP4A, epoxygenase CYP2C23 and soluble epoxide hydrolase enzyme expression in the kidney, accompanied by a reduction of 20-hydroxyeicosatetraenoic acid excretion in the urine. Moreover, it markedly reduced kidney oxidative and nitrosative stress accompanied by decreased expression of renal NAD(P)H oxidase and inducible nitric oxide synthase and increased expression of Cu/Zn superoxide dismutase, in the kidney of SHR. Moreover, angiotensin II receptor (AT) evaluation revealed a decrease in AT1 receptor expression and a restoration of AT2 receptor level in the kidney from PEA-treated SHR. Consistently, angiotensin converting enzyme expression was reduced, implying a decrease in angiotensin II synthesis. These results indicate that PEA treatment lowers blood pressure and can protect against hypertensive renal injury by increasing the antioxidant defense and anti-inflammatory response and modulating renin-angiotensin system.

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 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.