Emma Mitidieri

Sildenafil effect on the human bladder involves the L-cysteine/hydrogen sulfide pathway: a novel mechanism of action of phosphodiesterase type 5 inhibitors.

BACKGROUND Phosphodiesterase type 5 inhibitors (PDE5-Is) are effective in the treatment of lower urinary tract symptom (LUTS), although their mechanism of action is still unclear. PDE5-Is cause bladder detrusor relaxation, and this effect is partially independent of nitric oxide. Hydrogen sulfide (H(2)S) is a newly discovered transmitter with myorelaxant properties. It is predominantly formed from L-cysteine by cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE). OBJECTIVE To evaluate whether the L-cysteine/H(2)S pathway contributes to the relaxing effect of sildenafil on the human detrusor dome. DESIGN, SETTING, AND PARTICIPANTS Samples of bladders obtained from men undergoing open prostatectomy for benign prostatic hyperplasia (BPH) were used. The presence of CBS and CSE enzymes was assessed by western blot. H(2)S production was measured by a colorimetric assay in basal and stimulated conditions with L-cysteine and in response to sildenafil (1, 3, 10, and 30 μM), 8-bromo-cyclic guanosine monophosphate (8-bromo-cGMP; 100 μM) or dibutyryl-cyclic adenosine monophosphate (dibutyryl-cAMP; 100 μM). A curve concentration effect of sodium hydrosulfide (NaHS), H(2)S donor (0.1 μM to 10mM), L-cysteine (0.1 μM to 10mM), and sildenafil (0.1-10 μM) was performed on precontracted detrusor dome strips. To investigate H(2)S signaling in a sildenafil effect, CBS and CSE inhibitors were used. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS Analysis of variance was used, followed by the Bonferroni post hoc test. RESULTS AND LIMITATIONS CBS and CSE are present in the human bladder dome and efficiently convert L-cysteine into H(2)S. Both NaHS and L-cysteine relaxed human strips. Sildenafil caused (1) a relaxation of bladder dome strips and (2) a concentration-dependent increase in H(2)S production. Both effects were significantly reduced by CBS and CSE inhibitors. Similar to sildenafil, both 8-bromo-cGMP and dibutyryl-cAMP caused an increase in H(2)S production. CONCLUSIONS The sildenafil relaxant effect on the human bladder involves the H(2)S signaling pathway. This effect may account in part for the efficacy of PDE5-Is in LUTS. A better definition of the pathophysiologic role of the H(2)S pathway in the human bladder may open new therapeutic approaches.

Hydrogen Sulfide-Induced Dual Vascular Effect Involves Arachidonic Acid Cascade in Rat Mesenteric Arterial Bed

Hydrogen sulfide (H2S), a novel gaseous transmitter, is considered a physiological regulator of vascular homeostasis. Recent evidence suggests H2S as an endothelium-hyperpolarizing factor (EDHF) candidate. To address this issue, we evaluated the vascular effect of sodium hydrogen sulfide (NaHS), an H2S donor, on the rat mesenteric arterial bed. NaHS concentration-response curve was performed on preconstricted mesenteric arterial bed. To assess the contribution of EDHF, we performed a pharmacologic dissection using indomethacin, NG-nitro-l-arginine methyl ester (l-NAME), or apamin and charybdotoxin as cyclooxygenase, nitric-oxide synthase, and calcium-dependent potassium channel inhibitors, respectively. In another set of experiments, we used 4-(4-octadecylphenyl)-4-oxobutenoic acid, baicalein, or proadifen as phospholipase A2 (PLA2), lipoxygenase, and cytochrome P450 inhibitors, respectively. Finally, an immunofluorescence study was performed to support the involvement of PLA2 in mesenteric artery challenged by NaHS. NaHS promoted a dual vascular effect (i.e., vasoconstriction and vasodilation). l-NAME or baicalein administration affected neither NaHS-mediated vasodilation nor vasoconstriction, whereas apamin and charybdotoxin significantly inhibited NaHS-induced relaxation. Pretreatment with PLA2 inhibitor abolished both the contracting and the relaxant effect, whereas P450 cytochrome blocker significantly reduced NaHS-mediated relaxation. The immunofluorescence study showed that NaHS caused a migration of cytosolic PLA2 close to the nucleus, which implicates activation of this enzyme. Our data indicate that H2S could activate PLA2, which in turn releases arachidonic acid leading, initially, to vasoconstriction followed by vasodilation mediated by cytochrome P450-derived metabolites. Because EDHF has been presumed to be a cytochrome P450 derivative of the arachidonic acid, our results suggest that H2S acts through EHDF release.

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

Significance Here we have demonstrated that the l-cysteine/H2S pathway contributes to the increased thrombotic events associated with hyperhomocysteinemia. In particular, by using platelets harvested from hyperhomocysteinemic patients, H2S generated within the platelets in turn activates the arachidonic acid cascade by phosphorylating phospholipase A2. This cascade of events primes the platelet that becomes more responsive (reactive) to endogenous stimuli. Finally, our study also suggests that H2S could represent a unique biomarker in patients affected with hyperhomocystinemia to define the susceptibility of a cardiovascular event driven by platelets. 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 β-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 β-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.

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-β-synthase (CBS) and cystathionine-γ-lyase (CSE). It plays many physiological roles and contributes to cardiovascular function. Here we have evaluated the role played by 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.

Hydrogen sulphide induces mouse paw oedema through activation of phospholipase A2

BACKGROUND AND PURPOSE Hydrogen sulphide (H2S), considered as a novel gas transmitter, is produced endogenously in mammalian tissue from L‐cysteine by two enzymes, cystathionine β‐synthase and cystathionine γ‐lyase. Recently, it has been reported that H2S contributes to the local and systemic inflammation in several experimental animal models. We conducted this study to investigate on the signalling involved in H2S‐induced inflammation.

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.

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-β-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-1β (IL-1β)–induced mesenteric inflammation. AnxA1+/+ mice treated with NaHS (100 μmol/kg) displayed inhibition of IL-1β–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.

Urothelium muscarinic activation phosphorylates CBSSer227 via cGMP/PKG pathway causing human bladder relaxation through H2S production

The urothelium modulates detrusor activity through releasing factors whose nature has not been clearly defined. Here we have investigated the involvement of H2S as possible mediator released downstream following muscarinic (M) activation, by using human bladder and urothelial T24 cell line. Carbachol stimulation enhances H2S production and in turn cGMP in human urothelium or in T24 cells. This effect is reversed by cysthationine-β-synthase (CBS) inhibition. The blockade of M1 and M3 receptors reverses the increase in H2S production in human urothelium. In T24 cells, the blockade of M1 receptor significantly reduces carbachol-induced H2S production. In the functional studies, the urothelium removal from human bladder strips leads to an increase in carbachol-induced contraction that is mimicked by CBS inhibition. Instead, the CSE blockade does not significantly affect carbachol-induced contraction. The increase in H2S production and in turn of cGMP is driven by CBS-cGMP/PKG-dependent phosphorylation at Ser227 following carbachol stimulation. The finding of the presence of this crosstalk between the cGMP/PKG and H2S pathway downstream to the M1/M3 receptor in the human urothelium further implies a key role for H2S in bladder physiopathology. Thus, the modulation of the H2S pathway can represent a feasible therapeutic target to develop drugs for bladder disorders.

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.

Recombinant human erythropoietin prevents lipopolysaccharide-induced vascular hyporeactivity in the rat.

ABSTRACT Erythropoietin (EPO) is a hypoxia-inducible hormone that is essential for normal erythropoiesis in the bone marrow. Administration of recombinant human-EPO is currently being used for the therapy of anemia associated with chronic renal failure and cancer. Moreover, EPO reduces organ injury in experimental hemorrhagic as well as in splanchnic artery occlusion shock and preserves cardiac function after experimental cardiac I/R. Erythropoietin receptors are widely distributed in the cardiovascular system, including endothelial, smooth muscle, cardiac, and other cell types, and nonhematopoietic effects of EPO are increasingly recognized. Thus, the vasculature may be a biological target of EPO. Therefore, the aim of our study was to investigate whether EPO exerts a protective effect in septic shock by modulating vascular dysfunction and hyporeactivity. Rats received EPO (300 U/kg, i.v.) or vehicle 30 min before and 1 and 3 h after LPS (8 × 106 U/kg, i.v.). In vivo and ex vivo (aortic rings) experiments were performed to evaluate the vascular response to contracting and vasodilating agents. The expression of iNOS, intercellular adhesion molecule 1, poly(ADP)ribose polymerase, Bcl-xl, and Bcl-2 was evaluated by Western blot analysis in the rat aorta. We demonstrate that EPO significantly prevents LPS-induced vascular hyporeactivity and endothelial dysfunction. Interestingly, EPO inhibits the increase in iNOS, poly(ADP)ribose polymerase, and intercellular adhesion molecule 1 expression in the aorta of endotoxemic rats and attenuated the decline in the expression of both Bcl-xl and Bcl-2 caused by LPS. In conclusion, our data support the view that EPO has important nonerythropoietic effects protecting organ and tissue against injury and indicate that EPO may be useful in the therapy of patients with septic shock.