1,721,021 research outputs found
Tesi Dottorato (dott.ssa Mitidieri Emma): New pharmacological prospectives in erectile function
No full textErectile dysfunction (ED), defined as the consistent or recurrent inability of a man to attain and/or maintain a penile erection sufficient for sexual activity. It is well recognized that the causes of ED are frequently multiple, with psychological, neurological, endocrinological, vascular, traumatic and iatrogenic components. In the present study we investigated on physiological and physiopathological mechanisms involved in erectile function. The 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 ED. To date the PD is poorly understood mainly by the lack of an appropriate animal model. In the present study, we proposed a new animal model of spontaneous PD by using tight skin (Tsk) mice that present a mutation of fibrillin-1 gene associated with hypodermal fibrosis. The Tsk mice were followed up to 24 months of age and sacrificed at different ages in order to verify the time course of the development and progression of the disease. Gross anatomy of the Tsk mouse penis clearly showed fibrosis and bending of the penis that become more evident with age, reaching the maximum at 12 months. In addition, we demonstrated by molecular studies that Tsk mice displayed i) a gradual increase in synthesis and deposition of collagen of type I ii) an high level of TGFβ especially in the earlier stage of the disease iii) an increase of iNOS mRNA level during the development of the pathology. PD represents a particular condition associated with ED. However, ED is characterized by a lack of balance between vasorelaxant and vasoconstrictor factors. Thus, we investigated on urotensin II pathway (UII), an endogenous peptide previously described as an important vasoactive peptide, in erectile function by using human corpus cavernosum. Interestingly, we reported that i) UII receptor is expressed in rat and human corpus cavernosum (HCC) as protein and as well as mRNA and that it is localized on endothelial cells ii) HCC strips relaxed to UII in endothelium and nitric oxide (NO) dependent manner. Next, we evaluated the involvement of eNOS in UII-induced relaxation by using pharmacological inhibitors. Geldanamicin or wortmannin, Hsp 90 and Akt inhibitors respectively, abrogated UII induced relaxation. This latter result was also confirmed by western blot. Finally, the UII intracavernous administration in anaesthetized rats caused an increase of intracavernous pressure, thus penile erection. To date, the International Index of Erectile Function (IIEF) represents the primary endpoint in clinical studies on erectile dysfunction (ED). The IIEF demonstrates the sensitivity and specificity for detecting treatment-related changes in patients with ED. However, following the launch on the market of all three PDE5 inhibitors, clinical and practical studies have evidenced some limitations and/or variables related to the IIEF. Thus, the present study has been set to validate the measurement of platelet cGMP as possible effect measure of PDE5 inhibitors efficacy. 46 patients with ED were enrolled and were scheduled either on placebo or on vardenafil (5 mg/die bed time) for 6 weeks. All patients were asked to complete IIEF and SEP and were underwent to the rigiscan analysis as well as to donate blood samples, before starting the protocol and after 6 week of treatment. Washed platelet challenged with a nitric oxide donor were prepared, in order to measure cGMP in both groups of patients. Our data showed that platelet cGMP production was significantly increased in patients receiving vardenafil versus placebo. Vardenafil treatment caused an amelioration in IIEF-EF and SEP scores that was not significant compared with placebo group. Conversely, the rigiscan analysis revealed a marked amelioration only in Vardenafil group. The changes in platelet cGMP well correlated with rigiscan but not with IEEF-EF and SEP. The study clearly demonstrated that platelet cGMP can be a reliable measure of PDE5 inhibitors efficacy since it is not influenced by the placebo and it well correlates with the rigiscan clinical data. Thus, the measurement of platelet cGMP may represents an easy, not invasive, and objective biomarker of PDE5 inhibitors activity in ED management as well as in preference studies. In conclusion we proposed a naturally occurring model of PD contributing either to a better knowledge of the mechanisms involved or to asses new treatments. Then, we evidenced a novel pathway involved in human erectile function. Our results may help to unravel the complex mechanisms underlying the pathophysiology of human penile erection and may lead to the development of innovative therapeutic approaches in the treatment of ED. Finally, we suggested a relatively simple, reliable, and objective method to evaluate the PDE5 inhibitors efficacy in the ED management
Attività antinfiammatoria dei Glucocorticoidi: nuovi mediatori e regolazione dell'attività dei linfociti T
No full textPatologie gravi infiammatorie croniche o autoimmunitarie richiedono il trattamento con farmaci potentemente antinfiammatori ed immunosoppressivi, quali i Glucocorticoidi (GC). Nonostante la loro elevata efficacia terapeutica, un’ampia serie di effetti avversi ne compromette l’uso prolungato. La conoscenza fine dei meccanismi sottesi al loro funzionamento a livello molecolare permette di separare gli effetti terapeutici da quelli dannosi. I mediatori dell’azione dei GC sono in buona parte noti, ma essendo essi stessi ormoni in grado di attivare o reprimere centinaia di geni, la comprensione del ruolo di nuovi mediatori è assolutamente necessaria. Inoltre, considerando che nei processi infiammatori i linfociti T svolgono un ruolo importante con tutte le loro sottopopolazioni specializzate nella produzione di citochine specifiche, capaci anche di mantenere la “memoria immunologica” della malattia, il progetto ha come finalità quella di capire la correlazione fra vari sistemi proteici e gassosi partecipanti al processo infiammatorio e le cellule T CD4+ di tipo Th1, Th2, Th17 e Treg in topi geneticamente modificati per GILZ (GILZ-knock-out, KO o GILZ transgenici, TG), sottoposti a vari modelli in vivo di patologie autoimmunitarie ed infiammatorie.
GILZ (Glucocorticoid-Induced Leucine Zipper) è una proteina rapidamente indotta dai GC e capace di etero-dimerizzare con
molecole come NF-κB, Ras/Raf e SMAD, con conseguente blocco dell’attività trascrizionale di geni ad attività pro-infiammatoria e induzione di cellule T regolatorie, mimando, in tal modo, l’azione dei GC. Al processo infiammatorio prendono parte numerosi fattori, tra cui il sistema iNOS/NO, coinvolto nei disordini infiammatori delle articolazioni, dell’intestino e dei polmoni, e quello dell’H2S. L’H2S, è modulato dai GC e al pari dei GC sopprime il sistema iNOS/NO e modula l'attivazione delle cellule T. Altro importante fattore nell’infiammazione è la nicotinammide fosforibosil trasferasi (NAMPT), considerata un biomarcatore infiammatorio ed un possibile target terapeutico nell’artrite reumatoide (AR), in cui ne è stata descritta una sovra-espressione. Altrettanto rilevante è il sistema dell’Istamina; come noto i GC ne regolano la produzione e modulano l’espressione dei suoi recettori. Il nostro progetto si propone di indagare la relazione esistente fra GILZ, il sistema della iNOS/NO, l’H2S , la NAMPT e
l’istamina usando modelli murini di patologie infiammatorie/autoimmunitarie quali ad esempio l’AR, la colite, la fibrosi polmonare (FP), la dermatite atopica (DA) ed il dolore neuropatico conseguente al processo infiammatorio. I risultati porranno le basi per lo sviluppo di farmaci che, con un’azione mirata, agiscano selettivamente al fine di ottenere un effetto terapeutico con un esito simile a quello dei GC, mimandone gli effetti antinfiammatori/immunosoppressivi ma con minori effetti avversi
Editorial: Interplays and Functions of Gaseous Mediators: From Underlying Mechanisms to Therapeutic Approaches in Cardiovascular Diseases
No full textLinagliptin Rescues Vascular Response in Nonobese Diabetic Mice: Involvement of the L-arginine/eNOS Pathway
No full textChiarimento dei meccanismi molecolari alla base dell'effetto antidiabetico del Linagliptin
Breathing new in severe lung diseases: innovative therapeutic approaches based on inhalable formulations
No full textThe general aim of the BREATHE project is the integrated development of pulmonary delivery strategies useful to improve the efficacy of innovative oligonucleotide (ON)-based therapies for severe lung diseases, with a special emphasis to double stranded ONs. Upon positive peer-review of the European Science Foundation, the Italian Foundation Compagnia di San Paolothrough the so-called STAR program (i.e., Territorial Support to Research Activity) funded the Start-up phase of the project. it is a 2-year joint project involving Italian research teams from Dept. of Pharmacy of University of Naples Federico II (UNINA), Second University of Naples (SUN), National Cancer Institute of Naples G. Pascale (NCI), Giannina Gaslini Institute of Genoa (GASLINI) and a research team from Université Catholique de Louvain, Belgium (UNILV). The project is organized in 2 main work packages (WP), each characterized by specific objectives. In the context of each WP, the activities will be carried out in three steps, i.e. design and production of engineered dry powders (T1), evaluation of biological behavior and in vitro effects on cells of the dry powder formulations (T2), evaluation of in vivo biodistribution, toxicity and activity on rat models (T3). In particular, the feasibility of dry powders for inhalation of decoy ONs and siRNA, one of groundbreaking advances in pulmonary delivery over the past 30 years, will be investigate in different preclinical models
Involvement of nitric oxide and hydrogen sulfide in glucorticoid-induced hypertension in rat
No full textExcess of glucocorticoid, either endogenous, as in Cushing syndrome, or exogenous, via pharmacological administration of
glucorticoids, induces hypertension. This form of hypertension is commonly related to activation of the mineralcortocoids
receptor whereas it certainly has a role (Baid S et al., 2004). However, evidences indicate that glucocorticoid elevated
blood pressure independently by mineralcorticoid receptor, in both humans and animal model (Kalimi M et al., 1989;
Bertagna C et al., 1986). Furthermore, glucorticoid receptor is present in both vascular smooth muscle (Provencher PH et
al., 1995) and endothelium (Wallerath T et al., 1999). To date, the mechanism(s) underlying glucocorticoid induced
increase in blood pressure is still unclear. The gaseous transmitters nitric oxide (NO) and hydrogen sulphide (H2S) together
with endothelial-derived hyperpolarizing factor (EDHF) play a key role in the regulation of vascular homeostasis. In
cardiovascular system, NO derived from endothelial NO synthase (eNOS) while H2S is predominantly produced by
cisthationine-β synthase (CBS) and/or cystathionine-γ lyase (CSE). Published data suggest that H2S is EDHF
(d'Emmanuele di Villa Bianca et al., 2011; Tang G et al., 2013). Therefore, we investigated the involvement of NO and
H2S/EDHF signalling in glucocorticoid-induced hypertension. Male Wistar rats were treated with dexamethasone (DEX,
1.5mg/kg subcutaneously) or vehicle (saline) for 8 days. During treatment, blood pressure was recorded by using a tail cuff
apparatus, in conscious rats. Perfused arterial mesenteric plexus was used to evaluate the NO and the EDHF contribute.
Thereafter, western blot study was performed to appreciate phoshorylated-eNOS/eNOS ratio or CBS and CSE expression
in mesenteric tissue. The production of NO and H2S was also measured. DEX treatment caused a significant increase in
blood pressure. The contribute of NO mediated vasodilation was higher in the mesenteric bed of DEX-treated rats.
Conversely, EDHF-mediated vasodilatation resulted significantly reduced in DEX group. In line with these findings, the peNOS/
eNOS ratio as well as NO production were significantly increased in DEX group compared with vehicle. On the
other hand, CBS and CSE expression was markedly reduced in DEX group and well correlated with H2S production.
Consistently with the fact that H2S is EDHF, this latter result strongly support the reduction in EDHF-vasodilation
occurring in DEX-group. In conclusion, our data indicate that exists a cross-talk between NO and H2S in glucorticoidinduced
hypertension. In this scenario H2S pathway could represent a novel target opening new pharmacological strategies
in the control of blood pressure.
Baid S and Nieman LK. Curr Hypertens Rep 2004; 6:493-9.
d'Emmanuele et al., JPET 2011; 337:59-64.
Kalimi M et al., Am J Physiol 1989; 256:E682–E685.
Bertagna X et al., J Clin Endocrinol Metab 1986 63:639-43.
Wallerath T. et al., Proc Natl Acad Sci USA 1999; 13357-13362.
Provencher PH et al., J Steroid Biochem Mol Biol 1995; 2:219-25.
Tang G et al., Antioxid Redox Signal. 2013 [Epub ahead of print]
l-Cysteine/H2S pathway involvement in human platelet aggregation
No full textBackground
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] and [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 < 0.001). Neither L-cys nor NaHS (0.1 μM–10 mM) affected human washed platelets in resting conditions, but both significantly increased TRAP-6-induced aggregation (p < 0.001 for L-cys 0.1 mM and NaHS 0.1 mM and 10 μM; p < 0.01 for NaHS 1 μM and p < 0.05 for L-cys 10 μM). Besides, H2S did not modify platelet cAMP levels. Conversely, INDO, AACOCF3 and SQ29548 blocked the potentiating effect of H2S on platelet aggregation induced by TRAP-6. Interestingly, both NaHS and L-cys induced a significantly increase in TXA2production (p < 0.01).
Conclusions
Our data imply that the H2S endogenously produced within human platelets is involved in platelet aggregation through PLA2 activation. These findings may open a new pharmacological approache in platelet dependent disorders
Endogenous urotensin–II induced-penile erection trhough eNOS phosphorylation in human corpus cavernosum
No full textUrotensin II (U-II) is a cyclic peptide originally isolated from the neurosecretory system of the teleost fish and subsequently found in other species, including man. U-II was identified as the natural ligand of a G-protein coupled receptor (GPR 14), namely UT receptor (Romanic et al., 1999). To date, the source of U-II production in the human body remains to be elucidated. Both U-II and UT receptor are expressed widely within the cardiovascular system, and the expression is up-regulated in human cardiovascular disease, including congestive heart failure, hypertension, type II diabetes and diabetic nephropathy (Russell et al., 2004; Maguire et al., 2000).Recent evidence indicates the involvement of U-II/UT pathway in penile function in human, but the molecular mechanism is still unclear (d'Emmanuele di Villa Bianca et al., 2010).The aim of this study was to investigate the mechanism(s) of U-II-induced relaxation in human tissue and its relationship with L-arginine/Nitric oxide (NO) pathway. The human corpus cavernosum (HCC) was obtained from male to female surgical procedure. U-II mRNA by quantitative RT-PCR was assessed in human tissue. Nitrite content, as index of NO production, was fluorometrically determined in tissues challenged with U-II or vehicle. Western blots for phosphorylated-eNOS(Serine 1177) and eNOS were evaluated in HCC incubated with U-II (10 μM). HCC precontracted strips were challenged with U-II (0.1 nM–10 μM) in presence either wortmannin or geldanamycin, inhibitors of eNOS phosphorylation and Hsp90 coupling, respectively. A co-immunoprecipitation study between eNOS and UT receprtor was also performed following stimulation with U-II or vehicle. U-II as mRNA is expressed in HCC and promotes NO production. Wortmannin or geldanamycin inhibited significantly U-II-induced relaxation in HCC strips. Additionally, U-II significantly increased eNOS phosphorylation/eNOS ratio, supporting the functional study. UT receptor and eNOS co-immunoprecipitated following U-II challenge of HCC. In conclusion, U-II is endogenously synthesized and locally released in HCC. Thepro-erectile effect of U-II is strictly dependent upon NO generation by eNOSphosphorylation and Hsp90 recruitment. Thus, UII/UT pathway may contribute to the maintenance of full penile erection.
Romanic (1999). Nature. 401, 282–286.
Maguire (2000). Br J Pharmacol. 131, 441–446.
Russell (2004). Pharmacol Ther 103, 223-243.
d'Emmanuele di Villa Bianca (2010). J Sex Med.7, 1778-1786
Hydrogen sulphide enhances human platelet aggregation through phospholipase A2 activation
No full textHydrogen sulphide (H2S) is the third gaseous mediator alongside with nitric oxide and carbon
monoxide. It is endogenously produced in various mammalian tissues from L-cysteine (L-cys) by the
action of cysthationine-β-synthase (CBS) and/or cysthationine-γ-lyase (CSE), which are expressed in
tissue specific fashion (Zhao W et al., 2001, The EMBO Journal). As a biologically diffusible mediator,
H2S exerts physiological and pathological effects in many organs and systems. Several evidences
show the H2S involvement in the regulation of vascular homeostasis (Yang G. et al., 2008, Science),
including vascular smooth muscle cell relaxation and constriction (Hosoki R et al., 1997, Biochem
Biophys Res Commun.), pro- and anti-inflammatory actions (Li L et al., 2006, Curr Opin Pharmacol.),
modification of apoptosis (Sivarajah A et al. 2009, Shock), promotion of angiogenesis (Cai WJ et al.,
2007, Cardiovasc Res). To date, evidences for the role(s) of H2S in platelets is still lacking. Therefore,
we evaluated the effect of H2S on human platelets investigating its underlying mechanism. Human
washed platelets were collected from healthy volunteers. The expression of both CBS and CSE was
evaluated by western blot analysis. H2S levels were measured by a colorimetric assay for CBS and
CSE activity. Traditional light transmission aggregometry technique was used to analyse platelet
aggregation; H2S-induced effect was evaluated using both an exogenous source of H2S (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, 2μM) stimulus. In order to assess the H2S mechanism(s) we operated a
pharmacological modulation by using specific inhibithor of arachidonic acid cascade i.e. 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. In
addition, thromboxane (TXA2) and cAMP levels were evaluated. Our results clearly showed that
human washed platelets possess both CBS and CSE and generate detectable amounts of H2S,
significantly increased after incubation with L-cys (***p<0.001). Neither L-cys nor NaHS (0.1 μM–10
mM) affected human washed platelets in resting conditions, but both significantly increased
aggregations induced by TRAP-6 amide (p<0.001 for L-cys 0.1 mM and NaHS 0.1 mM and 10 μM;
p<0.01 for NaHS 1 μM and p<0.05 for L-cys 10 μM). H2S did not affect platelet cAMP levels. On the
other hand, INDO, AACOCF3 and SQ29548 blocked the potentiating effect of H2S on platelet
aggregation. Interestingly, both NaHS and L-cys induced an increase in TXA2 production. In
conclusion, our data suggest that H2S is endogenously produced within human platelets and is
involved in platelet aggregation through PLA2 activation. These findings may highlight new targets
for the development of interventions in platelet dependent disorders
Hydrogen sulphide enhances human platelet aggregation through PLA2 activation
No full textHydrogen sulphide (H2S) is emerging as an important endogenous mediator produced in various mammalian tissues from L-Cysteine by the actions of cysthationine β-synthase (CBS) and/or cysthationine-γ-lyase (CSE), which are expressed in tissue specific fashion (Zhao et al. 2001). H2S exerts physiological and pathological effects in many organs and systems, but attention has been focused mainly on the cardiovascular system. It is now well established that H2S is involved in vascular homeostasis, for example regulating blood pressureand producing biphasic effects in the regulation of vascular tone (Yang et al., 2008; d’Emmanuele di Villa Bianca et al., 2011; Lim et al. 2008). Evidence for the role(s) of H2S in platelets is still lacking. Here, we first evaluated the effect of H2S on human platelets and then we investigated the possible pathways involved. We evaluated the presence of both CBS and CSE by western blot analysis, and used an H2S quantification assay to assess CBS and CSE activity. In order to look at influences on platelet reactivity, human washed platelets were collected from healthy volunteers; thrombin receptor activator peptide 6 amide (TRAP-6, 2μM) was used in presence or absence of sodium hydrogen sulphide (NaHS, 0.1 μM – 10 mM), as an H2S donor, or L-Cysteine, as an H2S precursor (0.1 μM – 10 mM). D-Cysteine was used as negative control. These experiments were repeated in the presence of aminoxyacetic acid (AOAA, 1 mM, 15 min) or propargylglycine (PGG, 1 mM, 15 min), CBS and CSE inhibitors, respectively. In order to assess possible mechanism(s) underlying the effects of H2S, we measured cAMP levels and evaluated the effects of either indomethacin (INDO, 10 μM, 15 min),a COX inhibitor, or arachidonyl trifluoromethyl ketone (AACOCF3, 1 μM, 15 min), a PLA2 inhibitor. Western blot demonstrated the presence of both CBS and CSE in human platelets. L-Cysteine, but not D-Cysteine, significantly increased H2S production (p<0.001), and this effect was significantly reduced by either PGG or AOAA (p<0.001 and p<0.01, respectively). Neither L-Cysteine nor NaHS (both 0.1 μM – 10 mM) affected human washed platelets in resting conditions, but both significantly increased aggregations induced by TRAP-6 amide (p<0.001 for L-Cys 0.1 mM and NaHS 0.1 mM and 10 μM; p<0.05 for L-Cys 10 μM and NaHS 1 μM), effects that were completely abolished by pre-treatment with PGG or AOAA. H2S did not affect platelet cAMP levels. On the other hand , both INDO and AACOCF3 blocked the potentiating effects of H2S on platelet aggregation. In conclusion, our data suggest that H2S is endogenously produced within human platelets and is involved in platelet aggregation through PLA2activation. These findings may highlight new targets for the development of interventions in platelet dependent disorders.
d’Emmanuele di Villa Bianca (2011) J Pharmacol Exp Ther (in press).
Lim (2008)Am J Physiol Cell Physiol. 295,C1261-70.
Yang (2008) Science 322, 587-90.
Zhao (2001)The EMBO Journal20, 6008-16
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