Raffaella Sorrentino

Dynamic activation of endothelial nitric oxide synthase by Hsp90

Heat-shock protein 90 (Hsp90) coordinates the trafficking and regulation of diverse signalling proteins, but its precise role in regulating specific cellular targets is not known,. Here we show that Hsp90 associates with endothelial nitric oxide synthase (eNOS) and is rapidly recruited to the eNOS complex by agonists that stimulate production of nitric oxide, namely vascular endothelial growth factor, histamine and fluid shear stress. Moreover, the binding of Hsp90 to eNOS enhances the activation of eNOS. Inhibition of signalling through Hsp90 attenuates both agonist-stimulated production of nitric oxide and endothelium-dependent relaxation of isolated blood vessels. Our results indicate that Hsp90 facilitates signalling mediated by growth-factor, G-protein and mechanotransduction pathways that lead to the activation of eNOS. These observations indicate that in addition to its role as a molecular chaperone involved in protein folding and maturation, Hsp90 may also be recruited to cellular targets depending on the activation state of the cell.

Endothelial-derived relaxing factor released by endothelial cells of human umbilical vessels and its impairment in pregnancy-induced hypertension

The principal objective of this study was to determine and compare the capability of human umbilical vessels of normal and pregnancy-induced hypertensive parturients to produce and to release the endothelial-derived relaxing factor. A bolus of bradykinin injected in the perfusion system of human umbilical vessels induces a release of a relaxant factor, detectable by bioassay, that is pharmacologically similar to the endothelial-derived relaxing factor. Human umbilical cords were collected from normal and pregnancy-induced hypertensive parturients. In the latter group the release of endothelial-derived relaxing factor is extremely reduced. In fact, in umbilical vessels collected from normal parturients, bradykinin at a dose of 20 pmol produces a release of endothelial-derived relaxing factor equivalent to a relaxation induced by 59.9 +/- 11.0 and 30.8 +/- 11.4 pmol of glyceryl trinitrate for the artery and vein, respectively. The same dose of bradykinin in umbilical vessels, collected from pregnancy-induced hypertensive parturients, produces a release equivalent to 6.6 +/- 2.2 and 5.7 +/- 3.5 pmol of glyceryl trinitrate equivalent for the artery and vein, respectively. Neither an increasing bolus of exogenous bradykinin or an infusion of superoxide dismutase or L-arginine was able to restore the production of endothelial-derived relaxing factor to normal levels. Our results indicate a probable alteration of endothelial cell numbers or an alteration of the enzymatic pathway, probably due to cytotoxic endogenous factors produced in pregnancy.

NO-aspirins: a class of new anti-inflammatory and antithrombotic agents

Nitroaspirins are a new class of compounds that combine COX inhibitory activity with NO release. The release of NO accounts for both the safe GI profile and the ability of nitroaspirins to interfere with pathological processes, which most likely occur through two separate mechanisms. Furthermore, and most importantly, repeated administration of NCX4016 results in almost total inhibition of prostanoid production after five or six days of treatment, which is comparable to the effects of low-dose aspirin. The lack of GI side-effects, together with the potential for an increased therapeutic benefit due to their additional pharmacological activity, opens the possibility for a wide range of application for nitroaspirins that should be explored in depth.

Dry powders based on PLGA nanoparticles for pulmonary delivery of antibiotics: Modulation of encapsulation efficiency, release rate and lung deposition pattern by hydrophilic polymers

Although few experimental studies have been handled so far to exploit the potential of poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) in the production of dry powders for antibiotic inhalation, there has been no comprehensive study on the role played by NP composition. In this work, we try to shed light on this aspect by designing and developing a pulmonary delivery system for antibiotics, such as tobramycin (Tb), based on PLGA NPs embedded in an inert microcarrier made of lactose, referred to as nano-embedded micro-particles (NEM). At nanosize level, helper hydrophilic polymers were used to impart the desired surface, bulk and release properties to PLGA NPs prepared by a modified emulsion-solvent diffusion technique. Results showed that poly(vinyl alcohol) (PVA) and chitosan (CS) are essential to optimise the size and modulate the surface properties of Tb-loaded PLGA NPs, whereas the use of alginate (Alg) allows efficient Tb entrapment within NPs and its release up to one month. Optimized formulations display good in vitro antimicrobial activity against P. aeruginosa planktonic cells. Furthermore, spray-drying of the NPs with lactose yielded NEM with peculiar but promising flow and aerosolization properties, while preserving the peculiar NP features. Nonetheless, in vivo biodistribution studies showed that PVA-modified Alg/PLGA NPs reached the deep lung, while CS-modified NPs were found in great amounts in the upper airways, lining lung epithelial surfaces. In conclusion, PLGA NP composition appears to play a crucial role in determining not only the technological features of NPs but, once processed in the form of NEM, also their in vitro/in vivo deposition pattern.

Hydrogen sulfide as a mediator of human corpus cavernosum smooth-muscle relaxation

Hydrogen sulfide (H2S) is synthesized by 2 enzymes, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE). l-Cysteine (l-Cys) acts as a natural substrate for the synthesis of H2S. Human penile tissue possesses both CBS and CSE, and tissue homogenates efficiently convert l-Cys to H2S. CBS and CSE are localized in the muscular trabeculae and the smooth-muscle component of the penile artery, whereas CSE but not CBS is also expressed in peripheral nerves. Exogenous H2S [sodium hydrogen sulfide (NaHS)] or l-Cys causes a concentration-dependent relaxation of strips of human corpus cavernosum. l-Cys relaxation is inhibited by the CBS inhibitor, aminoxyacetic acid (AOAA). Electrical field stimulation of human penile tissue, under resting conditions, causes an increase in tension that is significantly potentiated by either propargylglycine (PAG; CSE inhibitor) or AOAA. In rats, NaHS and l-Cys promote penile erection, and the response to l-Cys is blocked by PAG. Our data demonstrate that the l-Cys/H2S pathway mediates human corpus cavernosum smooth-muscle relaxation.

Induction by endotoxin of nitric oxide synthase in the rat mesentery: lack of effect on action of vasoconstrictors

1 Male Sprague‐Dawley or Wistar rats were injected with bacterial lipopolysacharride (LPS; 5 mg kg−1, i.p.) and killed after 1, 3, 6, 15, and 24 h. The brains, mesenteries, spleens, lungs, livers, kidneys, hearts, aortae and diaphragms were removed and frozen immediately. Control rats were injected with sterile saline and killed after 6 h. 2 The organs were homogenized in a semi‐frozen state and NO synthase (NOS) activity measured in tissues from both LPS‐treated and saline‐treated groups by the ability of homogenates to convert [3H]‐l‐arginine to [3H]‐l‐citrulline in a NADPH‐dependent manner. 3 The NOS activity in all organs taken from control animals was found to be calcium‐dependent, with the highest activity being in the brain. After LPS‐treatment an induced calcium‐independent NOS was detected in all tissues tested, with the exception of the brain. The spleen, lung, mesentery and liver had the highest amounts of LPS‐induced NOS activity. No induction of calcium‐dependent NOS was detected. 4 Induction of NOS was maximum 6 h after administration of LPS and had returned to control levels in 24 h. 5 The constitutive NOS in brain and mesentery and the LPS‐induced activities in the spleen, lung, liver and mesentery were inhibited by NG‐monomethyl‐l‐arginine (l‐NMMA) or NG‐nitro‐l‐arginine methyl ester (l‐NAME) according to concentration. The IC50 for l‐NAME was 2.5 μm against the constitutive NOS from brain, and 20–25 μm against the inducible NOS. For l‐NMMA the IC50 was 20–25 μm against either NOS isoform. 7 The vascular responses to endothelin‐1 (ET‐1), the thromboxane A2‐mimetic 11α,9α‐epoxymethano prostaglandin F2α (U46619), phenylephrine (PE) or 5‐hydroxytryptamine (5‐HT) were measured in the simultaneously perfused arterial and venous mesenteric vascular beds from both control and LPS‐treated (6 h) rats. Vasoconstrictor responses to all agonists tested were unaffected by LPS treatment. In the presence of l‐NAME (100 μm) vasoconstrictor responses were potentiated in both the arterial and venous portion of the mesenteric beds from both control and LPS‐treated rats. The potentiation of responses to U46619 was significantly greater in beds from LPS‐treated rats. 8 Injection of LPS i.p. is associated with induction of NOS in all organs tested, except for the brain. In the mesentery this is not accompanied by a hyporesponsiveness to constrictor agents suggesting an increased sensitivity, particularly to U46619. This may explain the poor perfusion and tissue damage in the splanchnic circulation associated with sepsis.

Insulin-loaded PLGA/cyclodextrin large porous particles with improved aerosolization properties: In vivo deposition and hypoglycaemic activity after delivery to rat lungs

The aim of the present work is to develop large porous particles (LPP) of poly (lactide-co-glycolide) (PLGA) containing insulin with optimal aerodynamic properties and to test their in vivo potential, in pulmonary delivery. Insulin-loaded LPP were fabricated by a double emulsion method by aid of hydroxypropyl-beta-cyclodextrin (HPbetaCD). Conceiving this system for the controlled release of insulin to the lungs, the aerosolization properties and the release features in simulated lung fluids of PLGA/HPbetaCD/insulin LPP were investigated in depth. The technological results show that the combination of appropriate amounts of insulin and HPbetaCD plays a crucial role to achieve PLGA/HPbetaCD/insulin LPP with the desired bulk and aerodynamic properties, that is a highly porous structure, a very low density (0.1 g/ml), an experimental mass mean aerodynamic diameter (MMAD(exp)) ranging from 4.01 to 7.00 and a fine particle fraction (FPF) estimated to be 26.9-89.6% at the different airflow rates tested (i.e. 30-90 l/min). Confocal microscopy studies, performed after administration of labeled PLGA/HPbetaCD/insulin LPP to the rat lung by means of a low-scale dry powder inhaler (DPI), suggest that particles reach alveoli and remain in situ after delivery. The pharmacological effect of PLGA/HPbetaCD/insulin LPP was confirmed by dose-response studies performed on both normoglycaemic and streptozotocin-induced diabetic rats. While insulin solutions administered via pulmonary route are unable to cause a significant hypoglycaemic effect, insulin delivered through PLGA/HPbetaCD/insulin LPP at the same doses (0.5-4.0 IU/kg) significantly reduces blood glucose level as a function of the administered dose in both animal models. The developed LPP, tested in hyperglycaemic rats at evident pathological conditions, exerts a very significant and longer hypoglycaemic effect even at insulin doses as low as 0.5 IU/kg (about 0.5 mg of PLGA/HPbetaCD/insulin LPP per rat) as compared to a insulin solution. Taken together, our results support the viability of a dry powder formulation based on biodegradable LPP for the controlled release of insulin to the lungs. In vivo data show that PLGA/HPbetaCD/insulin LPP are able to reach alveoli, release insulin, which is absorbed in its bioactive form.

Protease-activated receptor-2 involvement in hypotension in normal and endotoxemic rats in vivo.

BACKGROUND The protease-activated receptor-2 (PAR-2) is expressed by vascular endothelial cells and upregulated by lipopolysaccharide (LPS) in vitro. PAR-2 is activated by a tethered ligand created after proteolytic cleavage by trypsin or experimentally by a synthetic agonist peptide (PAR-2AP) corresponding to the new amino terminus of the tethered ligand. METHODS AND RESULTS Intravenous administration of PAR-2AP (0.1, 0.3, and 1 mg/kg) to rats caused a dose-dependent hypotension. A scrambled peptide was without effect. A specific trypsin inhibitor, biotin-SGKR-chloromethylketone, inhibited trypsin-induced hypotension but not that stimulated by PAR-2AP. In animals treated with LPS 20 hours earlier, we found an increased sensitivity to trypsin and PAR-2AP in the hypotensive response. In particular, PAR-2AP caused hypotension at a low concentration of 30 ng/kg. Moreover, PAR-2 was immunolocalized to endothelial and smooth muscle cells in aorta and jugular vein in LPS-treated rats, and increased levels of PAR-2 mRNA were shown by reverse transcription-polymerase chain reaction analysis. CONCLUSIONS Our findings suggest that PAR-2 is important in the regulation of blood pressure in vivo. A functional upregulation of PAR-2 by LPS was demonstrated by the activity of concentrations of PAR-2AP that were inactive in normal animals. We conclude that PAR-2 may play an important role in the hypotension associated with endotoxic shock and may represent a new therapeutic target.

Involvement of β3-adrenergic receptor activation via cyclic GMP- but not NO-dependent mechanisms in human corpus cavernosum function

The β3-adrenoreceptor plays a major role in lipolysis but the role and distribution of β3-receptors in other specific sites have not been extensively studied. β3-adrenergic receptors are present not only in adipose tissue but also in human gall bladder, colon, prostate, and skeletal muscle. Recently, β3-adrenergic receptor stimulation was shown to elicit vasorelaxation of rat aorta through the NO–cGMP signal transduction pathway. Here we show that β3-receptors are present in human corpus cavernosum and are localized mainly in smooth muscle cells. After activation by a selective β3-adrenergic receptor agonist, BRL 37344, there was a cGMP-dependent but NO-independent vasorelaxation that was selectively blocked by a specific β3-receptor antagonist. In addition, we report that the human corpus cavernosum exhibits basal β3-receptor-mediated vasorelaxant tone and that β3-receptor activity is linked to inhibition of the RhoA/Rho-kinase pathway. These observations indicate that β3-receptors may play a physiological role in mediating penile erection and, therefore, could represent a therapeutic target for treatment of erectile dysfunction.

Systemic Administration of Sphingosine-1-Phosphate Increases Bronchial Hyperresponsiveness in the Mouse

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that plays important roles in allergic responses, including asthma. S1P acts on many cell types, such as mast cells, the airway epithelium, airway smooth muscle, and many immune cells. In this study we have evaluated whether a systemic administration of S1P to Balb/c mice modifies airway reactivity. Our data show that S1P (0.1-10 ng) given subcutaneously to Balb/c mice causes a specific and dose-dependent increase in cholinergic reactivity of bronchial tissues in vitro. This effect is (1) dose dependent, with a maximal effect of the dose of 10 ng of S1P; and (2) time dependent, reaching a maximal effect 21 days after S1P administration. Similarly, in the whole lung assay there is a dose- and time-dependent increase in lung resistance. Lungs isolated from S1P-treated mice displayed an increase in mast cell number. Furthermore, there is an increase of IL-4, IL-13, and IL-17 production. In conclusion, our data demonstrate that S1P signaling is involved in the complex pathway underlying airway hyperresponsiveness.