Fiorentina Roviezzo

In vivo delivery of the caveolin-1 scaffolding domain inhibits nitric oxide synthesis and reduces inflammation.

Caveolin-1, the primary coat protein of caveolae, has been implicated as a regulator of signal transduction through binding of its “scaffolding domain” to key signaling molecules. However, the physiological importance of caveolin-1 in regulating signaling has been difficult to distinguish from its traditional functions in caveolae assembly, transcytosis, and cholesterol transport. To directly address the importance of the caveolin scaffolding domain in vivo, we generated a chimeric peptide with a cellular internalization sequence fused to the caveolin-1 scaffolding domain (amino acids 82–101). The chimeric peptide was efficiently taken up into blood vessels and endothelial cells, resulting in selective inhibition of acetylcholine (Ach)-induced vasodilation and nitric oxide (NO) production, respectively. More importantly, systemic administration of the peptide to mice suppressed acute inflammation and vascular leak to the same extent as a glucocorticoid or an endothelial nitric oxide synthase (eNOS) inhibitor. These data imply that the caveolin-1 scaffolding domain can selectively regulate signal transduction to eNOS in endothelial cells and that small-molecule mimicry of this domain may provide a new therapeutic approach.

Aberrant inflammation and resistance to glucocorticoids in Annexin 1-/- Mouse

The 37‐kDa protein annexin 1 (Anx‐1; lipocortin 1) has been implicated in the regulation of phagocytosis, cell signaling, and proliferation and is postulated to be a mediator of glucocorticoid action in inflammation and in the control of anterior pituitary hormone release. Here, we report that mice lacking the Anx‐1 gene exhibit a complex phenotype that includes an altered expression of other annexins as well as of COX‐2 and cPLA2. In carrageenin‐ or zymosan‐induced inflammation, Anx‐1−/− mice exhibit an exaggerated response to the stimuli characterized by an increase in leukocyte emigration and IL‐1β generation and a partial or complete resistance to the antiinflammatory effects of glucocorticoids. Anx‐1−/− polymorphonuclear leucocytes exhibited increased spontaneous migratory behavior in vivo whereas in vitro, leukocytes from Anx‐1−/− mice had reduced cell surface CD 11b (MAC‐1) but enhanced CD62L (L‐selectin) expression and Anx‐1−/− macrophages exhibited anomalies in phagocytosis. There are also gender differences in activated leukocyte behavior in the Anx‐1−/−mice that are not seen in the wild‐type animals, suggesting an interaction between sex hormones and inflammation in Anx‐1−/− animals.

Hydrogen Sulfide Is an Endogenous Inhibitor of Phosphodiesterase Activity

Objective—Recent studies have demonstrated that hydrogen sulfide (H2S) is produced within the vessel wall from l-cysteine regulating several aspects of vascular homeostasis. H2S generated from cystathione &ggr;-lyase (CSE) contributes to vascular tone; however, the molecular mechanisms underlying the vasorelaxing effects of H2S are still under investigation. Methods and Results—Using isolated aortic rings, we observed that addition of l-cysteine led to a concentration-dependent relaxation that was prevented by the CSE inhibitors dl-propargylglyicine (PAG) and &bgr;-cyano-l-alanine (BCA). Moreover, incubation with PAG or BCA resulted in a rightward shift in sodium nitroprusside-and isoproterenol-induced relaxation. Aortic tissues exposed to PAG or BCA contained lower levels of cGMP, exposure of cells to exogenous H2S or overexpression of CSE raised cGMP concentration. RNA silencing of CSE expression reduced intracellular cGMP levels confirming a positive role for endogenous H2S on cGMP accumulation. The ability of H2S to enhance cGMP levels was greatly reduced by the nonselective phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. Finally, addition of H2S to a cell-free system inhibited both cGMP and cAMP breakdown. Conclusion—These findings provide direct evidence that H2S acts as an endogenous inhibitor of phosphodiesterase activity and reinforce the notion that this gasotransmitter could be therapeutically exploited.

Biosynthesis of H2S is impaired in non-obese diabetic (NOD) mice

Hydrogen sulphide (H2S) has been involved in cardiovascular homoeostasis but data about its role in animal models of diabetic pathology are still lacking. Here, we have analysed H2S signalling in a genetic model of diabetes, the non‐obese diabetic (NOD) mice.

Geldanamycin, an inhibitor of heat shock protein 90 (Hsp90) mediated signal transduction has anti‐inflammatory effects and interacts with glucocorticoid receptor in vivo

Histamine, vascular endothelial growth factor, acetylcholine, oestrogen as well as fluid shear stress activates a mechanism that recruits heat shock protein 90 to the endothelial nitric oxide synthase. The interaction between Hsp90 and eNOS enhances the activation of the enzyme in cells and in intact blood vessels leading to NO production. Intraplantar administration of carrageenan (50 μl paw−1) to mice causes an oedema lasting 72 h. Geldanamycin (0.1, 0.3, 1 mg kg−1), a specific inhibitor of Hsp‐90, that inhibits endothelium‐dependent relaxations of the rat aorta, mesentery and middle artery inhibits carrageenan‐induced mouse paw oedema in a dose dependent manner. Co‐administration to mice of dexamethasone (1 mg kg−1) with geldanamycin (0.3 mg kg−1) at anti‐inflammatory dose causes a loss of the total anti‐inflammatory effect of each agent alone. RU 486 (10 mg kg−1), a well known glucocorticoid receptorial antagonist, does not inhibit oedema formation but prevents the anti‐inflammatory action of dexamethasone (1 mg kg−1). Similarly, RU 486 prevents the anti‐inflammatory action of geldanamycin (0.3 mg kg−1). In conclusion we have described for the first time that geldanamycin, an inhibitor of Hsp90 dependent signal transduction, is anti‐inflammatory in vivo implying that Hsp90 is critical for pathways involved in carrageenan‐induced paw oedema. In addition the ability of GA to block NO release and reduce oedema formation suggests a therapeutic rationale for specific inhibitors of Hsp90 as potential anti‐inflammatory drugs.

Glucocorticoid Receptor Nitration Leads to Enhanced Anti-Inflammatory Effects of Novel Steroid Ligands

It has recently emerged that posttranslational modification of proteins via nitration of tyrosine residues can alter their function. In this study, we describe that specific nitration of the glucocorticoid receptor (GR) by NCX-1015, a novel NO-donating prednisolone derivative (prednisolone 21-[4′-(nitrooxymethyl)benzoate), results in an enhancement of GR-mediated events. Incubation of PBMC and U937 cells with 1–10 μM NCX-1015 caused faster activation of GR as assessed by augmented 1) binding to [3H]dexamethasone, 2) dissociation from heat shock protein 90, and 3) nuclear translocation. PBMCs treated with NCX-1015 contained GR that had undergone tyrosine nitration. The chemistry facilitating the increase in steroid binding capacity observed with NCX-1015 is specific, because changing the position of the NO-donating group or ubiquitous nitration by addition of an NO donor was unable to mimic this event. In vivo treatment with NCX-1015 provoked GR nitration and faster heat shock protein 90 dissociation as assessed in peritoneal cells. Accordingly, NCX-1015, but not prednisolone or other derivatives, produced a rapid inhibition of the early neutrophil recruitment and mediator generation in a model of peritonitis. In conclusion, we report here for the first time that posttranslational modification of GR by this novel nitrosteroid is associated with its enhanced anti-inflammatory activity.

Diabetic Mouse Angiopathy Is Linked to Progressive Sympathetic Receptor Deletion Coupled to an Enhanced Caveolin-1 Expression

Objective—Clinical studies have demonstrated that hyperglycaemia represents a major risk factor in the development of the endothelial impairment in diabetes, which is the first step in vascular dysfunction. Using non-obese diabetic mice, we have evaluated the role of the adrenergic system and eNOS on progression of the disease Methods and Results—When glycosuria is high (20 to 500 mg/dL), there is a selective reduction in the response to &agr;1 and &bgr;2 agonists but not to dopamine or serotonin. When glycosuria is severe (500 to 1000 mg/dL), there is a complete ablation of the contracture response to the &agr;1 receptor agonist stimulation and a marked reduced response to &bgr;2 agonist stimulation. This effect is coupled with a reduced expression of &agr;1 and &bgr;2 receptors, which is caused by an inhibition at transcriptional level as demonstrated by RT-PCR. In the severe glycosuria (500 to 1000 mg/dL), although eNOS expression is unchanged, caveolin-1 expression is significantly enhanced, indicating that high glucose plasma levels cause an upregulation of the eNOS endogenous inhibitory tone. These latter results correlate with functional data showing that in severe glycosuria, there is a significant reduction in acetylcholine-induced vasodilatation. Conclusions—Our results show that in diabetes development, there is a progressive selective downregulation of the &agr;1 and &bgr;2 receptors. At the same time, there is an increased expression of caveolin-1, the endogenous eNOS inhibitory protein. Thus, caveolin-1 could represent a new possible therapeutic target in vascular impairment associated with diabetes.

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.

17-β-oestradiol-induced vasorelaxation in vitro is mediated by eNOS through hsp90 and akt/pkb dependent mechanism

The L‐arginine‐NO pathway has been implicated in the vasorelaxant effect of 17‐β‐oestradiol. Here we have addressed the involvement of two distinct activation steps of endothelial nitric oxide synthase (eNOS) in the 17‐β‐oestradiol‐induced vasorelaxant effect on rat aortic rings. Rat aortic rings contracted with phenylephrine (PE) 1 μM relaxed in a concentration related fashion to 17‐β‐oestradiol water soluble cyclodextrin‐encapsulated (E2) only when endothelium was present. The pure anti‐oestrogen of E2 receptor ICI 182,780 (20 μM) significantly inhibited E2‐induced vasorelaxation. Geldanamycin (10 μM), a specific inhibitor of heat shock protein 90 (hsp90) and Nω‐nitro‐L‐arginine‐methyl ester (L‐NAME, 100 μM), a nitric oxide synthase inhibitor, significantly inhibited E2‐induced vasorelaxation. Incubation of rat aortic rings up to 6 h with LY 294002 (25 μM), a specific inhibitor of PI(3)K akt/pkb pathway reduced E2‐induced vasorelaxation. Incubation of rat isolated aorta with E2, induced prostacyclin (PGI2) release. PGI2 levels, measured as 6‐keto PGF1α, were abolished by ibuprofen (10 μM), both L‐NAME and GA did not influence basal or E2‐stimulated PGI2 confirming the specificity of these two compounds on eNOS pathway. In conclusion, we demonstrate that E2 interaction with its receptor is followed by a vasorelaxant effect in rat aortic rings mediated by eNOS activation through both hsp90 and akt/pkb dependent mechanisms.

Hydrogen sulphide is involved in testosterone vascular effect.

BACKGROUND Testosterone (T) induces a rapid relaxation in vascular tissues of different species due to a nongenomic effect of this steroid on vessels. Different mechanisms have been proposed to explain T-induced vasodilatation but the effective mechanism(s) and the mediators involved are still a matter of debate. OBJECTIVES We have evaluated if H(2)S pathway is involved in T vascular effects. DESIGN AND SETTING Male Wistar rats were sacrificed and thoracic aorta was rapidly dissected and cleaned from fat and connective tissue. Rings of 2-3 mm length were cut and placed in organ baths filled with oxygenated Krebs solution at 37 degrees C and mounted to isometric force transducers. H(2)S determination was performed on thoracic aortic rings incubated with T or vehicle and in presence of inhibitors. H2S concentration was calculated against a calibration curve of NaHS (3-250 microM). Results were expressed as nmoles/mg protein. MEASUREMENTS Vascular reactivity was evaluated by using isometric transducers. H(2)S determination was performed by using a cystathionine beta-synthetase (CBS) and cystathionine gamma lyase (CSE) activity assay. CSE and CBS protein levels were assessed by Western blot analysis. Statistical analysis was performed by using two-way ANOVA and unpaired Students t-test where appropriate. RESULTS T significantly increased conversion of L-cysteine to H(2)S. This effect was significantly reduced by PGG and BCA, two specific inhibitors of CSE. T (10 nM-10 microM) induced a concentration-dependent vasodilatation of rat aortic rings in vitro that was significantly and concentration-dependent inhibited by PGG, BCA, and glybenclamide. Incubation of aorta with T up to 1 h did not change CBS/CSE expression, suggesting that T modulates enzymatic activity. CONCLUSIONS Here we demonstrate that T vasodilator effect involves H(2)S, a novel gaseous mediator. T modulates H(2)S levels by increasing the enzymatic conversion of L-cysteine to H(2)S.