Mariarosaria Bucci

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.

Dual inhibitors of cyclooxygenase and 5-lipoxygenase. A new avenue in anti-inflammatory therapy?

Nonsteroidal anti-inflammatory drugs (NSAIDs) are a mainstay in the treatment of inflammatory disease and are among the most widely used drugs worldwide. They are anti-inflammatory, antipyretic, and analgesic and are prescribed as first choice for the treatment of rheumatic disorders and, in general, inflammation. The main limitation in using NSAIDs consists in their side-effects, including gastrointestinal ulcerogenic activity and bronchospasm. The mechanism of action of these drugs is attributed to the inhibition of cyclooxygenase (COX), and, consequently, the conversion of arachidonic acid into prostaglandins. It is hypothesized that the undesirable side-effects of NSAIDs are due to the inhibition of COX-1 (constitutive isoform), whereas the beneficial effects are related to the inhibition of COX-2 (inducible isoform). Arachidonic acid can also be converted to leukotrienes (LTs) by the action of 5-lipoxygenase (5-LOX). LTC(4,) LTD(4,) and LTE(4) are potent bronchoconstrictors, whereas LTB(4) is chemotactic for leukocytes and plays an important role in the development of gastrointestinal ulcers by contributing to the inflammatory process. Thus, developing dual inhibitor compounds that will simultaneously inhibit COX and 5-LOX could enhance their individual anti-inflammatory effects and reduce the undesirable side-effects associated with NSAIDs, especially of the gastrointestinal tract. The most promising COX/5-LOX inhibitor is ML3000 ([2,2-dimethyl-6-(4-chlorophenyl)-7-phenyl-2,3-dihydro-1H-pyrrolizine-5-yl]-acetic acid), now in Phase III clinical trials. This new approach will certainly help to unravel the mechanisms at the root of the undesirable effects of NSAIDs and to develop safer NSAIDs.

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.

Factor Xa as an interface between coagulation and inflammation. Molecular mimicry of factor Xa association with effector cell protease receptor-1 induces acute inflammation in vivo.

Coagulation proteases were tested in a rat model of acute inflammation. Subplantar injection of Factor Xa (10-30 microg) produced a time- and dose-dependent edema in the rat paw, and potentiated carrageenin-induced edema. In contrast, the homologous protease Factor IXa was ineffective. This inflammatory response was recapitulated by the Factor Xa sequence L83FTRKL88(G), which mediates ligand binding to effector cell protease receptor-1 (EPR-1), while a control scrambled peptide did not induce edema in vivo. Conversely, injection of the EPR-1-derived peptide S123PGKPGNQNSKNEPP137 (corresponding to the receptor binding site for Factor Xa) inhibited carrageenin-induced rat paw edema, while the adjacent EPR-1 sequence P136PKKRERERSSHCYP150 was without effect. EPR-1-Factor Xa-induced inflammation was characterized by fast onset and prominent perivascular accumulation of activated and degranulated mast cells, was inhibited by the histamine/serotonin antagonists cyproheptadine and methysergide, but was unaffected by the thrombin-specific inhibitor, Hirulog. These findings suggest that through its interaction with EPR-1, Factor Xa may function as a mediator of acute inflammation in vivo. This pathway may amplify both coagulation and inflammatory cascades, thus contributing to the pathogenesis of tissue injury in vivo.

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.

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.

Inflammation-coagulation network: are serine protease receptors the knot?

Following an injury, the body recruits a mechanism to delimit and repair tissue damage; this phenomenon is known as inflammation. Among the several different pathways that are activated during this process, which is necessary for survival, activation of the coagulation pathway is a key feature. In fact, clinical changes in blood fluidity have been closely related to ongoing inflammation. Recent evidence suggests that serine protease receptors might play a major role in the host defence mechanism at the interface between coagulation and inflammation.

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.

cGMP-dependent protein kinase contributes to hydrogen sulfide-stimulated vasorelaxation.

A growing body of evidence suggests that hydrogen sulfide (H2S) is a signaling molecule in mammalian cells. In the cardiovascular system, H2S enhances vasodilation and angiogenesis. H2S-induced vasodilation is hypothesized to occur through ATP-sensitive potassium channels (KATP); however, we recently demonstrated that it also increases cGMP levels in tissues. Herein, we studied the involvement of cGMP-dependent protein kinase-I in H2S-induced vasorelaxation. The effect of H2S on vessel tone was studied in phenylephrine-contracted aortic rings with or without endothelium. cGMP levels were determined in cultured cells or isolated vessel by enzyme immunoassay. Pretreatment of aortic rings with sildenafil attenuated NaHS-induced relaxation, confirming previous findings that H2S is a phosphodiesterase inhibitor. In addition, vascular tissue levels of cGMP in cystathionine gamma lyase knockouts were lower than those in wild-type control mice. Treatment of aortic rings with NaHS, a fast releasing H2S donor, enhanced phosphorylation of vasodilator-stimulated phosphoprotein in a time-dependent manner, suggesting that cGMP-dependent protein kinase (PKG) is activated after exposure to H2S. Incubation of aortic rings with a PKG-I inhibitor (DT-2) attenuated NaHS-stimulated relaxation. Interestingly, vasodilatory responses to a slowly releasing H2S donor (GYY 4137) were unaffected by DT-2, suggesting that this donor dilates mouse aorta through PKG-independent pathways. Dilatory responses to NaHS and L-cysteine (a substrate for H2S production) were reduced in vessels of PKG-I knockout mice (PKG-I−/−). Moreover, glibenclamide inhibited NaHS-induced vasorelaxation in vessels from wild-type animals, but not PKG-I−/−, suggesting that there is a cross-talk between KATP and PKG. Our results confirm the role of cGMP in the vascular responses to NaHS and demonstrate that genetic deletion of PKG-I attenuates NaHS and L-cysteine-stimulated vasodilation.

A new modified thrombin binding aptamer containing a 5′–5′ inversion of polarity site

The solution structure of a new modified thrombin binding aptamer (TBA) containing a 5′–5′ inversion of polarity site, namely d(3′GGT5′-5′TGGTGTGGTTGG3′), is reported. NMR and CD spectroscopy, as well as molecular dynamic and mechanic calculations, have been used to characterize the 3D structure. The modified oligonucleotide is characterized by a chair-like structure consisting of two G-tetrads connected by three edge-wise TT, TGT and TT loops. d(3′GGT5′-5′TGGTGTGGTTGG3′) is characterized by an unusual folding, being three strands parallel to each other and only one strand oriented in opposite manner. This led to an anti-anti-anti-syn and syn-syn-syn-anti arrangement of the Gs in the two tetrads. The thermal stability of the modified oligonucleotide is 4°C higher than the corresponding unmodified TBA. d(3′GGT5′-5′TGGTGTGGTTGG3′) continues to display an anticoagulant activity, even if decreased with respect to the TBA.