Abstract: Carrageenan-induced rat paw oedema is a widely used model to investigate the physiopathology of an acute local inflammation. Recently, much attention has been focused on the link between haemostasis and inflammation, and on the impact that inflammation might have on thrombotic events. It is known that the systemic response to inflammation is the "acute phase reaction" that represents a highly complex reaction of the organism to a variety of injuries, aimed to restore homeostasis; one important feature of the acute phase reaction is the hepatic synthesis of proteins involved in the coagulation cascade. Much attention has been focused on the role that systemic inflammation might have on thrombotic events, while there is not much information on the role played by an acute local inflammation on haemostasis, that can lead toward a pro-thrombotic state. The present study was conducted to evaluate the haemostatic balance in the early and the late phase of carrageenan-induced rat paw oedema; i.e. at 3 h, when paw inflammation is maximally expressed, and 24 h following carrageenan injection, when there is an almost complete absence of local inflammatory symptoms. We found that in inflamed animals, 24 h following oedema induction, there was an increase in plasma fibrinogen levels, antithrombin III activity and serum interleukin-6 levels, concomitant to a shortened prothrombin time and to an increased platelet responsiveness to ADP. Furthermore, in inflamed tissues at 3 h there was an increase in antithrombin III proteic expression. Our results demonstrate that a haemostatic imbalance occurs following carrageenan-induced rat paw oedema.
Abstract: The phytochemical investigation of the acetone and methanol extracts of the flowers of Magydaris tomentosa (Desf.) DC afforded six known coumarins as well as (+)-meranzin hydrate (7), not previously reported as a natural product. The antibacterial activity of umbelliprenin (1), osthol (2), imperatorin (3), citropten (4) and (+)-meranzin hydrate (7) was tested against Gram-positive and Gram-negative bacteria. All coumarins (1-7) isolated in this study inhibited growth of all bacterial strains tested (MIC between 16 and 256 microg/mL), the most active being imperatorin (3) (MICs between 32 and 128 microg/mL) and citropten (4) (MICs between 16 and 256 microg/mL). The anticoagulant activity of compounds 1-4 and 7 was also evaluated.
Abstract: The effect of a diterpenoid isolated from Salvia cinnabarina, 3,4-seicosopimar-4(18),7,15-triene-3-oic acid (SCB), on arterial blood pressure was evaluated in anaesthetized rats. Male Wistar rats, anaesthetized with urethane (sol. 10% p/v; 10 mL/kg), underwent surgery for continuous monitoring of arterial blood pressure. After preliminary experiments to evaluate the dose response (3, 10 and 30 mg/kg i.v.) of SCB, a dose of 3 mg/kg was chosen for all successive experiments. On different groups of rats treated with the ganglion-blocking agent chlorisondamine (2.5 mg/kg i.p.) the effect of SCB (3 mg/kg i.v.) was evaluated before and following an infusion of the nitric oxide synthase inhibitor L-NAME (0.3 mg/kg/min i.v.). Intravenous administration of SCB at doses of 3, 10 and 30 mg/kg led to a fall in mean arterial blood pressure (MABP) of 14.75 +/- 1.44 mmHg, 36.60 +/- 31.40 mmHg and 31.40 +/- 6.28 mmHg, respectively (n = 4-5), that was not modified by treatment of the rat with chlorisondamine nor with L-NAME. The results demonstrate a hypotensive effect of SCB - due to a peripheral mechanism but independent of endothelial nitric oxide release.
Abstract: Here we investigated the effect of the flavonoid galangin in isolated rat thoracic aortic rings. Galangin (0.1-100 microM) induced relaxation in rings pre-contracted with phenylephrine (PE 1 microM) or with KCl (100 mM) or pre-treated with the nitric oxide synthase inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME, 100 microM), the cyclooxygenase inhibitor indomethacin (10 microM) and the adenylate cyclase inhibitor, SQ 22,536 (100 microM). In another set of experiments, rat aortic rings were incubated with galangin (1-100 microM) and the contractile responses to PE (0.001-3 microM) or to KCl (60 mM) were evaluated. We also evaluated the effect of galangin (100 microM) on PE (10 microM)-induced contraction in a Ca2+-free medium. Galangin relaxed aortic rings with or without endothelium. Galangin effect was significantly inhibited by L-NAME. Galangin inhibited the contractile response to PE, either in presence or in absence of external calcium, and to KCl. In the end, we also found that galangin caused nitric oxide (NO) release from aortic rings and abolished the increase in [Ca2+]i triggered by PE or KCl in aortic smooth muscle cells, either in presence and in absence of external Ca2+. Our results suggest that galangin reduces the contractility of rat aortic rings through an endothelium-dependent mechanism, involving NO, and also through an endothelium-independent mechanism, inhibiting calcium movements through cell membranes.
Abstract: Adenosine is a potent endogenous regulator of airway inflammation that acts through specific receptor subtypes that can either cause constriction (A1R, A2BR, and A3R) or relaxation (A2AR) of the airways. We therefore examined the effects of key inflammatory mediators on the expression of the A2AR in a lung epithelial cell line (A549). IL-1beta and TNF-alpha increased the expression of the A2AR gene at the mRNA and protein levels. In contrast, LPS had no effect on A2AR gene expression. IL-1beta and TNF-alpha rapidly activated p50 and p65, but not C-Rel, RelB, or p52, and both IL-1beta- and TNF-alpha-stimulated A2AR expression was inhibited by the IkappaB kinase 2 inhibitor AS602868 in a concentration-dependent manner. Using chromatin immunoprecipitation assays, we demonstrate that IL-1beta can enhance p65 association with putative kappaB binding sites in the A2AR promoter in a temporal manner. In contrast, TNF-alpha failed to enhance p65 binding to these putative sites. Functionally, the two most 5' kappaB sites were important for IL-1beta-, but not TNF-alpha-, induced A2AR promoter reporter gene activity. Finally, neither TNF-alpha nor Il-1beta had any effect on A2AR mRNA transcript degradation. These results directly implicate a major role for NF-kappaB in the regulation of A2AR gene transcription by IL-1beta and TNF-alpha but suggest that the effects of TNF-alpha on A2AR gene transcription are not mediated through the proximal promoter.
Abstract: Proteinase activated receptor-2 (PAR2), a seven transmembrane domain G protein coupled receptor, is expressed on airway epithelium and smooth muscle cells and over-expressed in human airways under pathological conditions, such as asthma and chronic obstructive pulmonary disease (COPD). However, the role of PAR2 in airways has not yet been defined. Aim of the present study, was to evaluate the in vitro rat bronchial response to a synthetic peptide activating PAR2 (PAR2-AP; SLIGRL), following an in vivo treatment with bacterial lipopolysaccharide (LPS). Bronchi from LPS-treated animals showed an increased relaxant response to PAR2-AP, compared to naïve animals, the effect was maximum after 20-h pre-treatment and reduced by epithelium removal. Western blot analysis showed an increased PAR2 protein expression on bronchi removed 20h after LPS treatment. PAR2-AP-induced bronchorelaxation was inhibited by ibuprofen, by the selective cyclooxygenase2 (COX-2) inhibitor, diisopropyl fluorophosphate (DFP) and partially by the calcitonin gene related peptide (CGRP) antagonist, rat-CGRP([8-37]). Furthermore, there was a strong immunoreactivity for COX-2 on bronchial epithelium of LPS-treated rats. Prostaglandin E2 (PGE2) tissue release and CGRP tissue content were significantly increased following tissue incubation with PAR2-AP. The in vivo LPS treatment in rats strongly increases the bronchorelaxant effect of PAR2-AP, this effect correlates with an increased tissue protein receptor expression and the COX-2 localization on bronchial epithelium. Our work supports a role for PAR2 as a defence mechanism aimed to preserve bronchial functionality under systemic inflammatory conditions; both COX-2-derived PGE2 and CGRP are involved in this effect.
Abstract: 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 alpha1 and beta2 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 alpha1 receptor agonist stimulation and a marked reduced response to beta2 agonist stimulation. This effect is coupled with a reduced expression of alpha1 and beta2 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 alpha1 and beta2 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.
Abstract: OBJECTIVES: Protease-activated receptor 2 (PAR2) is a G-protein-coupled receptor proteolytically activated by trypsin, tryptase or factor Xa. Alternatively, PAR2 can be activated by synthetic peptides whose sequence mimics the tethered ligand exposed after receptor cleavage. It is known that PAR2 modulates vascular reactivity, both in vitro and in vivo. The present study was designed to investigate the role of basal nitric oxide and cyclic nucleotides, adenosine 3'5'cyclic monophosphate (cAMP) and guanosine 3'5' cyclic monophosphate (cGMP), in the vasorelaxation induced by a PAR2-activating peptide (PAR2-AP; SLIGRL-NH(2)) on rat aorta in vitro. METHODS: A concentration-response curve to PAR2-AP was performed on rat thoracic aorta with or without a functional endothelium, and the effect of inhibitors was evaluated. The effect of PAR2-AP (10(-7)-3 x 10(-5) M) on endothelium-denuded aorta was also evaluated after tissue incubation with sodium nitroprusside. RESULTS: PAR2-AP (10(-7)-3 x 10(-5) M) caused an endothelium-dependent relaxation abolished by N(omega)-nitro-L-arginine methyl ester, L-NAME, and by the guanylyl cyclase inhibitor, 1H-[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one (ODQ), but unaffected by geldanamycin. Vasorelaxant effect of PAR2-AP was only partially inhibited by the adenylyl cyclase inhibitor, 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ 22,536), and it was increased by tissue incubation with the phosphodiesterase inhibitor, rolipram. On tissue without endothelium, PAR2-AP did not cause vasorelaxation, up to a concentration of 10(-4) M. However, after tissue incubation with sodium nitroprusside (SNP, 3 x 10(-9) M), the vasorelaxant effect of PAR2-AP was restored. Following tissue incubation with PAR2-AP, cAMP levels were significantly increased compared to control values. CONCLUSIONS: Our results suggest that vasorelaxation induced by PAR2-AP is modulated by basal nitric oxide with an involvement of both cyclic nucleotides, cGMP and cAMP.
Abstract: This study was aimed to investigate the vascular activity of caffeic acid phenethylester (CAPE), one of the major components of honeybee propolis. Experiments were performed on rat thoracic aortic rings, mounted in an isolated organ bath and connected to an isometric force transducer. The effect of CAPE (0.1-300 microM) was evaluated on tissue pre-contracted with phenylephrine (PE, 1 microM) or with KCl (100 mM). In another set of experiments, tissue was incubated with CAPE (1-100 microM) and responses to PE (0.01-3 microM) or KCl (60 mM) were evaluated. The effect of CAPE on cytosolic Ca(2+) concentration in aortic smooth muscle cells stimulated with PE or KCl was also evaluated. CAPE (0.1-300 microM) caused a concentration-dependent relaxation (pEC(50) 4.99 +/- 0.19; Emax 100.75 +/- 1.65%; n = 4) of tissue pre-contracted with PE that was reduced by endothelium removal or by incubation with N(omega)-nitro-L-arginine methyl ester (L-NAME, 100 microM). CAPE also relaxed KCl-precontracted tissue (pEC(50) 4.40 +/- 0.08; n = 4). CAPE inhibited contractile responses to PE or to KCl, and also inhibited the contractile response to PE obtained in a Ca(2+)-free medium. In addition, CAPE inhibited the increase in cytosolic Ca(2+) concentration triggered by stimulation of aortic smooth muscle cells with PE or KCl. Our results demonstrate a vascular activity for CAPE, that is only partially dependent on nitric oxide. Indeed, at high concentrations, CAPE vasorelaxant effect occurs also in absence of endothelium and it is likely due to an inhibitory effect on calcium movements through cell membranes.
Abstract: 1. The L-arginine-NO pathway has been implicated in the vasorelaxant effect of 17-beta-oestradiol. Here we have addressed the involvement of two distinct activation steps of endothelial nitric oxide synthase (eNOS) in the 17-beta-oestradiol-induced vasorelaxant effect on rat aortic rings. 2. Rat aortic rings contracted with phenylephrine (PE) 1 microM relaxed in a concentration related fashion to 17-beta-oestradiol water soluble cyclodextrin-encapsulated (E2) only when endothelium was present. The pure anti-oestrogen of E2 receptor ICI 182,780 (20 microM) significantly inhibited E2-induced vasorelaxation. 3. Geldanamycin (10 microM), a specific inhibitor of heat shock protein 90 (hsp90) and N(omega)-nitro-L-arginine-methyl ester (L-NAME, 100 microM), a nitric oxide synthase inhibitor, significantly inhibited E2-induced vasorelaxation. 4. Incubation of rat aortic rings up to 6 h with LY 294002 (25 microM), a specific inhibitor of PI(3)K akt/pkb pathway reduced E2-induced vasorelaxation. 5. Incubation of rat isolated aorta with E2, induced prostacyclin (PGI(2)) release. PGI(2) levels, measured as 6-keto PGF(1alpha), were abolished by ibuprofen (10 microM), both L-NAME and GA did not influence basal or E2-stimulated PGI(2) confirming the specificity of these two compounds on eNOS pathway. 6. 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.
Abstract: Protease-activated receptor 2 (PAR-2) is involved in inflammatory, gastrointestinal, and vascular diseases. The aim of the present work was to elucidate the minimal structural features for PAR-2 agonist activity in short peptides. Our study resulted in the discovery of dipeptide derivatives of N(alpha)-benzoyl-Arg(NO(2))-Leu-NH(2) displaying a potency comparable to that of the full-length rat PAR-2 activating peptide (Ser-Leu-Ile-Gly-Arg-Leu-NH(2)).
Abstract: Protease-activated receptor-2 (PAR-2) is a member of seven transmembrane domain G protein-coupled receptors activated by proteolytic cleavage. PAR-2 is involved in inflammatory events and cardiac ischemic reperfusion injury. The objective of this study was to investigate the effects of PAR-2 in experimental myocardial ischemic preconditioning. To monitor the effects of PAR-2, Langendorff-perfused rat hearts were used. These hearts were treated with PAR-2-activating peptide (PAR-2AP) in various protocols. Hemodynamic parameters (left ventricular developed pressure, left ventricular diastolic pressure, coronary flow rate, and heart rate), several indexes of oxidative injury, and neutrophil accumulation were evaluated. We show for the first time that enhanced PAR-2 activation improves efficiency of ischemic preconditioning and reduces cardiac inflammation in the rat heart. Indeed, after PAR-2AP infusion we found that hemodynamic parameters, oxidative injury, infarct size, and neutrophil accumulation were involved. These data support the concept that PAR-2-dependent cell trafficking may regulate signaling responses to cardiac ischemia and inflammation.
Abstract: 1. Protease activated receptor-2 (PAR2) is a seven transmembrane domain G protein coupled receptor proteolytically activated. PAR2, together with other PARs, can be also activated by peptides mimicking the sequence of the receptor tethered ligand. We have evaluated the effect of systemic administration of a peptide activating PAR2 (PAR2-AP, SLIGRL) on histamine-induced increase in lung resistances in the guinea-pig. 2. Intravenous administration of PAR2-AP (1 mg kg(-1)) significantly inhibited histamine-induced increase in lung resistance in a time-dependent fashion that was not abolished by indomethacin or vagotomy. 3. Bronchoprotective effect of PAR2-AP was not reversed by the cyclo-oxygenase inhibitor, indomethacin, the nitric oxide synthetase inhibitor, L-NAME, nor by the non-selective beta-antagonist, propranolol. 4. Indomethacin augmented the bronchoconstriction to histamine which was inhibited by PAR2-AP. Furthermore, in vagotomized animals, the bronchial hyper-responsiveness to histamine was significantly reduced, and in these circumstances, PAR2-AP still retained the capacity to provide bronchoprotection against histamine. 5. PAR2-AP also produced a modest reduction in histamine-induced protein leakage in trachea and upper bronchi. 6. Our results indicated that PAR2 might have a bronchoprotective role in the guinea-pig in vivo independent of prostaglandin or nitric oxide release.
Abstract: A phytochemical investigation of the extracts obtained from bulbs of leek. Allium porrum L. has led to the isolation of five flavonoid glycosides based on the kaempferol aglycone. Two of them are new compounds and have been identified as kaempferol 3-O-[2-O-(trans-3-methoxy-4-hydroxycinnamoyl)-beta-D-galactopyranosyl]-(1-->4)-O-beta-D-glucopyranoside, and kaempferol 3-O-[2-O-(trans-3-methoxy-4-hydroxycinnamoyl)-beta-D-glucopyranosyl]-(1-->6)-O-beta-D-glucopyranoside, on the basis of spectroscopic methods, including 2D NMR. The isolated compounds have been evaluated for their human platelet anti-aggregation activity.
Abstract: BACKGROUND AND AIM: Platelet aggregation is involved in atherosclerosis and pharmacological inhibition of platelet activity may reduce the risk of coronary thrombosis and myocardial infarction. Red wine polyphenols may reduce platelet aggregability. This study evaluates the effect of de-alcoholated red wine (DRW) and its phenolic fractions on rat platelet aggregation and cyclic AMP (c-AMP) content. METHODS AND RESULTS: DRW was fractionated into four classes of phenolic compounds: phenolic acids (fraction 1), procyanidins, catechins and monomeric anthocyanidins (fraction 2), flavonols and resveratrol (fraction 3) and polymeric anthocyanidins (fraction 4). The effect of each fraction on ADP-induced rat platelet aggregation and c-AMP content was compared with that of DRW and pure phenolic compounds (quercetin, catechin, resveratrol, caffeic acid). DRW completely inhibited ADP-induced platelet aggregation. Fraction 2 also showed a significant anti-aggregating activity, whereas the effects of fractions 3 and 4 and the pure phenolics were not significant. A significant increase in platelet c-AMP content was observed after the addition of DRW and fraction 2. CONCLUSIONS: Our data indicate that DRW and its catechin-anthocyanidin fraction exert a significant effect on platelet aggregation in vitro, perhaps by enhancing platelet c-AMP levels.
Abstract: Thrombin (THR) plays a key role in the brain under physiological and pathological conditions. Several of the biological activities of thrombin have been shown to be mainly driven through activation of protease-activated receptor-1 (PAR-1)-type thrombin receptor. Here we have studied the effect of THR and PAR-1-activating peptide (PAR1-AP), SFLLRN, on cytokine-induced expression of inducible nitric oxide (iNOS), a prominent marker of astroglial activation using the rat C6 glioma cells. In this cell line, THR (1-10 U/mL) and PAR1-AP (1-100 microM) induced a significant concentration-dependent increase both of IFN-gamma- (250 U/mL) or TNF-alpha- (500 U/mL) induced NO release. The observed increase of NO production was related to an enhancement of iNOS expression as measured in cell lysates prepared from different treatments by using SDS-PAGE followed by western blot analysis. The effect of THR, but not that of PAR1-AP, was significantly inhibited by hirulog(TM) (60 microg/mL), a specific and stochiometric THR inhibitor or by cathepsin-G (40 mU/mL), an inhibitor of PAR-1. In conclusion our data suggest a role for THR through activation of PAR-1 in the induction of astroglial iNOS, and further support the hypothesis that THR may function as an important pathophysiological modulator of the inflammatory response.
Abstract: Protease-activated receptor-2 (PAR-2) is a member of seven transmembrane domain G protein-coupled receptors activated by proteolytic cleavage whose better known member is the thrombin receptor. The pathophysiological role of PAR-2 remains poorly understood. Because PAR-2 is involved in inflammatory and injury response events, we investigated the role of PAR-2 in experimental myocardial ischemia-reperfusion injury. We show for the first time that PAR-2 activation protects against reperfusion-injury. After PAR-2-activating peptide (2AP) infusion, we found a significant recovery of myocardial function and decrease in oxidation at reflow. Indeed, the glutathione cycle (glutathione and oxidized glutathione) and lipid peroxidation analysis showed a reduced oxidative reperfusion-injury. Moreover, ischemic risk zone and creatine kinase release were decreased after PAR-2AP treatment. These events were coupled to elevation of PAR-2 and tumor necrosis factor alpha (TNFalpha) expression in both nuclear extracts and whole heart homogenates. The recovery of coronary flow was not reverted by L-nitroarginine methylester, indicating a NO-independent pathway for this effect. Genistein, a tyrosine kinase inhibitor, did not revert the PAR-2AP effect. During early reperfusion injury in vivo not only oxygen radicals are produced but also numerous proinflammatory mediators promoting neutrophil and monocyte targeting. In this context, we show that TNFalpha and PAR-2 are involved in signaling in pathophysiological conditions, such as myocardial ischemia-reperfusion. At the same time, because TNFalpha may exert pro-inflammatory actions and PAR-2 may constitute one of the first protective mechanisms that signals a primary inflammatory response, our data support the concept that this network may regulate body responses to tissue injury.
Abstract: 1. Anti-inflammatory non steroidal drugs releasing NO (NO-NSAIDs) are a new class of anti-inflammatory drugs to which has been added an NO-releasing moiety. These compounds have been shown to retain the anti-inflammatory, analgesic and antipyretic activity of the parent compound but to be devoid of gastrointestinal (GI) toxicity. 2. Freund's adjuvant (FA) arthritis was induced in rats by a single intraplantar injection into the right hindpaw of 100 microl of mycobacterium butirricum (6 mg ml(-1)). The effect of equimolar doses of naproxen (1, 3 and 10 mg kg(-1)) and NO-naproxen (1.5, 4.5 and 16 mg kg(-1)) was evaluated using two dosage regimen protocols: (i) preventive, starting oral administration of the drugs at the time of induction of arthritis and for the following 21 days (day 1 - 21); (ii) therapeutic, starting oral administration of the drugs 7 days after adjuvant injection and for the following 14 days (day 7 - 21). 3. Hindpaw swelling (days 3, 7, 11, 14, 17, 21) and nociception (days 15 and 21) were measured. On day 22 rats were sacrificed, draining lymph nodes were removed and T cells isolated. In vitro proliferation of T cells following stimulation with concanavalin A (0.5 - 5 microg ml(-1)) was measured using a tritiated thymidine incorporation assay. IL-2 receptor expression on T cells was measured by FACS analysis. 4. Naproxen and NO-naproxen showed similar activity in reducing oedema formation in the non-injected (controlateral) hindpaw. Both drugs showed anti-nociceptive effect. NO-naproxen was anti-nociceptive at a dose of 4.5 mg kg(-1) while naproxen showed the same extent of inhibition only at a dose of 10 mg kg(-1). 5. T cells were isolated and characterized by FACS analysis. Stimulation of isolated T cells with concanavallin A in vitro caused a significant increase in thymidine uptake. NO-naproxen at a dose of 4.5 mg kg(-1) inhibited T cell proliferation to the same extent as 10 mg kg(-1) of naproxen. 6. Inhibition of T cell proliferation was well correlated with reduced IL-2 receptor expression on T cells. In addition, NO-naproxen reduced both IL-1beta and TNFalpha plasma levels whilst naproxen reduced IL-1beta levels only. 7. In conclusion, both naproxen and NO-naproxen reduce inflammation and nociception associated with arthritis. In addition NO-naproxen interferes to a larger extent with cellular mechanism involved in T cell activation in rat adjuvant arthritis indicating that introduction of the NO moiety in the naproxen structure increases the effect at the level of the immune system.
Abstract: 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 microl 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.
Abstract: 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.
Abstract: Rats injected in the hind paw with a mixture of Mycobacterium butirricum emulsified in mineral oil (FA) developed a severe polyarthritis that shared some immunological features with human rheumatoid arthritis. After this local administration, rats developed a secondary lesion (edema) in the contralateral paw, which is a hallmark of immune system activation. In vivo intravenous treatment with a monoclonal anti-very late antigen (VLA)-1 antibody (HA31/8) significantly reduced the edema formation in the contralateral paw. T cells isolated from contralateral paw draining lymph nodes of FA rats treated with HA31/8 showed a reduced cell proliferation in vitro, after stimulation with concanavalin A. Furthermore FACS analysis showed that the reduction in proliferation was concomitant to a reduction in the number of T cells positive to surface IL-2 receptor expression. Our data indicate that after in vivo treatment with a monoclonal anti-very late antigen-1 antibody, there is a beneficial effect on the development of the secondary lesion, which correlates to the reduced ability of T cells to proliferate in vitro as well as to a reduced surface expression of IL-2 receptor. The association of this antibody to other drugs interfering at other levels in rheumatoid arthritis may open a new therapeutic window.
Abstract: 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.
Abstract: The effects of angiotensin-converting enzyme (ACE)-inhibition with zofenopril on the development of atherosclerosis and low-density lipoprotein (LDL) oxidation were determined in Watanabe Heritable Hyperlipidemic (WHHL) rabbits. Rabbits received either placebo (n = 6) or 0.5 mg/kg/day of zofenopril (n = 6). After 6 weeks of treatment, the computer-assisted analysis revealed that zofenopril reduced the aortic and common carotid corrected cumulative lesion area by 34% and 39%, respectively (p < 0.05 vs placebo-treated group). The intimal/medial ratio of the largest fatty streaks was 0.426+/-0.158 in the zofenopril-treated group and 0.875+/-0.238 in the placebo-treated group (p < 0.05). Furthermore, we found in the zofenopril-treated group smaller lesions with an intimal/medial ratio of zofenopril also reduced plasmatic LDL oxidation, as shown by significant reduction of malondialdehyde content (p < 0.01) and relative agarose gel mobility (p < 0.05), as well as by the prolongation of the lag-time (p < 0.05). Compared to zofenopril-treated rabbits, arterial sections of the placebo-group had significant increase in the intimal presence of macrophages-derived foam cells (p < 0.05), ox-LDL (p < 0.01), and native LDL (p < 0.01) detected by immunocytochemistry with RAM-11, MDA2 and NP1533975 monoclonal antibodies, respectively. To investigate the amount of platelet accumulation in the atherosclerotic plaque we also measured platelet-associated radioactivity. Autologous platelets were labeled with 111Indiumoxine and injected intravenously. After 2 hours, WHHL were sacrificed and arterial sections were counted for platelet-associated radioactivity. In the placebo-treated group, platelet radioactivity was 0.52+/-0.12 equivalent of radioactivity per mg of tissue in the common carotid and 0.25+/-0.18 in the abdominal aorta; in contrast, rabbits treated by zofenopril had 0.20+/-0.12 in the common carotid and 0.06+/-0.01 in the abdominal aorta. These data indicate that ACE-inhibition with zofenopril has antiatherosclerotic and antioxidant effects in WHHL-rabbits. Our results also shows that these effects could be linked to a reduced wall-associated platelet deposition at the site of atherosclerotic lesions.
Abstract: 1. Several thrombin cellular effects are dependent upon stimulation of proteinase activated receptor-1 (PAR-1) localized over the cellular surface. Following activation by thrombin, a new N-terminus peptide is unmasked on PAR-1 receptor, which functions as a tethered ligand for the receptor itself. Synthetic peptides called thrombin receptor activating peptides (TRAPs), corresponding to the N-terminus residue unmasked, reproduce several thrombin cellular effects, but are devoid of catalytic activity. We have evaluated the bronchial response to intravenous administration of human alpha-thrombin or a thrombin receptor activating peptide (TRAP-9) in anaesthetized, artificially ventilated guinea-pigs. 2. Intravenous injection of thrombin (100 microkg(-1)) caused bronchoconstriction that was recapitulated by injection of TRAP-9 (1 mg kg(-1)). Animal pretreatment with the thrombin inhibitor Hirulog (10 mg kg(-1) i.v.) prevented thrombin-induced bronchoconstriction, but did not affect bronchoconstriction induced by TRAP-9. Both agents did not induce bronchoconstriction when injected intravenously to rats. 3. The bronchoconstrictor effect of thrombin and TRAP-9 was subjected to tolerance; however, in animals desensitized to thrombin effect, TRAP-9 was still capable of inducing bronchoconstriction, but not vice versa. 4. Depleting animals of circulating platelets prevented bronchoconstriction induced by both thrombin and TRAP-9. 5. Bronchoconstriction was paralleled by a biphasic change in arterial blood pressure, characterized by a hypotensive phase followed by a hypertensive phase. Thrombin-induced hypotension was not subject to tolerance and was inhibited by Hirulog; conversely, hypertension was subject to tolerance and was not inhibited by Hirulog. Hypotension and hypertension induced by TRAP-9 were neither subject to tolerance nor inhibited by Hirulog. 6. Our results indicate that thrombin causes bronchoconstriction in guinea-pigs through a mechanism that requires proteolytic activation of its receptor and the exposure of the tethered ligand peptide. Platelet activation might be triggered by the thrombin effect.
Abstract: Inflammation and coagulation cannot be considered as two separate processes, since there are several connecting points making them part of unique, defensive host response. The endothelium can be considered as the first link between inflammation and coagulation, since damaged endothelium during inflammation represents a surface where proteins involved in both coagulation and the development of inflammation are expressed. During inflammation, cytokines modulate the coagulation system by downregulating the expression of thrombomodulin and the activation of protein C pattern but, at the same time, they induce the expression of tissue factor, modifying, in this way, the balance between procoagulant and anticoagulant activities. At the same time, at the site of tissue injury, platelets become activated and release several mediators that modify tissue integrity. Thrombin, formed following activation of the coagulation cascade, is essential to promote haemostasis but also stimulates several cell functions, including chemotaxis and mitogenesis, which are responsible for the spreading of the lesion and the tissue repair process. Therefore, in the study of inflammation the involvement of the coagulation pathway has to be taken into account, since the interaction between coagulation and inflammation pathways is a critical issue.
Abstract: The signaling pathway initiated by factor Xa on vascular endothelial cells was investigated. Factor Xa stimulated a 5- to 10-fold increased release of nitric oxide (NO) in a dose-dependent reaction (0.1-2.5 microG/ml) unaffected by the thrombin inhibitor hirudin but abolished by active site inhibitors, tick anticoagulant peptide, or Glu-Gly-Arg-chloromethyl ketone. In contrast, the homologous clotting protease factor IXa or another endothelial cell ligand, fibrinogen, was ineffective. A factor Xa inter-epidermal growth factor synthetic peptide L (83)FTRKL(88) (G) blocking ligand binding to effector cell protease receptor-1 inhibited NO release by factor Xa in a dose-dependent manner, whereas a control scrambled peptide KFTGRLL was ineffective. Catalytically active factor Xa induced hypotension in rats and vasorelaxation in the isolated rat mesentery, which was blocked by the NO synthase inhibitor L-N(G)-nitroarginine methyl ester (L-NAME) but not by D-NAME. Factor Xa/NO signaling also produced a dose-dependent endothelial cell release of interleukin 6 (range 0.55-3.1 ng/ml) in a reaction inhibited by L-NAME and by the inter-epidermal growth factor peptide Leu(83)-Leu(88) but unaffected by hirudin. Maximal induction of interleukin 6 mRNA required a brief, 30-min stimulation with factor Xa, unaffected by subsequent addition of tissue factor pathway inhibitor. These data suggest that factor Xa-induced NO release modulates endothelial cell-dependent vasorelaxation and cytokine gene expression. This pathway requiring factor Xa binding to effector cell protease receptor-1 and a secondary step of ligand-dependent proteolysis may preserve an anti-thrombotic phenotype of endothelium but also trigger acute phase responses during activation of coagulation in vivo.
Abstract: An investigation of the extracts from Allium neapolitanum has led to the isolation of 13 flavonoid glycosides, based on kaempferol, quercetin and isorhamnetin. Four of them are new compounds and have been identified as: kaempferol 3-O-[[2-O-alpha-L-rhamnopyranosyl-4-O-beta-D-glucopyranosyl]-beta-D- glucopyranoside]], isorhamnetin 3-O-[[2-O-alpha-L-rhamnopyranosyl-6-O-beta- D-glucopyranosyl]-beta-D-glucopyranoside], isorhamnetin 3-O-[[2-O-alpha-L-rhamnopyranosyl-6-O-beta-D-glycopyranosyl] beta-D-glucopyranoside]-7-O-beta-D-glucopyranoside and isorhamnetin 3-O-[[2-O-alpha-L-rhamnopyranosyl-6-O-beta-D-gentiobiosyl]- beta-D-glucopyranoside]]. The isolated compounds were evaluated for their anti-aggregation human platelet activity.
Abstract: 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.
Abstract: The aim of the present study was to evaluate platelet responsiveness in rats following E.coli endotoxin administration. Injection of E.coli to rats caused a reduction in ADP-induced pulmonary 111In labelled platelet accumulation four hours later. Similarly, when platelet aggregation was evaluated on PRP obtained from rats four hours after endotoxin administration, we found that platelet response to both ADP and collagen was significantly reduced. When platelets obtained from endotoxemic rats were suspended in normal plasma, the aggregating response to ADP and collagen was not different from that obtained with control platelets. Similarly, platelets from control rats suspended in plasma from endotoxemic rats showed hyporesponsiveness to ADP and collagen. There was no difference in the aggregatory response to collagen or to thrombin of washed platelet suspension (WPS) obtained from endotoxemic and normal rats. In conclusion, by using an in vivo minimally invasive technique and an ex vivo platelet aggregation test we demonstrate that during endotoxemia platelet are functionally unaltered and the platelet hyporesponsiveness is only observed in presence of plasma.
Abstract: We studied the effects of i.v. administration of endotoxin (Escherichia coli, Serotype 0127:B8) on the kinetics of 111In-labelled platelets within the pulmonary, abdominal and splenic vascular beds of the rat, and on the radioactivity present in dissected samples of splenic and hepatic tissues. Bolus i.v. injection of endotoxin to anaesthetised rats caused a dose-dependent, transient accumulation of 111In-labelled platelets in the pulmonary vasculature. Increased radioactivity, suggestive of platelet sequestration, was detected in tissue samples from both the spleen and the liver at 4.5 h compared to the radioactivity detected in those organs in vehicle treated rats. The modulation of endotoxin-induced platelet accumulation within the lungs, spleen and liver by pharmacological agents was investigated. The pulmonary, hepatic and splenic platelet accumulation induced by endotoxin was unaffected by pre-treatment of the animals with indomethacin, Hirulog or L-NAME. Pre-treatment with dexamethasone significantly reduced the platelet accumulation within the liver and spleen, but not the lungs.
Abstract: From wild garlic Allium ursinum three new flavonoid glycosides were identified as kaempferol 3-O-beta-neohesperidoside-7-O-[2-O-(trans-p-coumaroyl)]-beta -D- glucopyranoside, kaempferol 3-O-beta-neohesperidoside-7-O-[2-O-(trans-feruloyl)]-beta-D- glucopyranoside, kaempferol 3-O-beta-neohesperidoside-7-O-[2-O-(trans-p-coumaroyl)-3-O-b eta-D- glucopyranosyl]-beta-D-glucopyranoside and characterized as the peracetates. Additionally, two known flavonoid glycosides kaempferol 3-O-beta-glucopyranoside and kaempferol 3-O-beta-neohesperidoside were isolated. The isolated compounds showed an inhibition of human platelet aggregation.
Abstract: A rat model of inflammation was used to investigate the biological effects of thrombin. The thrombin-specific inhibitor Hirulog markedly attentuated the carrageenin-induced edema of the paw of the rat. Injection of thrombin into the paw also produced edema. The effect of thrombin was due to activation of its receptor; a thrombin receptor activating peptide (TRAP) reproduced the effects of thrombin in causing edema. TRAP also increased vascular permeability as demonstrated by extravasation of Evans blue and 125I-labeled serum albumin. The release of bioactive amines played an important role in mediating the TRAP-induced edema; the serotonin/histamine antagonist cryproheptadine and the histamine H2 receptor antagonist cimetidine reduced significantly the edema caused by TRAP. Treatment of rats with the mast cell degranulator 48/80 to deplete these cells of their stores of histamine and serotonin abolished completely the ability of TRAP to produce edema. Histochemical examination confirmed that TRAP treatment led to mast cell degranulation. Thus, it has been possible to demonstrate that thrombin acts as an inflammatory mediator in vivo by activating its receptor, which in turn leads to release of vasoactive amines from mast cells.
Abstract: Lipopolysaccharide treated rats (25 mg/kg i.v.) were killed after 60 min and rat aortic rings were mounted in an isolated organ bath for measurement of isometric contractions in response to phenylephrine (0.01-10 microM) or potassium chloride (10 mM). Aortic rings from lipopolysaccharide-treated rats showed reduced contractility to phenylephrine and potassium chloride when compared to those from saline-treated rats. Indomethacin 10 microM, added in vitro further impaired phenylephrine-induced contraction of aortic rings from lipopolysaccharide ex vivo treated rats but was ineffective on aortic rings from saline treated rats. A similar pattern was observed when potassium chloride was used. Administration in vitro of thromboxane A2 receptor antagonist SQ29,548 gave a similar effect to indomethacin. Aortic rings collected from rat treated in vivo with dexamethasone (10 mg/kg) showed a reduction in phenylephrine induced contractions that was not further reduced by in vitro treatment with indomethacin (10 microM). Similarly, when rat aortic rings were incubated in vitro (60 min) with lipopolysaccharide (0.4 mg/ml) a reduction of phenylephrine- and potassium chloride-induced contraction was observed, but addition of either indomethacin or SQ29,548 did not further reduce contraction. Our results suggest that under these experimental conditions, in the early phase of endotoxin shock, synthesis of cyclooxygenase products (such as endoperoxides or thromboxane A2) occurs probably as a compensatory mechanism to lipopolysaccharide induced hypocontractility from the interaction, in vivo, between lipopolysaccharide, endothelium, circulating cells and vascular smooth muscles.
Abstract: Progressive lysis of bone in loose total hip replacement has been ascribed to the capacity of the synovial-like membrane present at the bone interface to produce prostaglandin E2 and cytokines such as tumor necrosis factor (TNF alpha). Phospholipase A2 (PLA2) produces rate limiting precursor i.e. arachidonic acid in the biosynthesis of various types of biologically active lipids including prostaglandins. It has been shown that extracellular human group II phospholipase A2 is present in large amount in synovial fluid of patients with synovitis and that the expression of this enzyme is under the control of cytokines such as TNF alpha. Furthermore, the human extracellular enzyme has been also shown to induce in an experimental animal model to cause disruption of a synovial like membrane without increasing prostaglandin production. Here we have evaluated PLA2 and TNF alpha levels in the supernatant of homogenate of the synovial-like membrane present at the bone interface retrieved from six patients with a loose non septic failed total hip replacement. In all the membranes examined were found high levels of both TNF alpha (856 +/- 211 units/ml) and extracellular phospholipase A2 (2616 +/- 862 ng/ml). These findings suggest that extracellular PLA2 may play a major role in the process that cause disruption of the membrane at the bone-prosthesis interface.
Abstract: We have recently shown that the introduction of a nitroxybutylester moiety into flurbiprofen, to form Flurbi-NO, results in a compound with markedly reduced undesired effects in the gastrointestinal tract. This effect has been shown to be linked to nitric oxide release from the Flurbi-NO. Here we have investigated whether this is associated with a reduction in platelet aggregability in vivo, as assessed in a mouse model of thromboembolism and a rat model of platelet aggregation, and found in both models that Flurbi-NO is more potent than flurbiprofen at inhibiting collagen-induced platelet aggregation. Further in vitro studies using human washed platelets and cells in culture suggest that this is due to the release of NO from Flurbi-NO following the action of (possibly plasma) esterases. Together with our earlier data, these results strongly suggest that Flurbi-NO and other members of this class of drugs, have particular potential as anti-thrombotic agents devoid of gastrointestinal side effects.
Abstract: Hirulog is a thrombin catalytic site inhibitor which exhibits specificity for the anionic binding exosite of alpha thrombin. Here, we have evaluated the effect of Hirulog (1, 5 and 10 mg/kg, 30 min pretreatment) in a rat model of endotoxemia. Intravenous injection of lipopolysaccharide from E. coli (25 mg/kg; serotype 0127:B8) caused decreases in blood pressure which were significantly reduced (about 60%) in animals pretreated with Hirulog. Rat survival to endotoxin was significantly increased in Hirulog pretreated group (5 and 10 mg/kg) up to 24 hours. Hirulog at the dose of 10 mg/kg inhibited both endotoxin-induced leukopenia at 30 and 60 minute points and thrombocytopenia at 30 minute point but not at 90 and 120 minute points. Fibrinogen levels were significantly reduced after 2 hours following endotoxin administration. Pretreatment with Hirulog (5-10 mg/kg i.v.) 30 min prior to administration of endotoxin prevented changes in fibrinogen plasma levels. These results demonstrate that Hirulog-induced inhibition of thrombin is effective in reducing toxic and lethal effects of endotoxin.
Abstract: In this study by using the human recombinant non-pancreatic-secreted platelet PLA2 (r-hnps-PLA2) and rabbit polyclonal antibodies directed against either the human (group II) or the porcine enzyme (group I), we have shown a possible involvement of platelet PLA2 in poly-L-arginine (25 kDa)-induced rat paw edema. Local treatment of rats with the anti-platelet-PLA2 antibody (anti-hnps-PLA2) but not with anti-porcine-PLA2 antibody (anti-porc-PLA2) significantly reduced the edema induced by a maximal dose of poly-L-arginine (1 mg/paw). Furthermore when r-hnps-PLA2 (1-10 micrograms) was injected together with a subliminal dose of poly-L-arginine (50 micrograms/paw), a dose-dependent increase in both edema and protein leakage was observed. This effect was selectively inhibited by the anti-hnps-PLA2 (10-100 micrograms/paw) but not anti-porc-PLA2 (10-100 micrograms paw). Thus, platelets seem to be involved in both vascular and cellular components of the inflammatory response by contributing, most likely in the early phase, to the edema formation through secretion of PLA2.
Abstract: Platelets contain a phospholipase A2 in their granules which can be released in response to various activating stimuli in vitro as well as in vivo. Human recombinant phospholipase A2 (1-10 micrograms/ml) had no direct effect on platelet aggregation in vitro using rabbit platelet rich plasma. In contrast human recombinant phospholipase A2 (1-20 micrograms/ml) was able to inhibit aggregation of washed rabbit platelet in vitro induced by collagen (0.250-2.0 micrograms/ml). When rabbit platelet rich plasma was recalcified with CaCl2 1 M in the presence of the thrombin inhibitor hirulog (10 micrograms/ml), human recombinant phospholipase A2 (10-40 micrograms/ml) was able to inhibit platelet aggregation. The anti-aggregatory effect was removed by incubation of platelet rich plasma with a monoclonal anti-human recombinant phospholipase A2 antibody. Human recombinant phospholipase A2 (1-10 micrograms) inhibited 111In-labelled platelet accumulation within the thoracic region of rats and guinea pigs induced by i.v. administration of submaximal doses of collagen or adenosine diphosphate. Phospholipase A2 (1-20 micrograms/ml) from Naja mocambique mocambique snake venom had no direct effect on platelet aggregation in vitro. However, Naja phospholipase A2 administered i.v. to rats or guinea pigs was able to induce a dose related accumulation of 111In-labelled platelet within the thoracic region. The inhibitory effect of exogenously added human recombinant phospholipase A2 on platelet aggregation in vivo suggests a possible pathophysiological role for the extracellular form of phospholipase A2 but this property is not a feature of all phospholipase A2 preparations.
Abstract: Platelet-activating factor (PAF) is thought to play an important role in pathogenesis of endotoxin shock. Here, using essential fatty acid deficient (EFAD) rats, we have evaluated changes in mean arterial blood pressure, PAF levels and damage in both stomach and duodenum following intravenous administration of endotoxin (LPS). In EFAD rats the second phase of LPS-induced hypotension was strongly reduced. Similarly, PAF levels in stomach and duodenum of EFAD rats were also reduced and correlated to the diminished damage. Our study confirms a direct involvement of PAF in LPS-induced gastrointestinal damage.
Abstract: In this study, we assessed the effects of addition of a nitroxybutyl moiety to diclofenac on its ulcerogenic properties. The diclofenac derivative, 'nitrofenac', was examined in terms of its ability to induce acute gastric erosions and chronic-type gastric ulcers in rats and rabbits, respectively. The effects of these compounds on prostaglandin synthesis in the stomach and at a site of peripheral inflammation were also assessed, as were their anti-inflammatory properties in a model of acute inflammation. Diclofenac dose-dependently caused acute gastric mucosal injury in the rat at all doses tested (10-40 mg/kg), that was significantly greater in severity than that observed with the same doses of nitrofenac. In rabbits, twice-daily administration of diclofenac induced penetrating antral ulcers and small intestinal damage. No damage was observed in the stomach or small intestine of rabbits receiving nitrofenac. Diclofenac and nitrofenac exerted similar inhibitory effects on prostaglandin E2 synthesis in the stomach and in a carrageenan-sponge model of peripheral inflammation. These compounds exerted similar inhibitory effects on carrageenan-induced paw edema. Nitrofenac, but not diclofenac, caused a significant increase in plasma levels of nitrate/nitrite. These results suggest that the addition of a nitroxybutyl moiety to diclofenac markedly reduces the ulcerogenic properties of this compound without interfering with its ability to inhibit cyclo-oxygenase activity or to reduce acute inflammation.
Abstract: OBJECTIVE. The aim of this study was to test the proinflammatory action of human secreted phospholipase A2 (sPLA2) in an animal model of synovitis-like inflammation and to compare it with a Group 1 (porcine pancreatic) and a Group 2 (Naja mocambique mocambique) PLA2. METHODS. The subcutaneous injection of air in the rat pouch induced the formation of a connective tissue cavity lined with cells closely resembling a synovium. sPLA2 (0.01-10 micrograms/pouch) porcine pancreatic (10 micrograms/pouch), PLA2 or Naja mocambique mocambique PLA2 (10 micrograms/pouch) were injected in a 6-day rat air pouch. RESULTS. Our data show that recombinant human enzyme PLA2 (0.01-10 micrograms/pouch), but not Naja mocambique mocambique (Group 2 PLA2) and porcine pancreatic PLA2 (Group 1 PLA2), was able to exacerbate the inflammatory response without increasing eicosanoid release. CONCLUSION. Only sPLA2 was able to exacerbate rat air pouch and its effect was unrelated to eicosanoid release.
Abstract: BACKGROUND/AIMS: The use of nonsteroidal anti-inflammatory drugs (NSAIDs) is limited by their ability to induce gastrointestinal injury. Two NSAIDs were modified by incorporation of an nitroxybutyl moiety. The short-term ulcerogenic and anti-inflammatory properties of these derivatives were compared with the native NSAIDs. METHODS: Rats were given flurbiprofen, ketoprofen, or their respective derivatives, and the extent of gastric damage and effect on gastric prostaglandin E2 synthesis was assessed. The damage-promoting effects of these compounds were also compared following twice-daily administration for 1 week. Anti-inflammatory properties were examined using a carrageenan-induced paw edema model. RESULTS: The derivatives of flurbiprofen and ketoprofen caused significantly less short-term gastric mucosal injury at all doses tested, despite producing comparable suppression of prostaglandin synthesis. The NSAID derivatives also showed comparable anti-inflammatory activity to the native compounds. The flurbiprofen derivative inhibited collagen-induced platelet aggregation significantly more than the native NSAID. Plasma nitrate/nitrite levels increased significantly following administration of the flurbiprofen derivative, consistent with release of a nitrogen oxide. CONCLUSIONS: Addition of a nitroxybutyl moiety to two NSAIDs markedly reduced the ability of these agents to induce short-term gastric injury but did not interfere with their ability to suppress inflammatory processes, inhibit prostaglandin synthesis, or inhibit platelet aggregation. These NSAID derivatives may therefore represent a novel class of anti-inflammatory drugs with markedly less ulcerogenic effects on the stomach.
Abstract: 111Indium-labelled platelets were continuously monitored in the cranial vasculature of anaesthetised rabbits and thrombin inhibitors and anti-coagulants were tested on the sustained platelet accumulation induced by intracarotid injection of thrombin (90 U/kg). Pretreatment, commencing 30 min prior to thrombin, with a 1-h intracarotid infusion of D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone (PPACK; 0.25-1.0 micrograms/kg per min), unfractionated heparin (Multiparin; 5-20 U/kg bolus + 0.75-3.0 U/kg per min infusion) or low molecular weight heparin (Fragmin; 2.4-9.6 U/kg per min) produced dose-related reductions in platelet accumulation. Continuous infusion of acetyl-D-phenylalanyl-prolyl-boroarginine (DuP-714 ester; 30 micrograms/kg per min) for 30 min induced marked accumulation of platelets in the pulmonary circulation in the absence of thrombin. Bolus intracarotid injection, 1 min before thrombin, of Hirulog (0.05-0.2 mg/kg), PPACK (10-30 micrograms/kg), Multiparin (25-100 U/kg), Fragmin (150 U/kg) or DuP-714 ester (15-30 micrograms/kg) caused significant reductions in platelet accumulation. When injected 1 min after thrombin, Hirulog (1 mg/kg), PPACK (100 micrograms/kg), Fragmin (150 U/kg) and DuP-714 ester (30 micrograms/kg) had no significant effect and Multiparin (100 U/kg) increased platelet accumulation. The results demonstrate that pretreatment with a range of thrombin inactivators, acting via different mechanisms, can inhibit thrombin-induced cerebral thromboembolism in the rabbit.
Abstract: 1. An increase in circulating levels of extracellular group II phospholipase A2 (PLA2) has been detected in patients with septic shock as well as in rats, rabbits and human volunteers following intravenous endotoxin administration. 2. Group II PLA2 from Naja mocambique mocambique (NajaPLA2) snake venom (1-10 micrograms i.v.) induced a dose-dependent hypotension and leucopenia in the rat similar to that induced by intravenous administration of endotoxin. 3. NajaPLA2 did not aggregate rat platelet, but did aggregate purified rat neutrophils in vitro. However intravenous PLA2 caused rat platelets to aggregate in vivo. 4. The hypotensive effect was reduced either by inactivating NajaPLA2 in vitro with para-bromophenacyl bromide or by infusing in vivo polyclonal rabbit antiPLA2 antisera. Neither the hypotension nor the leucopenia was affected by several agonists and inhibitors. 5. The leukotriene D4 antagonist L-649,923 produced a dose-related (5-20 mg/kg i.v.) inhibition of NajaPLA2-induced hypotension while the dual inhibitor of lipo- and cyclooxygenase BW755c (5-50 mg/kg i.v.) was ineffective. 6. In rats rendered leucopenic with methotrexate the L-649,923 was ineffective implying that the L-649,923 effect could be partially mediated through its action on neutrophils.
Abstract: Extracellular phospholipase A2 is secreted from the platelets upon activation by a stimulus such as thrombin. The secreted enzyme has been recently cloned and the recombinant protein produced. Snake venom PLA2 effect on platelet and coagulation has been extensively studied (for review see 3,4) and it has been proposed that the anticoagulant phospholipases may inhibit coagulation by competing with clotting proteins for the lipid surface. Structure function relationship for PLA2s with anticoagulant activity indicates that the activity is conferred by positively charged aminoacids between residues 54 and 77. The corresponding segment of human recombinant secreted platelet PLA2 (r-hnps-PLA2) possesses five positively charged aminoacids in this region being at the identical positions to those of PLA2s with known anticoagulant activities. Here using human and rat plasma we have demonstrated for the first time that the recombinant human extracellular secreted platelet PLA2 increases activated partial thromboplastin time but not prothrombin time.
Abstract: Lipocortin 1 is a member of a group of calcium and phospholipid binding proteins named lipocortins (Di Rosa et al. (1984) Prostglandins 28, 441-442) and also known as annexins (Crumpton (1990) Nature 345, 212). In this study we have shown that human recombinant lipocortin-1 can increase the activated partial thromboplastin time but not the prothrombin time of human plasma in vitro. This effect is dose and time dependent and is reversed by polyclonal anti-lipocortin-1 antibodies. To our knowledge, this is the first demonstration that the human recombinant protein has the same activity as the native protein on human plasma.
Abstract: An acetylated polypeptide corresponding to residues 2-26 of human lipocortin 1 was synthesized and the anti-inflammatory activity assessed in three models of acute inflammation in rat and mouse. In the carrageenin rat paw oedema test, the peptide produced a maximal inhibition of approximately 41% at the 3 h time point with a 10 micrograms dose. When rat paw oedema was induced by the injection of venom phospholipase A2, the peptide produced a significant inhibition (31%) at the top dose of 20 micrograms per paw. In the mouse air-pouch model, systemic treatment with the peptide produced a dramatic reduction in cytokine-induced leukocyte migration with an ID50 of approximately 40 micrograms per mouse. The N-terminal peptide 2-26 shares the actions of lipocortin 1 in these acute models of inflammation.
Abstract: The ability of tepoxalin to render the gastric mucosa susceptible to injury by a topically applied irritant was compared to that of indomethacin, naproxen and diclofenac. While the three NSAIDs significantly increased the extent of mucosal damage at doses in the 1-30 mg/kg range, tepoxalin failed to significantly augment damage at doses of up to 300 mg/kg. Daily treatment with tepoxalin (10-100 mg/kg) for 4 days also did not significantly affect the susceptibility of the gastric mucosa to damage. The absence of ulcerogenic properties of tepoxalin at doses previously shown to be anti-inflammatory may be related to its relative lack of activity as an inhibitor of gastric prostaglandin synthesis, or to its 5-lipoxygenase inhibitory activity.
Abstract: 1. The rat paw oedema produced by a local injection of phospholipase A2 from Naja mocambique mocambique has been shown to be mainly driven by the liberation of serotonin and kinins. 2. Using specific bradykinin receptor antagonists we have shown that kinins are acting through B2 receptors. 3. Using endothelium-derived relaxing factor (EDRF) synthesis inhibitors NG-monomethyl-L-arginine and L-NG-nitro arginine we have tested the possible envolvement of EDRF as mediator. 4. Our work supports the view that extracellular phospholipases A2 are involved in inflammation, and suggests a role for EDRF as mediator of extravasation in this model of inflammation.
Abstract: The effects of the constituent sesquiterpene glycosides 1-3 and polyhydroxylated triterpenoids 5-6 isolated by MeOH extraction of Eriobotrya japonica were studied in genetically diabetic mice (C57BL/KS-db/db/Ola) and normoglycemic rats. The sesquiterpene glycoside 3 and the polyhydroxylated triterpenoids 5 and 6 produced a marked inhibition of glycosuria. Furthermore, 5 and 6 were able to reduce blood glucose levels in normoglycemic rats. While there are already some data reported on hypoglycemic activity of polyhydroxylated triterpenoids, there are no previous data showing hypoglycemic activity of sesquiterpene glycosides.
