Eosinophils have long been considered to play solely crucial role in the pathogenesis of aspirin-induced asthma, however increasing evidence suggest that the bronchial epithelium is also involved in the initiation and maintenance of allergic inflammation. Epithelial cells and eosinophils retained within airways interact reciprocally to mount and sustain inflammatory response. Recently, we have shown that eosinophil-epithelial cell interactions are capable of amplifying the production of cysteinyl leukotrienes (Cys-LTs). The aim of this study was to investigate if there is any influence of aspirin (ASA) on Cys-LTs and prostaglandin E2 (PGE2) production in the model of co-cultured human epithelial cells (line BEAS-2B) and human eosinophils. Synthesis of Cys-LTs in eosinophils was increased after incubation with ASA. At the same time the production of PGE2 was decreased by aspirin (n=32). BEAS-2B cells barely formed Cys-LTs; addition of ASA increased this production, while production of PGE2 was inhibited by aspirin (n=32). Synthesis of Cys-LTs by eosinophils co-incubated with BEAS-2B was nearly 7-fold higher than that of activated eosinophils alone (1631.5 pg/ml ± 154 vs. 258 pg/ml ± 31; p<0.05; n=32). Surprisingly, in the eosinophil-epithelial cell co-culture, aspirin inhibited both augmentation of Cys-LTs synthesis (from 1631.5 pg/ml ± 154 to 1458 pg/ml ± 137; p<0.05; n=32) and the production of PGE2 (from 2640 pg/ml ± 231 to 319 pg/ml ± 27; p<0.05; n=32). In summary, we have demonstrated that interactions between non-atopic eosinophils and epithelial cells result in augmentation of Cys-LTs production, and this augmentation could be inhibited by aspirin.
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Nitric oxide (NO), depending on the amount, time and source of generation may exert both, protective and deleterious actions during endotoxic acute lung injury (ALI). Evaluation of the expression and localization of NOS isoforms in the lung of lipopolysaccharide (LPS) - treated rats may contribute to understanding the role of NO in pathogenesis of ALI. Tissue samples (lung, heart, liver, kidney and spleen) as well as peripheral blood polymorphonuclear cells (PMNs) were collected from control male Wistar rats and LPS - treated animals, 15, 30, 60, 120 and 180 min after LPS injection (2 mg kg-1 min-1 for 10 minutes, i.v.). Levels of NOS-2 and NOS-3 mRNA and protein in tissues and PMNs were estimated by RT-PCR, Northern blotting and Western blotting. Additionally, myeloperoxidase (MPO) activity in tissue samples was assayed. NOS-3 mRNA as well as protein were detected in lungs of control animals; pulmonary NOS-3 expression was not influenced by LPS. The induction of NOS-2 mRNA in rat lungs and in PMNs isolated from peripheral blood was observed 15 minutes after LPS challenge. In contrast, increase of NOS-2 mRNA in the heart, kidneys, liver and spleen was observed 2-3 hours after LPS injection. In all tissues rise in NOS-2 mRNA was followed after 1-2 hours by increase of NOS-2 protein. Importantly, progressive leukocyte sequestration in the lung parenchyma that started as early as 15 min after LPS injection was revealed only in the lungs; in other organs no significant changes in MPO activity were detected up to 180 min after LPS injection. In conclusion, infusion of LPS caused much more rapid expression of NOS-2 in lungs as compared to the heart, kidneys, liver and spleen. Early induction of NOS-2 may depend on the LPS-stimulated rapid neutrophil sequestration within lung vasculature and fast induction of NOS-2 in sequestrated neutrophils.
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Recently, we have shown that MK-886 - an inhibitor of five lipoxygenase activating protein (FLAP) inhibits atherosclerosis in apolipoprotein E / LDL receptor - double knockout mice. We, therefore, wanted to find out if other FLAP inhibitor - BAYx1005 given at a dose of 1.88 mg per 100 mg of body weight per day during 16 weeks, could also attenuate atherogenesis. In apoE/LDLR - DKO mouse model BAYx1005 inhibited atherogenesis, measured both by "en face" method (23.84 ± 2.7% vs. 15.16 ± 1.4%) and "cross-section" method (497236 ± 31516 µm2 vs. 278107 ± 21824 µm2). This is the first report that shows the effect of BAYx1005 on atherogenesis in gene-targeted mice.
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Prostaglandin endoperoxide H2 (PGH2) is generated from arachidonic acid by either constitutive (COX-1) or inducible (COX-2) cyclooxygenases. In arterial wall PGH2 is converted by PGI2 synthase (PGI-S) to prostacyclin (PGI2), and in platelets by thromboxane synthase (TX-S) to thromboxane (TXA2). Other prostanoids as PGD2, PGF2alpha or PGE2 were believed to arise non-enzymatically from PGH2. Only recently, human prostaglandin E synthase (PGE-S) has been identified and cloned as a membrane bound, microsomal, glutathione-dependent inducible enzyme. Here we demonstrated that interleukin 1ß (IL-1ß) is an inducer of COX-2 and PGE-S in human umbilical vein endothelial cells (HUVEC). Functional expression of PGE-S was measured at the level of specific mRNA by semi-quantitative RT-PCR, PGE-S protein was detected by Western blot in HUVEC, while PGE2 was measured by immunoassay in the supernatant. Actinomycin D, a classical transcription inhibitor, was used to prove that indeed IL-1ß induced the functional PGE-S enzyme. PGE2 generation in HUVEC was inhibited by indomethacin, acetamoniphen and dexamethasone. In conclusion, we found that in cultured endothelial cells IL-1ß induced as evidenced by the appearance of its transcript and its functional enzyme. The induction of endothelial PGE-S and COX-2 appeared to be and their transcripts were induced as fast as one might expect from immediate early genes. It means that IL-1ß-triggered-PGE2 biosynthesis in endothelial cells is probably regulated by induction of both COX-2 and PGE-S. This is way we hypothesise the existence of at least two distinct pools of COX-2: the first selectively coupled to PGE-S and the second one that is coupled to PGI-S yielding the main endothelial product - PGI2.
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Production of arachidonic acid (AA) metabolites - prostacyclin (PGI2) in large vessels and prostaglandin E2 (PGE2) in microcirculation is intrinsically involved in maintenance of vascular wall homeostasis. EA.hy 926 is a hybrid cell line, is derived by fusion of HUVEC with A549 cells. The aim of this study was to examine the production of prostacyclin and PGE2 in resting and IL-1ß-stimulated EA.ha 926 cells, in comparison with its progenitor cells. Non-stimulated EA.hy 926 cells has been found to produce much lower amounts of prostacyclin than resting HUVEC. Resting hybrid cells produced more PGE2 than prostacyclin, despite they expressed high levels of COX-1 and PGI2 synthase. On the contrary to HUVEC and A549, EA.hy 926 cells did not respond to IL-1ß with COX-2 induction and increase of prostaglandin production, however they did it in response to lysophosphatidylcholine (LPC). The characteristics of EA.hy 926 cells in terms of the pattern of prostanoid formation could facilitate studies on endothelial metabolism and role of these important lipid mediators.
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Although lipopolysaccharide (LPS) is recognized to induce a biphasic cardiovascular response its mechanism is not fully elucidated. In this study we analysed the involvement of PAF, TXA2 and cysteinyl leukotrienes (cysLTs) in the acute cardiovascular effects of LPS in the isolated rat heart as well as in delayed phase of LPS response using a surrogate cellular model of the induction of NOS-2 by LPS in mouse macrophages. Perfusion of rat hearts with LPS resulted, in an immediate fall in heart contractility and coronary flow by 2.5 ± 0.59 ml min-1 and 560 ± 81 mmHg sec-1, respectively. This response was fully blocked by platelet activating factor (PAF) antagonist - WEB 2170 and partially inhibited, by inhibitor of cyclooxygenase (indomethacin) or by inhibitor of thromboxane synthase (camonagrel). The inhibition of leukotriene synthesis (BAY x1005) or cysLTs receptors (BAY x7195) was without effect. Administration of stable PAF analog (methylcarbamyl-PAF - MC-PAF) alone, mimicked heart response to LPS. In cultured mouse macrophages, MC-PAF did not induce NOS-2 expression and when given with LPS it slightly potentiated NOS-2 induction by LPS. However, in presence of WEB 2170 NOS-2 induction by LPS was inhibited in a dose-dependent manner. Inhibition of cyclooxygenase and leukotriene pathways had no effect on NOS-2 induced by LPS. These results indicate that PAF and TXA2 but not cysLTs mediate the instant heart response induced by LPS, while PAF alone mediates a delayed NOS-2 induction by LPS. Accordingly, PAF may constitute the mediator that links acute and delayed phases of LPS-induced cardiovascular response.
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Increased generation of reactive oxygen species contribute to endothelial dysfunction in atherosclerosis, hypertension and heart failure. Recently, it was suggested that bursts of superoxide anions may inactivate endothelial surface-bound enzymes such as angiotensin converting enzyme (ACE). Here, we tested effects of xanthine/xanthine oxidase-derived superoxide anions on vascular responses and ACE activity in the isolated guinea pig heart. We analysed effects of intracoronary infusion of low concentration of xanthine oxidase (10 mU/ml) in the presence of xanthine (0,5 mM) (X/XO) on bradykinin, other endothelium-dependent and independent vasodilators (acetylcholine, ADP, SNAP), as well as vasoconstrictor responses to angiotensin I and angiotensin II. Surprisingly, X/XO significantly augmented coronary response to bradykinin without an effect on responses to ADP, acetylcholine, SNAP, angiotensin I and angiotensin II. In contrast, inhibition of ACE by perindoprilate (100 nM) resulted in augmentation of bradykinin-induced vasodilatation as well as diminution of angiotensin I-evoked vasoconstriction without an influence on other responses. In summary, in the isolated guinea pig heart, X/XO-derived free radicals selectively augmented coronary vasodilator response to bradykinin, which cannot be explained by X/XO- induced derangement of ACE. The mechanism of this paradoxical phenomenon, which might represent a defensive response of the coronary circulation to oxidative stress requires further investigations.
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Induction of NOS-2 in macrophages and smooth muscles within vascular wall with concomittant suppression of endothelial NOS-3 activity is considered to be a hallmark of vascular inflammation that triggers atherogenesis. Accordingly, drugs designed to reverse these changes should not only support vaning function of NOS-3 but also suppress proinflammatory NO production by NOS-2. It means that using selective inhibitors of induction of NOS-2 (they spare ex definitione constitutive activity of NOS-3) is a more rational approach than using isselectivel. inhibitors of activity of previously induced NOS-2. First of all, those drugs are never sufficiently selective. In our work we tried to identify inhibitors of NOS-2 induction within the group of flavonoids, known stimulators of NOS-3 with putative antiatherogenic effects. Representatives of four main groups of flavonoids: flavonols (kaempferol, quercetin, rutin), flavones (apigenin, primuletin), flavanols (catechine) and flavanones (hesperetin, hesperidin, naringenin) were tried on NOS-2 induction and activity in the in vitro model of LPS-treated macrophages (cell line J774.2). While none of these compounds inhibited activity of NOS-2, all with unexpectedly scattered potencies inhibited induction of NOS-2 protein in LPS-treated J774.2 cells, as evidenced by Western blotting technique. Subsequently, RT-PCR and Northern blotting methods revealed that so far the most potent compounds, kaempferol and apigenin, at micromolar concentrations did inhibit NOS-2 induction at the level of NOS-2 gene transcription. We conclude that some of flavonoids are potent inhibitors of NOS-2 induction. At the same time they may increase endothelial NOS-3 activity. Could these flavonoids become natural parents of future drugs, which will be used for reversal of inflammatory component of atherothrombosis?
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Eosinophils accumulation in the airways and sustained eosinophil-derived cysteinyl leukotrienes production represent key elements of the inflammatory response seen in asthma.However,it is not known whether activated epithelial cells influence cysteinyl leukotrienes production by eosinophils from healthy valunteers.The aim of the present study was therefore to analyse the effects of interactions between non-atopic eosinophils and epithelial cells on cysteinyl leukotrienes production in vitro .We measured cysteinyl leukotrienes released by phorbol 12-myristate 13-acetate (PMA)–activated human eosinophils or epithelial cells (human bronchial epithelial cell line -BEAS-2B)cultured alone or together.While activated BEAS-2B cells barely formed leukotrienes (1.39 pg/ml ± 0.2))(n=32),activated eosinophils produced considerable amount of them (62.25 pg/ml ± 10.29))(n=32).Interestingly,when activated eosinophils and epithelial cells were co-incubated,production of cysteinyl leukotrienes increased substantially (571.1 pg/ml ± 80.9))(n=32).Thus,eosinophil-epithelial cell interactions,when occur,are associated with increased biosythesis of cysteinyl leukotrienes.
Activation of both poly (ADP-ribose) polymerase (PARP) and inducible nitric oxide synthase (NOS-2) have been implicated in the pathogenesis of various forms of inflammation, therefore compounds which may simultaneously inhibit both pathways are of potential therapeutic interest. We tested the influence of potent inhibitor of PARP, 1, 5-isoquinolinediol (ISO), on NOS-2 induction in model of mouse macrophages (cell line J774.2) stimulated with lipopolysaccharide (1 µg/ml). Pretreatment with ISO (1-300 µM) resulted in dose-dependent inhibition of accumulation of NOS-2-derived nitrite in culture medium (IC50 = 9,3 µM) as well as inhibition of NOS-2 protein induction in cultured J774.2 cells; ISO given 10 hours after LPS did not influence activity of NOS-2. Interestingly, another PARP inhibitor, 3-aminobenzamide (3-AB, 10-3000 µM), did not influence 24-hr nitrite accumulation in J774.2 cell culture, either administered 15 minutes prior to LPS or 10 hrs after LPS. Scavenging of reactive oxygen species by use of mixture of SOD and catalase (SOD/Cat, 100/300 - 1000/3000 U/ml) as well as cell permeable SOD-mimetic [Mn(III)TBAP, 1- 100 µM], did not influence NOS-2 induction in J774.2 cells. In summary, we identified 1, 5-isoquinoline as potent inhibitor of induction of NOS-2 in LPS-treated mouse macrophages. The exact mechanism of inhibitory action of this compound on NOS-2 induction requires further investigation.
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Our view of renin-angiotensin system (RAS) has changed over the past two decades: new metabolites and pathways have been described; also the importance of local renin-angiotensin systems became more clearly understood. However, there is relatively scarce information about formation and action of angiotensin peptides in gastrointestinal tract, especially in the stomach. Here, using LC-ESI-MS method we assessed the metabolism of Ang I in organ bath of rat stomach wall. Additionally we compared the expression of mRNA of angiotensin converting enzymes (ACE, ACE2) and neprilysin (NEP) in the stomach, aorta and renal artery in rats. Despite, similar levels of expression of ACE and ACE2 mRNA in stomach wall, aorta and renal artery, the absolute amounts of main Ang I metabolites produced by stomach wall (in ng/mg of dry tissue) were much lower than that produced by aorta and renal artery. Also, the pattern of angiotensin I metabolites was different: opposite to aorta and renal artery, incubation of Ang I with stomach wall fragments resulted in predominant formation of Ang-(1-7) and relatively lower production of Ang II. In stomach wall both, perindoprilat and tiorphan decreased production of Ang II, but did not influence generation of Ang-(1-7). In conclusion, we identified Ang-(1-7) as the main product of Ang I conversion in rat stomach wall. The biological role of prevalence of Ang-(1-7) formation in stomach require further investigation.
Alterations in the formation and metabolism of bradykinin (Bk) are hypothesized to play a role in the pathophysiology of hypertension, atherosclerosis and vascular complications of diabetes. However, despite its prominent role in cardiovascular regulation, studies on bradykinin have been limited by various difficulties in accurate measurements of this peptide in biological samples. In this study, using the LC-ESI-MS method we estimated the conversion of exogenous Bk to its main metabolites - Bk-(1-5) and Bk-(1-7) - in endothelial cell culture and in fragments of aorta of normotensive (WKY) and hypertensive rats (SHR). The effects of angiotensin converting enzyme (ACE) and neutral endopeptidase (NEP) inhibitors were more pronounced in SHR: perindoprilat inhibited Bk-(1-5) formation by 49 % and 76 % in WKY and SHR rats, respectively, and tiorphan tended to decrease formation of Bk-(1-5) in both groups of animals. The degradation of bradykinin and generation of both metabolites were significantly higher in the aorta of SHR rats than in WKY controls. Our results show that even in relatively early hypertension (in 4-month old SHR rats) inactivation of Bk by aorta wall is enhanced.
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We have shown that inhibitors of five lipoxygenase activating protein (FLAP) - MK-886 and BAYx1005 inhibit atherosclerosis in apolipoprotein E / LDL receptor - double knockout mice. We, therefore, investigated whether cysteinyl leukotrienes receptor inhibitor - montelukast, given at a dose of 0.125 µg per 100 mg of body weight per day during 16 weeks, could also attenuate atherogenesis. In apoE/LDLR - DKO mouse model montelukast significantly decreased atherogenesis, measured both by "en face" method (25.5±2.% vs. 17.23 ± 1.8%) and "cross-section" method (455 494 ± 26 477 µm2 vs. 299 201 ± 20 373 µm2). The results were, however, less pronounced, comparing to FLAP inhibitors. This is the first report showing the effect of montelukast on atherogenesis in gene-targeted mice.
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Inhibition of angiotensin converting enzyme (ACE) has proved to be beneficial in the treatment of various cardiovascular disorders. The aim of this study was to evaluate ACE inhibitory potential of two polyphenolic compounds with different structures: resveratrol (present in high quantities in French wine) and kaempferol (abundant in greens), using method of liquid chromatography coupled with electrospray ionization mass spectrometry (LC-ESI-MS) for ex vivo measurement of angiotensin I to angiotensin II conversion by ACE in aortic tissue of Wistar-Kyoto rats. In this setting, kaempferol (10-30-100 µM), but not resveratrol (10-30-100 µM) appeared to inhibit dose-dependently conversion of Ang I to Ang II. Although the mechanism of ACE inhibition by kaempferol remains to be elucidated, this observation may help in search or designing of new classes of ACE inhibitors.
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