Cysteinyl leukotriene receptor 1 antagonism prevents experimental abdominal aortic aneurysm.

Cysteinyl-leukotrienes (cys-LTs) are 5-lipoxygenase-derived lipid mediators involved in the pathogenesis and progression of inflammatory disorders, in particular asthma. We have previously found evidence linking these mediators to increased levels of proteolytic enzymes in tissue specimens of human abdominal aortic aneurysm (AAA). Here we show that antagonism of the CysLT1 receptor by montelukast, an established antiasthma drug, protects against a strong aorta dilatation (>50% increase = aneurysm) in a mouse model of CaCl2-induced AAA at a dose comparable to human medical practice. Analysis of tissue extracts revealed that montelukast reduces the levels of matrix metalloproteinase-9 (MMP-9) and macrophage inflammatory protein-1α (MIP-1α) in the aortic wall. Furthermore, aneurysm progression was specifically mediated through CysLT1 signaling since a selective CysLT2 antagonist was without effect. A significantly reduced vessel dilatation is also observed when treatment with montelukast is started days after aneurysm induction, suggesting that the drug not only prevents but also stops and possibly reverts an already ongoing degenerative process. Moreover, montelukast reduced the incidence of aortic rupture and attenuated the AAA development in two additional independent models, i.e., angiotensin II- and porcine pancreatic elastase-induced AAA, respectively. Our results indicate that cys-LTs are involved in the pathogenesis of AAA and that antagonism of the CysLT1 receptor is a promising strategy for preventive and therapeutic treatment of this clinically silent and highly lethal disease.

Neutralizing blood-borne polyphosphate in vivo provides safe thromboprotection.

Polyphosphate is an inorganic procoagulant polymer. Here we develop specific inhibitors of polyphosphate and show that this strategy confers thromboprotection in a factor XII-dependent manner. Recombinant Escherichia coli exopolyphosphatase (PPX) specifically degrades polyphosphate, while a PPX variant lacking domains 1 and 2 (PPX_Δ12) binds to the polymer without degrading it. Both PPX and PPX_Δ12 interfere with polyphosphate- but not tissue factor- or nucleic acid-driven thrombin formation. Targeting polyphosphate abolishes procoagulant platelet activity in a factor XII-dependent manner, reduces fibrin accumulation and impedes thrombus formation in blood under flow. PPX and PPX_Δ12 infusions in wild-type mice interfere with arterial thrombosis and protect animals from activated platelet-induced venous thromboembolism without increasing bleeding from injury sites. In contrast, targeting polyphosphate does not provide additional protection from thrombosis in factor XII-deficient animals. Our data provide a proof-of-concept approach for combating thrombotic diseases without increased bleeding risk, indicating that polyphosphate drives thrombosis via factor XII.

Evaluation of Molecular Markers and Analytical Methods Documenting the Occurrence of Mustard Gas and Arsenical Warfare Agents in Soil.

The chemicals warfare agents (CWAs) are an extremely toxic class of molecules widely produced in many industrialized countries for decades, these compounds frequently contained arsenic. The plants where the CWAs have been produced or the plants where they have been demilitarized after the Second World War with unacceptable techniques can represent a serious environmental problem. CWAs standards are difficult to find on market so in present work an environmental assessment method based on markers has been proposed. Triphenylarsine, phenylarsine oxide and thiodiglycol have been selected as markers. Three reliable analytical methods based on gaschromatography and mass detection have been proposed and tested for quantitative analysis of markers. Methods performance have been evaluated testing uncertainty, linearity, recovery and detection limits and also comparing detection limits with exposure limits of reference CWAs. Proposed assessment methods have been applied to a case study of a former industrial plant sited in an area characterized by a high background of mineral arsenic.

Defective glycosylation of coagulation factor XII underlies hereditary angioedema type III.

Hereditary angioedema type III (HAEIII) is a rare inherited swelling disorder that is associated with point mutations in the gene encoding the plasma protease factor XII (FXII). Here, we demonstrate that HAEIII-associated mutant FXII, derived either from HAEIII patients or recombinantly produced, is defective in mucin-type Thr309-linked glycosylation. Loss of glycosylation led to increased contact-mediated autoactivation of zymogen FXII, resulting in excessive activation of the bradykinin-forming kallikrein-kinin pathway. In contrast, both FXII-driven coagulation and the ability of C1-esterase inhibitor to bind and inhibit activated FXII were not affected by the mutation. Intravital laser-scanning microscopy revealed that, compared with control animals, both F12-/- mice reconstituted with recombinant mutant forms of FXII and humanized HAEIII mouse models with inducible liver-specific expression of Thr309Lys-mutated FXII exhibited increased contact-driven microvascular leakage. An FXII-neutralizing antibody abolished bradykinin generation in HAEIII patient plasma and blunted edema in HAEIII mice. Together, the results of this study characterize the mechanism of HAEIII and establish FXII inhibition as a potential therapeutic strategy to interfere with excessive vascular leakage in HAEIII and potentially alleviate edema due to other causes.

Autocrine activity of cysteinyl leukotrienes in human vascular endothelial cells: Signaling through the CysLT2 receptor.

We evaluated the autocrine activities of cysteinyl leukotrienes (cysteinyl-LTs) in HUVEC and studied the signaling and the pharmacological profile of the CysLT2 receptor (CysLT2R) expressed by ECs, finally assessing the role of the CysLT2R in permeability alterations in a model of isolated brain. Cysteinyl-LTs and their precursor LTA4 contracted HUVEC and increased permeability to macromolecules, increasing the formation of stress fibers through the phosphorylation of myosin light-chain (MLC) following Rho and PKC activation. Accordingly, in an organ model of cerebral vasculature with an intact intima, neutrophils challenge leaded to significant formation of cysteinyl-LTs and edema. Pretreatment with a selective CysLT2R antagonist prevented cytoskeleton rearrangement and HUVEC contraction, along with edema formation in the brain preparation, while leaving the synthesis of cysteinyl-LTs unaffected. We also demonstrate here that the CysLT1R antagonist zafirlukast, pranlukast, pobilukast and iralukast also possess CysLT2R antagonistic activity, which could help in reconsidering previous data on the role of cysteinyl-LTs in the cardiovascular system. The results obtained are further supporting a potential role for CysLT2R in cardiovascular disease.

Plasma contact system activation drives anaphylaxis in severe mast cell-mediated allergic reactions.

BACKGROUND: Anaphylaxis is an acute, potentially lethal, multisystem syndrome resulting from the sudden release of mast cell-derived mediators into the circulation. OBJECTIVES AND METHODS: We report here that a plasma protease cascade, the factor XII-driven contact system, critically contributes to the pathogenesis of anaphylaxis in both murine models and human subjects. RESULTS: Deficiency in or pharmacologic inhibition of factor XII, plasma kallikrein, high-molecular-weight kininogen, or the bradykinin B2 receptor, but not the B1 receptor, largely attenuated allergen/IgE-mediated mast cell hyperresponsiveness in mice. Reconstitutions of factor XII null mice with human factor XII restored susceptibility for allergen/IgE-mediated hypotension. Activated mast cells systemically released heparin, which provided a negatively charged surface for factor XII autoactivation. Activated factor XII generates plasma kallikrein, which proteolyzes kininogen, leading to the liberation of bradykinin. We evaluated the contact system in patients with anaphylaxis. In all 10 plasma samples immunoblotting revealed activation of factor XII, plasma kallikrein, and kininogen during the acute phase of anaphylaxis but not at basal conditions or in healthy control subjects. The severity of anaphylaxis was associated with mast cell degranulation, increased plasma heparin levels, the intensity of contact system activation, and bradykinin formation. CONCLUSIONS: In summary, the data collectively show a role of the contact system in patients with anaphylaxis and support the hypothesis that targeting bradykinin generation and signaling provides a novel and alternative treatment strategy for anaphylactic attacks.

Targeting leukotriene B4 in inflammation.

INTRODUCTION: Leukotriene (LT) B(4) is a powerful proinflammatory lipid mediator and triggers adherence to the endothelium, activates and recruits leukocytes to the site of injury. When formed in excess, LTB(4) plays a pathogenic role and may sustain chronic inflammation in diseases such as asthma, rheumatoid arthritis, and inflammatory bowel disease. Recent investigations have also indicated that LTB(4) is involved in cardiovascular diseases. AREAS COVERED: As the 5-lipoxygenase pathway involves several discrete, tightly coupled, enzymes, which convert the substrate, 'step by step', into bioactive products, several different strategies have been used to target LTB(4) as a means to treat inflammation. Here, we discuss recent findings regarding the development of selective enzyme inhibitors and antagonists for LTB(4) receptors, as well as their application in preclinical and clinical studies. EXPERT OPINION: Components of the 5-lipoxygenase pathway have received considerable attention as candidate drug targets resulting in one new class of medications against asthma, that is, the antileukotrienes. However, efforts to specifically target LTB(4) have not yet been fruitful in the clinical setting, in spite of very promising preclinical data. Recently, crystal structures along with hitherto unknown functions of key enzymes in the leukotriene cascade have emerged, offering new opportunities for drug development and, with time, pharmacological intervention in LTB(4)-mediated pathologies.

Cathelicidin LL-37 induces angiogenesis via PGE2-EP3 signaling in endothelial cells, in vivo inhibition by aspirin.

OBJECTIVE: LL-37, the unique cathelicidin expressed in humans, in addition to acting as an endogenous antibiotic, is an important cell-signaling molecule upregulated in ovarian, breast, and lung tumors. However, the role of LL-37 in tumor microenvironment and its specific actions on the endothelial compartment remain elusive. Prostanoids are key regulators of inflammation, and cyclooxygenases (COXs) display proangiogenic activity in vitro and in vivo, mediated principally through prostaglandin E2 (PGE2). Here, we provide evidence for a novel proangiogenic role of LL-37, exerted via activation of endothelial cells and subsequent PGE2 biosynthesis. APPROACH AND RESULTS: LL-37 triggers PGE2 synthesis in endothelial cells in a dose-dependent manner with maximal induction after 4 hours. Endothelial PGE2 biosynthesis was dependent on COX-1, rather than COX-2, as judged by pharmacological inhibition and gene silencing. In vitro matrigel assays supported these findings because LL-37-induced cord formation was abolished by COX-1, but not COX-2, small interfering RNA, and the angiogenic phenotype could be rescued by addition of exogenous PGE2. We find that LL-37 acts on endothelial cells as a potent calcium agonist, inducing phosphorylation and activation of cytosolic phospholipase A2 (cPLA2), promoting a cPLA2→COX-1→PGE2 biosynthetic pathway and subsequent signaling via PGE2 receptor EP3. Moreover, cathelicidin-related antimicrobial peptide, which is the murine ortholog of LL-37, induced prostaglandin-dependent angiogenesis in vivo, which could be blocked by aspirin. CONCLUSIONS: Our results identify a novel proangiogenic role of LL-37, suggesting that the axis LL-37/COX-1/PGE2 followed by EP3 signaling is amenable to therapeutic intervention in pathological angiogenesis, for instance by aspirin.

The leukotrienes: immune-modulating lipid mediators of disease.

The leukotrienes are important lipid mediators with immune modulatory and proinflammatory properties. Classical bioactions of leukotrienes include chemotaxis, endothelial adherence, and activation of leukocytes, chemokine production, as well as contraction of smooth muscles in the microcirculation and respiratory tract. When formed in excess, these compounds play a pathogenic role in several acute and chronic inflammatory diseases, such as asthma, rheumatoid arthritis, and inflammatory bowel disease. An increasing number of diseases have been linked to inflammation implicating the leukotrienes as potential mediators. For example, recent investigations using genetic, morphological, and biochemical approaches have pointed to the involvement of leukotrienes in cardiovascular diseases including atherosclerosis, myocardial infarction, stroke, and abdominal aortic aneurysm. Moreover, new insights have changed our previous notion of leukotrienes as mediators of inflammatory reactions to molecules that can fine-tune the innate and adaptive immune response. Here, we review the most recent understanding of the leukotriene cascade with emphasis on recently identified roles in immune reactions and pathophysiology.

Increased expression of leukotriene C4 synthase and predominant formation of cysteinyl-leukotrienes in human abdominal aortic aneurysm.

Leukotrienes (LTs) are arachidonic acid-derived lipid mediators involved in the pathogenesis and progression of diverse inflammatory disorders. The cysteinyl-leukotrienes LTC(4), LTD(4), and LTE(4) are important mediators of asthma, and LTB(4) has recently been implicated in atherosclerosis. Here we report that mRNA levels for the three key enzymes/proteins in the biosynthesis of cysteinyl-leukotrienes, 5-lipoxygenase (5-LO), 5-LO-activating protein (FLAP), and LTC(4) synthase (LTC(4)S), are significantly increased in the wall of human abdominal aortic aneurysms (AAAs). In contrast, mRNA levels of LTA(4) hydrolase, the enzyme responsible for the biosynthesis of LTB(4), are not increased. Immunohistochemical staining of AAA wall revealed focal expression of 5-LO, FLAP, and LTC(4)S proteins in the media and adventitia, localized in areas rich in inflammatory cells, including macrophages, neutrophils, and mast cells. Human AAA wall tissue converts arachidonic acid and the unstable epoxide LTA(4) into significant amounts of cysteinyl-leukotrienes and to a lesser extent LTB(4). Furthermore, challenge of AAA wall tissue with exogenous LTD(4) increases the release of matrix metalloproteinase (MMP) 2 and 9, and selective inhibition of the CysLT1 receptor by montelukast blocks this effect. The increased expression of LTC(4)S, together with the predominant formation of cysteinyl-leukotrienes and effects on MMPs production, suggests a mechanism by which LTs may promote matrix degradation in the AAA wall and identify the components of the cysteinyl-leukotriene pathway as potential targets for prevention and treatment of AAA.

Leukotriene B4-induced changes in vascular permeability are mediated by neutrophil release of heparin-binding protein (HBP/CAP37/azurocidin).

In humans, heparin-binding protein (HBP) and the potent chemotactic lipid leukotriene B(4) (LTB(4)) are important mediators of innate immune responses. Here we show that human neutrophils (PMNs) challenged with LTB(4) (30 s to 5 min) release HBP as determined by Western blot analysis. This response peaks at 100 nM of agonist and is mediated by the BLT1 receptor. Protein phosphatase-1 (30 microM) and wortmannin (0.5 microM) block the LTB(4)-mediated HBP release from PMNs, which suggests involvement of the 1-phosphatidylinositol 3-kinase intracellular pathway during degranulation. Furthermore, postsecretory supernatants from LTB(4)-stimulated PMNs induce intracellular calcium mobilization in endothelial cells in vitro and increase in vascular permeability in vivo, as assessed in a mouse model of pleurisy. Selective removal of HBP from the supernatant significantly reduces these activities attributing a key role to HBP in the LTB(4)-induced change in vascular permeability. This lipid-protein axis could offer novel opportunities for pharmacological intervention in key steps of the vascular response to inflammation.

From field to health: a simple way to increase the nutraceutical content of grape as shown by NO-dependent vascular relaxation.

Polyphenolic grapevine components involved in plant resistance against pathogens possess various pharmacological properties that include nitric oxide (NO)-dependent vasodilation and anti-inflammatory and free radical scavenging activities, which may explain the protective effect of moderate red wine consumption against cardiovascular disease. The aim of this work was (a) to verify the possibility that preharvest treatments of grapevine with a plant activator, benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH), could lead to an enriched nutraceutical potential of wine and (b) to characterize the profile of metabolites responsible for pharmacological activity. Plant spraying at the end of veraison, with a water suspension of BTH (0.3 mM), led to increased whole anthocyanin content as confirmed by HPLC comparative analysis. Extracts from berry skins of BTH-treated grapevines caused NO-dependent vasorelaxation, with a concentration-response curve that was significantly shifted to the left of the control non-BTH-treated curve. Moreover, 1:1000 dilutions of berry extracts from BTH-treated plants significantly increased basal production of guanosine 3',5'-cyclic monophosphate (cGMP) in human vascular endothelial cells when compared to the corresponding extracts of untreated plants. These results show that BTH treatment increases anthocyanin content of grape extracts, as well as their ability to induce NO-mediated vasoprotection. No increase of anthocyanin content was observed in the wine extracts from BTH-treated vines. It is concluded that BTH treatment could be exploited to increase the nutraceutical potential of grapes.

Pharmacological characterization of 2NTX-99 [4-methoxy-N1-(4-trans-nitrooxycyclohexyl)-N3-(3-pyridinylmethyl)-1,3-benzenedicarboxamide], a potential antiatherothrombotic agent with antithromboxane and nitric oxide donor activity in platelet and vascular preparations.

Thromboxane (TX) A(2), prostacyclin (PGI(2)), and nitric oxide (NO) regulate platelet function and interaction with the vessel wall. Inhibition of TXA(2), implemented synthesis of PGI(2), and supply of exogenous NO may afford therapeutic benefit. 2NTX-99 [4-methoxy-N(1)-(4-trans-nitrooxycyclohexyl)-N(3)-(3-pyridinylmethyl)-1,3-benzenedicarboxamide], a new chemical entity related to picotamide, showed antithromboxane activity and NO donor properties. 2NTX-99 relaxed rabbit aortic rings precontracted with norepinephrine or U46619 (9,11-dideoxy-9alpha,11alpha-methanoepoxy-prosta-5Z,13E-dien-1-oic acid; EC(50), 7.9 and 17.1 microM, respectively), an effect abolished by 10 microM 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ). 2NTX-99 inhibited arachidonic acid (AA)-induced washed platelet aggregation (EC(50), 9.8 microM) and TXB(2) formation (-71% at 10 microM), and its potency increased in the presence of aortic rings (EC(50), 1.4 microM). In whole rabbit aorta incubated with homologous platelets, AA caused contraction and TXA(2) formation, reduced by 2NTX-99 (10-40 microM): contraction, -28 and -47%, TXA(2) formation, -37 and -75.4%, respectively, with concomitant increase in PGI(2). 2NTX-99 (20-40 microM) inhibited U46619-induced aggregation in rabbit platelet-rich plasma (PRP) (-74 +/- 6.7 and -96 +/- 2.4%, respectively) and inhibited collagen-induced aggregation in human PRP (-48.2 +/- 10 and -79.2 +/- 6%), whereas ozagrel was ineffective. In human embryonic kidney 293 cells transfected with the TXA(2) receptor isophorm alpha receptor, 2NTX-99 did not compete with the ligand, [(3)H]SQ29,548 ([(3)H][1S-[1alpha,2beta(5Z),3beta,4alpha]]-7-[3-[[2-(phenylamino)-carbonyl]hydrazino]methyl]-7-oxabicyclo[2,2,1]-hept-2-yl]-5-heptanoic acid), or prevent inositol phosphate accumulation. After oral administration (50-250 mg/kg), 2NTX-99 inhibited TXA(2) production in rat clotting blood (-71 and -91%); at 250 mg/kg, an area under the curve, 0 to 16 h, of 149.5 h/microg/ml and a t(1/2) of 6 h were calculated, with a C(max) value of 31.8 +/- 8.2 microg/ml. An excellent correlation between plasma concentrations and TXA(2) inhibition occurs. 2NTX-99 controls platelet function and vessel wall interaction by multifactorial mechanisms and possesses therapeutic potential.

Lipid peroxidation and 5-lipoxygenase activity in chronic obstructive pulmonary disease.

We studied the urinary excretion of the isoprostane 8-iso-prostaglandin F(2alpha) as an index of in vivo oxidant stress, and the production of leukotriene (LT) B(4) (LTB(4)) by neutrophils in subjects with chronic obstructive pulmonary disease (COPD) and normal subjects. Overnight urinary excretion of the isoprostane was significantly higher in patients with COPD than in control subjects, and LTB(4) production by challenge of neutrophils obtained from patients with COPD was also significantly higher than that observed in control neutrophils. Treatment with a standardized polyphenol extract caused a significant decrease in isoprostane excretion, accompanied by a statistically significant increase of Pa(O(2)). Furthermore, changes in FEV(1) significantly correlated with the changes in isoprostane urinary excretion observed from enrollment to the end of treatment. The results of this study suggest that enhanced oxidative stress in subjects with COPD is paralleled by the increased ability of neutrophils to synthesize the chemotactic factor LTB(4), and may ultimately contribute to the infiltration/activation of neutrophils into the airways of subjects with COPD. Antioxidant treatment in subjects with COPD is effective in reducing oxidant stress as shown by the decrease of urinary isoprostane, a reduction that correlates with the severity of the disease, as indicated by changes in Pa(O(2)) and FEV(1).

Cysteinyl-leukotrienes receptor activation in brain inflammatory reactions and cerebral edema formation: a role for transcellular biosynthesis of cysteinyl-leukotrienes.

We studied the effect of intravascular activation of human neutrophils on the synthesis of cysteinyl leukotrienes (cysLT) and the formation of cerebral edema in guinea-pig brains. Challenge with the chemotactic formylated tripeptide fMLP (0.1 microM) of neutrophil-perfused brain in vitro resulted in blood-brain barrier disruption associated with a significant increase of cysLT. Both events were completely prevented by neutrophil pretreatment with a specific 5-lipoxygenase (5-LO) inhibitor. Perfusion with the 5-LO metabolite leukotriene B4 (10 nM), together with neutrophils treated with the 5-LO inhibitor, did not restore the alteration in permeability observed upon perfusion with untreated and activated neutrophils. The dual cysLT1-cysLT2 receptor antagonist BAYu9773 was more potent and more effective than a selective cysLT1 antagonist in preventing the brain permeability alteration induced by neutrophil activation. RT-PCR showed significant expression of cysLT2 receptor mRNA in human umbilical vein endothelial cells. Intravital microscopy in mice showed that inhibition of leukotriene synthesis significantly reduced firm adhesion of neutrophils to cerebral vessels without affecting rolling. These data support the hypothesis that neutrophil and endothelial cells cooperate toward the local synthesis of cysLT within the brain vasculature and, acting via the cysLT2 receptor on endothelial cells, may represent a contributing pathogenic mechanism in the development of cerebral inflammation and edema.