Mechanistic aspects of inducible nitric oxide synthase-induced lung injury in burn trauma.

INTRODUCTION: Although the beneficial effects of inducible nitric oxide synthase (iNOS) inhibition in acute lung injury secondary to cutaneous burn and smoke inhalation were previously demonstrated, the mechanistic aspects are not completely understood. The objective of the present study is to describe the mechanism(s) underlying these favourable effects. We hypothesised that iNOS inhibition prevents formation of excessive reactive nitrogen species and attenuates the activation of poly(ADP) (poly(adenosine diphosphate)) ribose polymerase, thus mitigating the severity of acute lung injury in sheep subjected to combined burn and smoke inhalation. METHODS: Adult ewes were chronically instrumented for a 24-h study and allocated to groups: sham: not injured, not treated, n = 6; control: injured, not treated, n = 6; and BBS-2: injured treated with iNOS dimerisation inhibitor BBS-2, n = 6. Control and BBS-2 groups received 40% total body surface area 3rd-degree cutaneous burn and cotton smoke insufflation into the lungs under isoflurane anaesthesia. RESULTS: Treatment with iNOS inhibitor BBS-2 significantly improved pulmonary gas exchange (partial pressure of oxygen in the blood/fraction of inspired oxygen (PaO2/FiO2) 409 ± 43 mmHg vs. 233 ± 50 mmHg in controls, p < 0.05) and reduced airway pressures (peak pressure 20 ± 1 cm H2O vs. 28 ± 2 cm H2O in controls, p < 0.05) and lung water content (lung wet-to-dry ratio 4.1 ± 0.3 vs. 5.2 ± 0.2 in controls, p < 0.05) 24h after the burn and smoke injury. BBS-2 significantly reduced the increases in lung lymph nitrite/nitrate (10 ± 3 µM vs. 26 ± 6 µM in controls, p < 0.05) and 3-nitrotyrosine (109 ± 11 (densitometry value) vs. 151 ± 18 in controls, p < 0.05). Burn/smoke-induced increases in lung tissue nitrite/nitrate, poly(ADP)ribose polymerase, nuclear factor-κB (NF-κB) activity, myeloperoxidase activity and malondialdehyde formation and interleukin (IL)-8 expression were also attenuated with BBS-2. CONCLUSIONS: The results provide strong evidence that BBS-2 ameliorated acute lung injury by inhibiting the inducible nitric oxide synthase/reactive nitrogen species/poly(ADP-ribose) polymerase (iNOS/RNS/PARP) pathway.

Mechanisms of intimal hyperplasia learned from a murine carotid artery ligation model.

The murine carotid artery ligation (CAL) model has been widely used in the research of intimal hyperplasia, a major pathological process in vascular diseases, such as atherosclerosis and restenosis after angioplasty. Using a variety of gene knockout or transgenic mice and different pharmacological interventions, these studies have yielded significant new findings that contribute not only to unraveling the basic molecular mechanisms involved in the pathogenesis of intimal hyperplasia, but also to the identification of novel targets for intervention of these diseases. The current review outlines the findings derived from the murine CAL model, including studies run by the authors, covering the impacts of hyperlipidemia, pro-inflammatory factors, endothelial dysfunction, protease activity and growth mediators on neointimal hyperplasia.

Lipoxin A4 stable analogs reduce allergic airway responses via mechanisms distinct from CysLT1 receptor antagonism.

Cellular recruitment during inflammatory/immune responses is tightly regulated. The ability to dampen inflammation is imperative for prevention of chronic immune responses, as in asthma. Here we investigated the ability of lipoxin A4 (LXA4) stable analogs to regulate airway responses in two allergen-driven models of inflammation. A 15-epi-LXA4 analog (ATLa) and a 3-oxa-15-epi-LXA4 analog (ZK-994) prevented excessive eosinophil and T lymphocyte accumulation and activation after mice were sensitized and aerosol-challenged with ovalbumin. At <0.5 mg/kg, these LXA4 analogs reduced leukocyte trafficking into the lung by >50% and to a greater extent than equivalent doses of the CysLT1 receptor antagonist montelukast. Distinct from montelukast, ATLa treatment led to marked reductions in cysteinyl leukotrienes, interleukin-4 (IL-4), and IL-10, and both ATLa and ZK-994 inhibited levels of IL-13. In cockroach allergen-induced airway responses, both intraperitoneal and oral administration of ZK-994 significantly reduced parameters of airway inflammation and hyper-responsiveness in a dose-dependent manner. ZK-994 also significantly changed the balance of Th1/Th2-specific cytokine levels. Thus, the ATLa/LXA4 analog actions are distinct from CysLT1 antagonism and potently block both allergic airway inflammation and hyper-reactivity. Moreover, these results demonstrate these analogs' therapeutic potential as new agonists for the resolution of inflammation.

The rational design of inhibitors of nitric oxide formation by inducible nitric oxide synthase.

A series of compounds was rationally designed as inhibitors of dimer formation of the inducible isoform of nitric oxide synthase, and subsequent nitric oxide production. The conformation of two fragments obtained from a crystal structure was utilized to design a tether connecting those same two fragments. The resulting compounds were potent dimerization inhibitors that bound to the enzyme in a similar conformation as the fragments.

Development of a homogeneous time-resolved fluorescence leukotriene B4 assay for determining the activity of leukotriene A4 hydrolase.

Leukotriene A4 (LTA4) hydrolase catalyzes a rate-limiting final biosynthetic step of leukotriene B4 (LTB4), a potent lipid chemotactic agent and proinflammatory mediator. LTB4 has been implicated in the pathogenesis of various acute and chronic inflammatory diseases, and thus LTA4 hydrolase is regarded as an attractive therapeutic target for anti-inflammation. To facilitate identification and optimization of LTA4 hydrolase inhibitors, a specific and efficient assay to quantify LTB4 is essential. This article describes the development of a novel 384-well homogeneous time-resolved fluorescence assay for LTB4 (LTB4 HTRF assay) and its application to establish an HTRF-based LTA4 hydrolase assay for lead optimization. This LTB4 HTRF assay is based on competitive inhibition and was established by optimizing the reagent concentration, buffer composition, incubation time, and assay miniaturization. The optimized assay is sensitive, selective, and robust, with a Z' factor of 0.89 and a subnanomolar detection limit for LTB4. By coupling this LTB4 HTRF assay to the LTA4 hydrolase reaction, an HTRF-based LTA4 hydrolase assay was established and validated. Using a test set of 16 LTA4 hydrolase inhibitors, a good correlation was found between the IC50 values obtained using LTB4 HTRF with those determined using the LTB enzyme-linked immunoassay (R = 0.84). The HTRF-based LTA4 hydrolase assay was shown to be an efficient and suitable assay for determining compound potency and library screening to guide the development of potent inhibitors of LTA4 hydrolase.

The inhibition of inducible nitric oxide synthase in ovine sepsis model.

Excessive NO has been shown to play a major role in the pathogenesis of multiple organ dysfunctions in septic condition. Burn injury, especially if it is associated with smoke inhalation, is often complicated by subsequent development of pneumonia or sepsis that determine the outcome. In the present study, we developed an ovine sepsis model, created by exposing sheep to smoke inhalation followed by instillation of bacteria into the airway, that closely mimics human sepsis and pneumonia. We hypothesized that the inhibition of iNOS-derived excessive NO might be beneficial in treating the cardiopulmonary derangement in this model. Female sheep (n = 18) were surgically prepared for the study and given a tracheostomy. This was followed by insufflation of 48 breaths of cotton smoke (< 40 degrees C) into the airway of each animal and subsequent instillation of live Pseudomonas aeruginosa (5 x 10(11) colony forming units) into each sheep's lung. All sheep were mechanically ventilated using 100% O2. Continuous infusion of BBS-2 (100 microg/kg/h), an iNOS inhibitor, was started 1 h after insult. The administration of BBS-2 improved pulmonary gas exchange (PaO2/FiO2 and pulmonary shunt fraction) and partially reduced airway obstruction and an increase in ventilatory pressures. The lung water content was not affected by iNOS inhibition. The hypotension seen in nontreated animals was not ameliorated either. The increase in plasma concentration of nitrate and nitrite was inhibited by BBS-2. The results of present study show that iNOS may be partially involved in the pathogenesis of acute lung injury induced by smoke inhalation followed by bacterial instillation in the airway.

Lipoxin and synthetic lipoxin analogs: an overview of anti-inflammatory functions and new concepts in immunomodulation.

Lipoxin A4 (LXA4) and lipoxin B4 (LXB4) were first identified in 1984 by Serhan and colleagues as 5- and 15-lipoxygenase interaction products of activated leukocytes. Endogenous transcellular biosynthesis of LXA4 and LXB4 occurs via interaction of leukocytes with epithelium, endothelium or platelets. Acetylation of cyclooxygenase-2 (COX-2) by aspirin can trigger 15-epi-LXA4 (ATL) biosynthesis. Elucidating the pharmacological actions of lipoxins and ATL was facilitated by total synthesis of LXA4 in 1988 by Nicolaou and colleagues. In 1994, Fiore and colleagues used [3H]-LXA4 to identify the cDNA for a human G-protein-coupled, high affinity LXA4 and ATL receptor (ALX-R/FPRL-1), providing the first hints for the molecular basis of lipoxin actions. The recognition that lipoxins and ATL undergo rapid, prostaglandin dehydrogenase (PGDH)-mediated metabolic inactivation led do the design and synthesis of first-generation PGDH-resistant LXA4, LXB4 and ATL analogs in 1995-1998 by Serhan, Petasis and colleagues. These relatively stable pharmacological agents, together with myeloid-specific ALX-R-expressing transgenic mice, have provided powerful tools to explore lipoxin functions in vivo. Here we briefly review the substantial body of evidence supporting the lipoxin --> ALX-R pathway as a novel and potent mechanism for preventing/resolving acute inflammation. Emphasis will also be placed on recent findings that lipoxins play new roles in "immunomodulation" via regulation of macrophage, dendritic cell, and T-lymphocyte effector functions in the setting of polarized T-helper cell responses (Th1 and Th2). These studies suggest roles for lipoxins as novel regulators of allergy and adaptive immunity and that lipoxins may have therapeutic potential in chronic immune disorders.

Visualizing inducible nitric-oxide synthase in living cells with a heme-binding fluorescent inhibitor.

The study of nitric-oxide synthase (NOS) physiology is constrained by the lack of suitable probes to detect NOS in living cells or animals. Here, we characterized a fluorescent inducible NOS (iNOS) inhibitor called PIF (pyrimidine imidazole FITC) and examined its utility for microscopic imaging of iNOS in living cells. PIF binding to iNOS displayed high affinity, isoform selectivity, and heme specificity, and was essentially irreversible. PIF was used to successfully image iNOS expressed in RAW264.7 cells, HEK293T cells, human A549 epithelial cells, and freshly obtained human lung epithelium. PIF was used to estimate a half-life for iNOS of 1.8 h in HEK293T cells. Our work reveals that fluorescent probes like PIF will be valuable for studying iNOS cell biology and in understanding the pathophysiology of diseases that involve dysfunctional iNOS expression.

Second-generation beta-oxidation resistant 3-oxa-lipoxin A4 analogs.

Lipoxin A4 (LXA4) and aspirin-triggered 15-epi-LXA4 are structurally and functionally distinct eicosanoids, with potent anti-inflammatory and immunomodulatory actions. Therapeutic use of LXA4 is greatly limited by its rapid metabolism in vivo and chemical instability. First-generation synthetic LXA4 analogs such as methyl (5R,6R,7E,9E,11Z,13E,15S)-16-(4-fluorophenoxy)-5,6,15-trihydroxy-7,9,11,13-hexadecatetraenoate (2, ATLa), were designed to minimize metabolism from the omega-end of the molecule. Pharmacokinetic analysis of ATLa revealed beta-oxidation as a novel route for LXA4 metabolism, prompting the development of second-generation 3-oxa-LXA4 analogs with improved pharmacokinetic disposition. Second-generation 3-oxa-LXA4 analogs such as (5R,6R,7E,9E,11Z,13E,15S)-16-(4-fluorophenoxy)-3-oxa-5,6,15-trihydroxy-7,9,11,13-hexadecatetraenoic acid (3), have shown potency and efficacy comparable to ATLa in diverse animal models after topical, intravenous or oral delivery. These include several acute (2-24 h) inflammatory reactions: calcium ionophore-induced skin edema and inflammation (topical), LTB4/PGE2-induced skin inflammation and vascular leak (topical), zymosan A-induced peritonitis (i.v. and oral) and ischemia-reperfusion-induced secondary organ injury (i.v.). Remarkably, 3-oxa-LXA4 analogs have potent once daily oral efficacy in preventing and promoting the resolution of established colitis induced by the hapten trinitrobenzene sulphonic acid (TNBS), an acute/chronic 7-14-day model of Crohn's disease. The second-generation 3-oxa-LXA4 analogs thus provide new stable pharmacophores with which to explore the emerging role of lipoxins as a new therapeutic principle for regulating inflammation, allergy and immune dysfunction in preclinical and clinical research.

A beta-oxidation-resistant lipoxin A4 analog treats hapten-induced colitis by attenuating inflammation and immune dysfunction.

Lipoxins and aspirin-triggered 15-epi-lipoxins (ATL) are counter-regulatory eicosanoids with potent antiinflammatory actions. Oral efficacy and mechanism of action of ZK-192, a beta-oxidation-resistant 3-oxa-ATL analog, were examined in trinitrobenzenesulphonate (TNBS)-induced colitis. When dosed orally once daily, 300 and 1,000 mug/kg ZK-192 markedly attenuated TNBS colitis in rodents both in preventive and therapeutic regimens. ZK-192 attenuated weight loss, macroscopic and histologic colon injury, mucosal neutrophil infiltration, and colon wall thickening. ZK-192 was as effective as 3-10 mg/kg oral prednisolone. ZK-192 decreased mucosal mRNA levels for several inflammatory mediators: inducible nitric oxide synthase, cyclooxygenase 2, and macrophage inflammatory protein 2. ZK-192 also decreased mucosal mRNA and protein levels of T helper 1 effector cytokines: tumor necrosis factor alpha, IL-2, and IFN-gamma. Systemic levels of these cytokines were also dramatically attenuated. CD3/CD28-mediated costimulation of T helper 1 effector cytokine release in lamina propria mononuclear cells was markedly inhibited by ZK-192 ex vivo and in vitro. ZK-192 also prevented colitis in lymphocyte-deficient severe combined immunodeficient mice, with approximately 75% inhibition of mucosal tumor necrosis factor alpha and IL-2 levels. The results are further evidence that innate immune cells function as triggers for hapten-induced colitis. The combined antiinflammatory and immunomodulatory effects of ZK-192 in TNBS colitis suggest that ATL analogs may be an attractive oral treatment approach for inflammatory bowel diseases.

Lipoxins and novel 15-epi-lipoxin analogs display potent anti-inflammatory actions after oral administration.

1. Lipoxins (LX) and aspirin-triggered 15-epi-lipoxins (ATL) exert potent anti-inflammatory actions. In the present study, we determined the anti-inflammatory efficacy of endogenous LXA(4) and LXB(4), the stable ATL analog ATLa2, and a series of novel 3-oxa-ATL analogs (ZK-996, ZK-990, ZK-994, and ZK-142) after intravenous, oral, and topical administration in mice. 2. LXA(4), LXB(4), ATLa2, and ZK-994 were orally active, exhibiting potent systemic inhibition of zymosan A-induced peritonitis at very low doses (50 ng kg(-1)-50 microg kg(-1)). 3. Intravenous ZK-994 and ZK-142 (500 microg kg(-1)) potently attenuated hind limb ischemia/reperfusion-induced lung injury, with 32+/-12 and 53+/-5% inhibition (P<0.05), respectively, of neutrophil accumulation in lungs. The same dose of ATLa2 had no significant protective action. 4. Topical application of ATLa2, ZK-994, and ZK-142 ( approximately 20 microg cm(-2)) prevented vascular leakage and neutrophil infiltration in LTB(4)/PGE(2)-stimulated ear skin inflammation. While ATLa2 and ZK-142 displayed approximately equal anti-inflammatory efficacy in this model, ZK-994 displayed a slower onset of action. 5. In summary, native LXA(4) and LXB(4), and analogs ATLa2, ZK-142, and ZK-994 retain broad anti-inflammatory effects after intravenous, oral, and topical administration. The 3-oxa-ATL analogs, which have enhanced metabolic and chemical stability and a superior pharmacokinetic profile, provide new opportunities to explore the actions and therapeutic potential for LX and ATL.

Novel 3-oxa lipoxin A4 analogues with enhanced chemical and metabolic stability have anti-inflammatory activity in vivo.

Lipoxin A(4) (LXA(4)) is a structurally and functionally distinct natural product called an eicosanoid, which displays immunomodulatory and anti-inflammatory activity but is rapidly metabolized to inactive catabolites in vivo. A previously described analogue of LXA(4), methyl (5R,6R,7E,9E,11Z,13E,15S)-16-(4-fluorophenoxy)-5,6,15-trihydroxy-7,9,11,13-hexadecatetraenoate (2, ATLa), was shown to have a poor pharmacokinetic profile after both oral and intravenous administration, as well as sensitivity to acid and light. The chemical stability of the corresponding E,E,E-trien-11-yne analogue, 3, was improved over 2 without loss of efficacy in the mouse air pouch model of inflammation. Careful analysis of the plasma samples from the pharmacokinetic assays for both 2 and 3 identified a previously undetected metabolite, which is consistent with metabolism by beta-oxidation. The formation of the oxidative metabolites was eliminated with the corresponding 3-oxatetraene, 4, and the 3-oxatrien-11-yne, 5, analogues of 2. Evaluation of 3-oxa analogues 4 and 5 in calcium ionophore-induced acute skin inflammation model demonstrated similar topical potency and efficacy compared to 2. The 3-oxatrien-11-yne analogue, 5, is equipotent to 2 in an animal model of inflammation but has enhanced metabolic and chemical stability and a greatly improved pharmacokinetic profile.

A selective inducible NOS dimerization inhibitor prevents systemic, cardiac, and pulmonary hemodynamic dysfunction in endotoxemic mice.

Increased nitric oxide (NO) production by inducible NO synthase (NOS2), an obligate homodimer, is implicated in the cardiovascular sequelae of sepsis. We tested the ability of a highly selective NOS2 dimerization inhibitor (BBS-2) to prevent endotoxin-induced systemic hypotension, myocardial dysfunction, and impaired hypoxic pulmonary vasoconstriction (HPV) in mice. Mice were challenged with Escherichia coli endotoxin before treatment with BBS-2 or vehicle. Systemic blood pressure was measured before and 4 and 7 h after endotoxin challenge, and echocardiographic parameters of myocardial function were measured before and 7 h after endotoxin challenge. The pulmonary vasoconstrictor response to left mainstem bronchus occlusion, which is a measure of HPV, was studied 22 h after endotoxin challenge. BBS-2 treatment alone did not alter baseline hemodynamics. BBS-2 treatment blocked NOS2 dimerization and completely inhibited the endotoxin-induced increase of plasma nitrate and nitrite levels. Treatment with BBS-2 after endotoxin administration prevented systemic hypotension and attenuated myocardial dysfunction. BBS-2 also prevented endotoxin-induced impairment of HPV. In contrast, treatment with NG-nitro-l-arginine methyl ester, which is an inhibitor of all three NOS isoforms, prevented the systemic hypotension but further aggravated the myocardial dysfunction associated with endotoxin challenge. Treatment with BBS-2 prevented endotoxin from causing key features of cardiovascular dysfunction in endotoxemic mice. Selective inhibition of NOS2 dimerization with BBS-2, while sparing the activities of other NOS isoforms, may prove to be a useful treatment strategy in sepsis.

The inducible nitric oxide synthase inhibitor BBS-2 prevents acute lung injury in sheep after burn and smoke inhalation injury.

In this study we examined the role of inducible nitric oxide synthase (iNOS) in acute respiratory distress syndrome (ARDS) in sheep with severe combined burn and smoke inhalation injury. BBS-2, a potent and highly selective iNOS dimerization inhibitor, was used to exclude effects on the endothelial and neuronal NOS isoforms. Seven days after surgical recovery, sheep were given a burn (40% of total body surface, 3rd degree) and insufflated with cotton smoke (48 breaths, < 40 degrees C) under anesthesia. BBS-2 was provided by constant infusion at 100 microg/kg/hour, beginning 1 hour after injury. During 48 hours, control sheep developed multiple signs of ARDS. These included decreased pulmonary gas exchange, increased pulmonary edema, abnormal lung compliance, and extensive airway obstruction. These pathologies were associated with a large increase in tracheal blood flow and elevated plasma NO2-/NO3- (NOx) levels. These variables were all stable in sham animals. Treatment of injured sheep with BBS-2 attenuated the increases in tracheal blood flow and plasma NOx levels, and significantly attenuated all the pulmonary pathologies that were noted. The results provide definitive evidence that iNOS is a key mediator of pulmonary pathology in sheep with ARDS resulting from combined burn and smoke inhalation injury.

Pharmacological interference with dimerization of human neuronal nitric-oxide synthase expressed in adenovirus-infected DLD-1 cells.

A recombinant adenovirus containing the cDNA of human neuronal nitric-oxide synthase (nNOS) was constructed to characterize the interaction of nNOS with N-[(1,3-benzodioxol-5-yl)methyl]-1-[2-(1H-imidazole-1-yl)pyrimidin-4-yl]-4-(methoxycarbonyl)-piperazine-2-acetamide (BBS-1), a potent inhibitor of inducible NOS dimerization [Proc Natl Acad Sci USA 97:1506-1511, 2000]. BBS-1 inhibited de novo expression of nNOS activity in virus-infected cells at a half-maximal concentration (IC(50)) of 40 +/- 10 nM in a reversible manner. Low-temperature gel electrophoresis showed that BBS-1 attenuated the formation of SDS-resistant nNOS dimers with an IC(50) of 22 +/- 5.2 nM. Enzyme inhibition progressively decreased with increasing time of addition after infection. BBS-1 did not significantly inhibit dimeric nNOS activity (IC(50) > 1 mM). Long-term incubation with BBS-1 of human embryonic kidney cells stably transfected with nNOS or endothelial NOS revealed a slow time- and concentration-dependent decrease of NOS activity with half-lives of 30 and 43 h and IC(50) values of 210 +/- 30 nM and 12 +/- 0.5 microM, respectively. These results establish that BBS-1 interferes with the assembly of active nNOS dimers during protein expression. Slow inactivation of constitutively expressed NOS in intact cells may reflect protein degradation and interference of BBS-1 with the de novo synthesis of functionally active NOS dimers. As time-dependent inhibitors of NOS dimerization, BBS-1 and related compounds provide a promising strategy to develop a new class of selective and clinically useful NOS inhibitors.

An aspirin-triggered lipoxin A4 stable analog displays a unique topical anti-inflammatory profile.

Lipoxins and 15-epi-lipoxins are counter-regulatory lipid mediators that modulate leukocyte trafficking and promote the resolution of inflammation. To assess the potential of lipoxins as novel anti-inflammatory agents, a stable 15-epi-lipoxin A(4) analog, 15-epi-16-p-fluorophenoxy-lipoxin A(4) methyl ester (ATLa), was synthesized by total organic synthesis and examined for efficacy relative to a potent leukotriene B(4) (LTB(4)) receptor antagonist (LTB(4)R-Ant) and the clinically used topical glucocorticoid methylprednisolone aceponate. In vitro, ATLa was 100-fold more potent than LTB(4)R-Ant for inhibiting neutrophil chemotaxis and trans-epithelial cell migration induced by fMLP, but was approximately 10-fold less potent than the LTB(4)R-Ant in blocking responses to LTB(4). A broad panel of cutaneous inflammation models that display pathological aspects of psoriasis, atopic dermatitis, and allergic contact dermatitis was used to directly compare the topical efficacy of ATLa with that of LTB(4)R-Ant and methylprednisolone aceponate. ATLa was efficacious in all models tested: LTB(4)/Iloprost-, calcium ionophore-, croton oil-, and mezerein-induced inflammation and trimellitic anhydride-induced allergic delayed-type hypersensitivity. ATLa was efficacious in mouse and guinea pig skin inflammation models, exhibiting dose-dependent effects on edema, neutrophil or eosinophil infiltration, and epidermal hyperproliferation. We conclude that the LXA(4) and aspirin-triggered LXA(4) pathways play key anti-inflammatory roles in vivo. Moreover, these results suggest that ATLa and related LXA(4) analogs may have broad therapeutic potential in inflammatory disorders and could provide an alternative to corticosteroids in certain clinical settings.

Lipoxin a4 analogs attenuate induction of intestinal epithelial proinflammatory gene expression and reduce the severity of dextran sodium sulfate-induced colitis.

The anti-inflammatory eicosanoid lipoxin A(4) (LXA(4)), aspirin-triggered 15-epi-LXA(4), and their stable analogs down-regulate IL-8 secretion and subsequent recruitment of neutrophils by intestinal epithelia. In an effort to elucidate the mechanism by which these lipid mediators modulate cellular proinflammatory programs, we surveyed global epithelial gene expression using cDNA microarrays. LXA(4) analog alone did not significantly affect expression of any of the >7000 genes analyzed. However, LXA(4) analog pretreatment attenuated induction of approximately 50% of the 125 genes up-regulated in response to the gastroenteritis-causing pathogen Salmonella typhimurium. A major subset of genes whose induction was reduced by LXA(4) analog pretreatment is regulated by NF-kappaB, suggesting that LXA(4) analog was influencing the activity of this transcription factor. Nanomolar concentrations of LXA(4) analog reduced NF-kappaB-mediated transcriptional activation in a LXA(4) receptor-dependent manner and inhibited induced degradation of IkappaBalpha. LXA(4) analog did not affect earlier stimulus-induced signaling events that lead to IkappaBalpha degradation, such as S. typhimurium-induced epithelial Ca(2+) mobilization or TNF-alpha-induced phosphorylation of IkappaBalpha. To establish the in vivo relevance of these findings, we examined whether LXA(4) analogs could affect intestinal inflammation in vivo using the mouse model of DSS-induced inflammatory colitis. Oral administration of LXA(4) analog (15-epi-16-para-fluoro-phenoxy-LXA(4), 10 microg/day) significantly reduced the weight loss, hematochezia, and mortality that characterize DSS colitis. Thus, LXA(4) analog-mediated down-regulation of proinflammatory gene expression via inhibition of the NF-kappaB pathway can be therapeutic for diseases characterized by mucosal inflammation.

Mechanistic studies with potent and selective inducible nitric-oxide synthase dimerization inhibitors.

A series of potent and selective inducible nitric-oxide synthase (iNOS) inhibitors was shown to prevent iNOS dimerization in cells and inhibit iNOS in vivo. These inhibitors are now shown to block dimerization of purified human iNOS monomers. A 3H-labeled inhibitor bound to full-length human iNOS monomer with apparent Kd approximately 1.8 nm and had a slow off rate, 1.2 x 10(-4) x s(-1). Inhibitors also bound with high affinity to both murine full-length and murine oxygenase domain iNOS monomers. Spectroscopy and competition binding with imidazole confirmed an inhibitor-heme interaction. Inhibitor affinity in the binding assay (apparent Kd values from 330 pm to 27 nm) correlated with potency in a cell-based iNOS assay (IC50 values from 290 pm to 270 nm). Inhibitor potency in cells was not prevented by medium supplementation with l-arginine or sepiapterin, but inhibition decreased with time of addition after cytokine stimulation. The results are consistent with a mechanism whereby inhibitors bind to a heme-containing iNOS monomer species to form an inactive iNOS monomer-heme-inhibitor complex in a pterin- and l-arginine-independent manner. The selectivity for inhibiting dimerization of iNOS versus endothelial and neuronal NOS suggests that the energetics and kinetics of monomer-dimer equilibria are substantially different for the mammalian NOS isoforms. These inhibitors provide new research tools to explore these processes.