Altered Colonic Mucosal Polyunsaturated Fatty Acid (PUFA) Derived Lipid Mediators in Ulcerative Colitis: New Insight into Relationship with Disease Activity and Pathophysiology

Objectives

Ulcerative colitis (UC) is a relapsing inflammatory disorder of unconfirmed aetiology, variable severity and clinical course, characterised by progressive histological inflammation and with elevation of eicosanoids which have a known pathophysiological role in inflammation. Therapeutic interventions targetting eicosanoids (5-aminosalicylates (ASA)) are effective first line and adjunctive treatments in mild-moderate UC for achieving and sustaining clinical remission. However, the variable clinical response to 5-ASA and frequent deterioration in response to cyclo-oxygenase (COX) inhibitors, has prompted an in depth simultaneous evaluation of multiple lipid mediators (including eicosanoids) within the inflammatory milieu in UC. We hypothesised that severity of inflammation is associated with alteration of lipid mediators, in relapsing UC.

Design

Study was case-control design. Mucosal lipid mediators were determined by LC-MS/MS lipidomics analysis on mucosal biopsies taken from patients attending outpatients with relapsing UC. Univariate and multivariate statistical analyses were used to investigate the association of mucosal lipid mediators, with the disease state and severity graded histologically.

Results

Levels of PGE₂, PGD₂, TXB₂, 5-HETE, 11-HETE, 12-HETE and 15-HETE are significantly elevated in inflamed mucosa and correlate with severity of inflammation, determined using validated histological scoring systems.

Conclusions

Our approach of capturing inflammatory mediator signature at different stages of UC by combining comprehensive lipidomics analysis and computational modelling could be used to classify and predict mild-moderate inflammation; however, predictive index is diminished in severe inflammation. This new technical approach could be developed to tailor drug treatments to patients with active UC, based on the mucosal lipid mediator profile.     

15-Hydroxyeicosatetraenoic Acid Inhibits Superoxide Anion Generation by Human Neutrophils: Relationship to Lipoxin Production

Human neutrophils can aggregate, degranulate, and release mediators of inflammation including oxygen radicals and lipoxygenase (LO)-derived products of arachidonic acid. The regulation of 5– and 15-lipoxy-genases appears to be important since their products (e.g. leukotrienes and lipoxins) display unique spectra of bioactions. Addition of 15-HETE. a product of the 15-LO, to neutrophils in suspension dramatically shifted the LO products generated and led to a dose-dependent increase in lipoxins, while the production of leukotriene B₄ and its μ-oxidation products (i.e. 20-COOH-LTB₄ and 20-OH-LTB₄) was inhibited. Exogenous 15-HETE also dose-dependently inhibited the generation of superoxide anions induced by either the chemotactic peptide f-met-leu-phe or the divalent cation ionophore A23187. Neither lipoxin A, nor lipoxin B₄ (10^?8?10^?6M) inhibited O₂^?_? generation induced by either f-met-leu-phe or A23187. These results indicate that in addition to serving as a substrate for lipoxin generation, 15-HETE also inhibits superoxide anion generation by human neutrophils. Together they provide further evidence to suggest that products of the 15-lipoxygenase may serve a regulatory role at inflammatory loci.     

Role of leukotrienes and platelet activating factor in gastric mucosal damage and repair.

Gastric mucosal integrity depends upon the balance between "aggressive" factors and "defensive" mechanisms. The formation of mucosal lesions results from the disruption of defense lines, including the breaking of unstirred mucus layer, the reduction of surface hydrophobicity, extensive exfoliation of surface epithelium, penetration of offending agents deeply into the mucosa and damage to the microvessels. The release of proinflammatory and vasoactive mediators such as leukotrienes (LT), thromboxanes, platelet activating factor (PAF), endothelins and others has been thought to be involved in the pathomechanism of mucosal injury, especially damage to the microvascular endothelium, increased vascular permeability, reduction in mucosal blood flow, vascular stasis, tissue ischemia and glandular cell necrosis. This paper reviews the mechanisms and possible pathogenetic implication of two related compounds, LT and PAF in acute mucosal injury by topical irritants such as ethanol, aspirin, bile salts and by stress. LT and PAF arise from similar membrane phospholipids and may regulate the biosynthesis of one another in the damaged mucosa. Although pharmacological studies have clearly demonstrated the noxious effects of cysteinyl LT and PAF on the mucosa, especially when exposed to topical irritants, recent publications have challenged the primary role of these mediators in the pathogenesis of mucosal lesions and ulcerations because the treatment with agents that selectively antagonize their biosynthesis or the receptor sites at the target cells did not always interrupt the chain of events leading to mucosal injury. The role of these mediators in the mucosal repair processes has been little studied but both cysteinyl LT and PAF seem to delay the restitution and healing of the mucosa. Further studies are necessary to clarify to what extent the biosynthesis of LT and PAF and the pharmacological inhibition of their action on the target tissues is related to noxious, protective and reparative events in the mucosa exposed to mild irritants and ulcerogens.     

Role of histamine and platelet-activating factor in allergic rhinitis

This review is focused on the effects of histamine and platelet-activating factor (PAF) in allergic rhinitis and the plausible implications for therapy. Rhinitis is defined as a heterogeneous disorder resulting from an IgE-mediated reaction associated with nasal inflammation of variable intensity. Two phases of response are triggered by an IgE/allergen cross-linking event: the first is the release of preformed mediators such as histamine or interleukins from mast cells and basophils; the second begins when cells start producing lipid-derived mediators. One of these mediators is PAF. Apart from leukotrienes, PAF is perhaps the most potent inflammatory mediator in allergic rhinitis for inducing vascular leakage, a response that may contribute to the appearance of rhinorrhea and nasal congestion.     

Celiac disease

Clinically, celiac disease has always been regarded as a wasting, malabsorptive disorder due to disease of the small intestinal mucosa. It has been difficult for clinicians to recognize that this condition is primarily due to sensitization of mesenteric T lymphocytes to wheat protein (gluten) in genetically predisposed (DQ2⁺) individuals. On contact with dietary-derived gluten in the upper intestine, these sensitized T lymphocytes are activated leading to inflammation of and morphologically altered mucosal architecture: the latter reverts to normal with a gluten-free diet. The circulation of sensitized T lymphocytes to other parts of the intestinal mucosa explains why identical immunopathological inflammation can be induced in ileal and rectal mucosa. It appears, then, that in predisposed DQ2⁺ subjects, measenteric T lymphocytes recognize gluten as foreign (non-self) antigen, thereby inducing mucosal pathology secondary to the intiating lymphocyte-protein interaction, analogously to the mucosal lesions that typify graft-vs-host reactions, or nematode or Giaraia infestations. Today, as this article describes, we recognize that celiac disease often exists in a subclinical, or “compensated-latent,” form, or with symptoms that do not immediately suggest an origin in the gastrointestinal tract.     

On the relationship between leukotriene and lipoxin production by human neutrophils: evidence for differential metabolism of 15-HETE and 5-HETE

Lipoxygenase (LO) products generated by human PMN were examined utilizing a gradient-HPLC and rapid spectral detector which permitted continuous UV-spectral monitoring of leukotrienes, lipoxins and related oxygenated products of arachidonic acid. When exposed to the ionophore A23187, PMN generated LTB4 and its omega-oxidation products as well as LXA4, LXB4, and 7-cis-11-trans-LXA4 from endogenous sources. Addition of 15-HETE changed the profile of products generated by activated PMN and led to a time- and dose-dependent increase in lipoxins and related compounds while the production of LTB4 and its omega-oxidation products was inhibited. Results of time-course and radiolabel studies revealed that 15-HETE is rapidly transformed within 15 s to 5,15-DHETE and conjugated tetraene-containing products, and that the inhibition of leukotriene formation followed a similar time-course. In contrast, PMN did not generate either lipoxins or related products from 5-[3H]HETE, nor did 5-HETE block leukotriene formation. Stimulated PMN generated 5,15-DHETE from exogenous 5-HETE, while in the absence of ionophore, 5-HETE was transformed to 5,20-HETE. These results indicate that PMN can generate lipoxins and related products from endogenous sources and that 15-HETE and 5-HETE are transformed by different routes.     

Epidemiology of coeliac disease in children in one Croatian county: possible factors that could affect the incidence of coeliac disease and adherence to a gluten-free diet (Part II)

Coeliac disease is a life-long disorder characterised by small-intestinal mucosal damage caused by gluten. The treatment, gluten-free diet, leads to mucosal restoration and reduces the risk of malignant and non-malignant complications. According to our recent results, cumulative incidence in Croatian children is 1:519 life-births. Coeliac disease presents early, mostly with typical symptoms. We investigated possible influences of breast-feeding duration and time of gluten introduction on such presentation of the disease. Study included coeliacs born in Medimurje between 1985 and 1994 (N = 31). Patients' adherence to treatment was also studied. Although short breast-feeding (mean 2.9 months) and early gluten introduction (mean 4.9 months) were shown, no correlation was found between those factors and time of the disease presentation. Concerning adherence to gluten-free diet, only 50% of patients maintained it strictly, 23% irregularly, while 27% were on normal diet. In order to improve it, regular follow-ups are recommended.     

Alpha gliadin antibody levels: a serological test for coeliac disease.

The diagnostic value in coeliac disease of circulating antibodies to casein, crude gliadin, and alpha gliadin was assessed using an adaption of the enzyme linked immunosorbent assay system. alpha Gliadin was the only antigen which consistently separated 26 patients with untreated coeliac disease from 26 normal controls and 13 patients with chronic inflammatory bowel disease. The mean assay index for the 26 patients was 3.1 (SD 1.2) compared with 1.05 (0.5) for the normal controls and 1.1 (0.6) for patients with chronic inflammatory bowel disease. The alpha gliadin antibody levels of six patients with coeliac disease who had maintained a gluten free diet for at least two years were not significantly higher than normal (1.0 (0.4)). The validity of the test was determined in 90 consecutive patients who were being investigated for the presence of coeliac disease. Levels of alpha gliadin antibody were raised in 36 out of 44 patients found to have histologically proved coeliac disease and in six out of 46 subjects whose jejunal mucosa was normal. Serial alpha gliadin concentrations were measured in 12 patients with coeliac disease who had repeat jejunal biopsies performed six months after starting a gluten free diet. The levels of antibody fell in seven of the eight patients whose jejunal mucosa improved on maintaining the diet. They remained raised in four patients who did not adhere to the diet and whose mucosa did not improve. Although a test measuring alpha gliadin antibodies is unlikely to replace jejunal biopsy in the diagnosis of coeliac disease it may be useful in screening for the disease among outpatients.     

Tissue transglutaminase in celiac disease: role of autoantibodies

In celiac disease (CD), gluten, the disease-inducing toxic component in wheat, induces the secretion of IgA-class autoantibodies which target tissue transglutaminase (tTG). These autoantibodies are produced in the small-intestinal mucosa, and, during gluten consumption, they can also be detected in patients' serum but disappear slowly from the circulation on a gluten-free diet. Interestingly, after adoption of a gluten-free diet the serum autoantibodies disappear from the circulation more rapidly than the small-intestinal mucosal autoantibody deposits. The finding of IgA deposits on extracellular tTG in the liver, kidney, lymph nodes and muscles of patients with CD indicates that tTG is accessible to the gut-derived autoantibodies. Although the specific autoantibody response directed against tTG is very characteristic in celiac patients, their role in the immunopathology of the celiac mucosal lesion is a matter of debate. Here we report a brief summary of anti-tTG antibody effects demonstrating that these antibodies are functional and not mere bystanders in the disease pathogenesis. In fact, they inhibit intestinal epithelial cell differentiation, induce intestinal epithelial cell proliferation, increase epithelial permeability and activate monocytes and disturb angiogenesis.     

Gastrointestinal inflammation: a central component of mucosal defense and repair

The mucosal layer of the gastrointestinal (GI) tract is able to resist digestion by the endogenous substances that we secrete to digest foodstuffs. So-called "mucosal defense" is multi-factorial and can be modulated by a wide range of substances, many of which are classically regarded as inflammatory mediators. Damage to the GI mucosa, and its subsequent repair, are also modulated by various inflammatory mediators. In this article, we provide a review of some of the key inflammatory mediators that modulate GI mucosal defense, injury, and repair. Among the mediators discussed are nitric oxide, polyamines, the eicosanoids (prostaglandins and lipoxins), protease-activated receptors, and cytokines. Many of these endogenous factors, or the enzymes involved in their synthesis, are considered potential therapeutic targets for the treatment of diseases of the digestive tract that are characterized by inflammation and ulceration.     

Expression of clusterin in Crohn's disease of the terminal ileum

Crohn's disease (CD) is a chronic inflammatory intestinal disorder with disturbance and injury of the intestinal mucosal barrier, in which various proinflammatory molecules as well as molecules with antiinflammatory activity and cytoprotective function are found to be expressed. We investigated whether clusterin, a multifunctional cytoprotective protein, is upregulated in Crohn's disease, because augmented expression of clusterin is seen in many organs following various forms of tissue injury. Human actively and inactively inflamed ileal tissues from CD patients as well as normal intestinal specimens from control patients (normal ileum) were investigated by Western blot analysis, immunohistochemisty and in situ hybridization. As compared with controls, a strongly enhanced expression of clusterin was found in CD tissues, correlating with disease activity. Immunohistochemistry and in situ hybridization analysis revealed foci of crypts almost completely lined by clusterin expressing enterocytes in CD, a feature that was never seen in controls. Such crypts appeared especially within the morphologically intact mucosa apart from erosive or ulcerative lesions. Besides epithelia, clusterin was also expressed by inflammatory mononuclear cells. Enhanced expression of clusterin by crypt epithelia might reflect a cytoprotective function of the protein in order to prevent further injury of the intestinal mucosal barrier in CD.     

Cytokine responses during mucosal infections: role in disease pathogenesis and host defence

Mucosal pathogens use diverse and highly specific molecular mechanisms to activate mucosal inflammation. It may even be argued that their virulence depends on the inflammatory response that they induce. Some bacteria target epithelial cells and trigger them to produce inflammatory mediators but others cross the mucosa and activate macrophages or dendritic cells. Although systemic release of inflammatory mediators causes many symptoms and signs of infection, local chemokine production leads to the recruitment of inflammatory cells and lymphocytes that participate directly in the clearance of bacteria from mucosal sites. In this way, mucosal inflammation is a two-edged sword responsible for disease associated tissue destruction and crucial for the antimicrobial defence. Understanding of these pathways should create tools to enhance the defence and interfere with disease.     

A PPAR-gamma ligand, 15-deoxy-Delta12,14-prostaglandin J(2), inhibited gastric mucosal injury induced by ischemia-reperfusion in rats

INTRODUCTION: Recent studies have demonstrated the anti-inflammatory action of 15-deoxy-Delta12,14-prostaglandin J(2) (15d-PGJ(2)), a derivative of the PGD(2) metabolic pathway. Acute inflammation, including neutrophil activation, plays a critical role in the pathogenesis of ischemia-reperfusion (I/R). The aim of the present study was to determine the effect of 15d-PGJ(2) on I/R-induced gastric mucosal injury in rats.METHODS: Gastric mucosal damage was induced in male Wistar rats by clamping the celiac artery for 30 min followed by reperfusion. 15d-PGJ(2) (0.01-1.0 mg/kg) was given to the rats intraperitoneally 1 h before the vascular clamping. The area of gastric mucosal erosions (erosion index) was measured. Thiobarbituric acid reactive substances (TBARS) and tissue-associated myeloperoxidase (MPO) activity were measured in the gastric mucosa as indices of lipid peroxidation and neutrophil infiltration. The expression of tumor necrosis factor-alpha (TNF-alpha) in gastric mucosa was measured by ELISA. In addition, to elucidate whether the protective effects of 15d-PGJ(2) are related to the activation of the PPAR-gamma receptor, we also investigated the effects of a PPAR-gamma antagonist, GW9662.RESULTS: After 60 min of reperfusion, the area of gastric erosion index had significantly increased from the mean basal levels. The increase in the erosion index was significantly inhibited by pretreatment with 15d-PGJ(2) in a dose-dependent manner. On the other hand, GW9662 reversed the protective effect of 15d-PGJ(2). The concentration of TBARS and MPO activity in the gastric mucosa were both significantly increased after I/R, and pretreatment with 15d-PGJ(2) significantly reduced these increases. The TNF-alpha content was significantly higher in the I/R group than in the sham-operated group. However, the increase in TNF-alpha was significantly inhibited by pretreatment with 15d-PGJ(2).CONCLUSIONS: 15d-PGJ(2) significantly inhibited the severity of acute gastric mucosal injury induced by I/R in rats through PPAR-gamma-dependent mechanisms. This effect may be due, in part, to a reduction in the infiltration of neutrophils into the gastric mucosa, possibly via the inhibition of inflammatory cytokine.     

Metabolomic profiling of regulatory lipid mediators in sputum from adult cystic fibrosis patients

Retained respiratory tract (RT) secretions, infection, and exuberant inflammatory responses are core abnormalities in cystic fibrosis (CF) lung disease. Factors contributing to the destructive CF airway inflammatory processes remain incompletely characterized. The pro-oxidative inflammatory CF RT milieu is known to contain enzymatically and nonenzymatically produced regulatory lipid mediators, a panel of structurally defined oxidized metabolites of polyunsaturated fatty acids known to play a role in pathology related to inflammation. Using an extraction protocol that maximizes recoveries of sputum-spiked deuterated standards, coupled with an LC/MS/MS detection system, this study presents a metabolomic method to assess a broad spectrum of regulatory lipid mediators in freshly obtained sputum from CF patients. A broad range of both proinflammatory and anti-inflammatory lipid mediators was detected, including PGE2, PGD2, TXB2, LTB4, 6-trans-LTB4, 20-OH-LTB4, 20-COOH-LTB4, 20-HETE, 15-HETE, 11-HETE, 12-HETE, 8-HETE, 9-HETE, 5-HETE, EpETrEs, diols, resolvin E1, 15-deoxy-PGJ2, and LXA4. The vast majority of these oxylipins have not been reported previously in CF RT secretions. Whereas direct associations of individual proinflammatory lipid mediators with compromised lung function (FEV-1) were observed, the relationships were not robust. However, multiple statistical analyses revealed that the regulatory lipid mediators profile taken in aggregate proved to have a stronger association with lung function in relatively stable outpatient adult CF patients. Our data reveal a relative paucity of the anti-inflammatory lipid mediator lipoxin A4 in CF sputum. Patients displaying detectable levels of the anti-inflammatory lipid mediator resolvin E1 demonstrated a better lung function compared to those patients with undetectable levels. Our data suggest that comprehensive metabolomic profiling of regulatory lipid mediators in CF sputum should contribute to a better understanding of the molecular mechanisms underlying CF RT inflammatory pathobiology. Further studies are required to determine the extent to which nutritional or pharmacological interventions alter the regulatory lipid mediators profile of the CF RT and the impact of potential modulations of RT regulatory lipid mediators on the clinical progression of CF lung disease.     

Endogenous anti-inflammatory mediators from arachidonate in human neutrophils.

Eicosanoids have been historically involved in the pathogenesis of various inflammatory diseases. Lipoxins (LXs) and epi-LXs show physiological effects relevant to inflammation regulation. In this study, we focused on LX precursors based on the hypothesis that their entrance and metabolism into the cell may facilitate their targeting at the inflammation site. Because compound chirality is of considerable importance in the efficacy of therapeutic agents, our aim was to study the anti-inflammatory effects of various epimers of LXA(4) precursors compared to LXA(4). Blood polymorphonuclear cells (PMNs) were incubated with 15(S)- or 15(R)-hydroxyeicosatetraenoic acid (HETE), 14(R)-,15(S)-, or 14(S),15(S)-diHETE, and LXA(4) and then stimulated with the calcium ionophore A23187. We found that 15(R)-HETE rather than 15(S)-HETE was preferentially metabolized and that 15-epi-LXs were produced in larger amounts than LXs. In contrast, when PMNs were incubated with the diastereoisomers of 14,15(S)-diHETE, 14-epi-LXB(4) was produced in lower amounts than LXB(4). Enantiomers of 15-HETE and diastereoisomers of 14,15-diHETE and LXA(4) were able to significantly decrease LTB(4) release by PMNs. These results suggest a potential resolution of the inflammatory process through endogenous anti-inflammatory mediators released by the way of trans-cellular metabolism.     

Expression of 15-lipoxygenase type-1 in human mast cells

Mast cells play a key role in the pathophysiology of asthma. These cells exert their effector functions by releasing a variety of proinflammatory and immunoregulatory compounds. Mast cells infiltrate the bronchial epithelium and smooth muscle to a higher degree in patients with asthma compared to control subjects. 15-Lipoxygenase type-1 (15-LO-1) is a prooxidant enzyme which is expressed in asthmatic lungs leading to formation of pro- and anti-inflammatory mediators. Here we report that interleukin-4 (IL-4) induced the expression of 15-LO-1 in human cord blood derived mast cells (CBMC) as demonstrated by RT-PCR, western blot and immunocytochemistry. The major metabolite of arachidonic acid formed via the 15-LO pathway in IL-4 treated CBMC was identified as 15-ketoeicosatetraenoic acid (15-KETE, also named 15-oxo-ETE) with smaller amounts of 15-hydroxyeicosatetraenoic acid (15-HETE) as identified by HPLC and mass spectrometry (MS/MS). Furthermore, immunohistochemical stainings demonstrated the expression of 15-LO-1 in mast cells in lung and skin in vivo. Osmotic activation of CBMC with mannitol resulted in activation of the 15-LO-1 pathway. In conclusion, the expression of 15-LO-1 and release of 15-LO-1 derived products by mast cells may contribute to the role of these cells in asthma and other inflammatory diseases.     

Development of a radioimmunoassay for 15-HETE and its application to 15-HETE production by reticulocytes

Mono-hydroxy-eicosatetraenoic acids (HETE's) are frequently the principal lipoxygenase-derived products in a number of cell types. This paper describes the development of a selective and sensitive radioimmunoassay procedure for 15-HETE, a metabolite which has previously been shown to be both an activator and inhibitor of leukotriene formation in various cells. Initially, rabbits were immunized with 15-HETE conjugated to bovine serum albumin. After seven months, the anti-plasma showed significant binding of tritiated 15-HETE (40-45% binding with a 1:600 dilution of the anti-plasma) which was displaceable by cold 15-HETE. The sensitivity of the assay was approximately 20 pg. of 15-HETE. The anti-plasma exhibited very little (less than 1%) cross-reactivity with arachidonic acid, 5-, 8-, 9-, 11- and 12-HETE's, HHT, TXB2, PGE2 and 6-Keto-PGF1 alpha. Significant cross-reactivity was observed with 5,15-diHETE (53%), 8, 15-diHETE (6.6%), and several other 15-hydroxy-eicosanoids. Rabbit reticulocytes have a very active 15S-lipoxygenase and converted arachidonic acid (final concentration 7 microM) principally to 15-HETE. Unstimulated reticulocytes were found to release negligible amounts of 15-HETE as determined by radioimmunoassay. Treatment of these cells with the calcium ionophore A23187 (0.16 to 4.0 micrograms/ml) elicited a level of 15-HETE release (8 - 14 ng/ml) that was twenty to forty times less than that obtained with exogenous arachidonic acid (2.5 micrograms/ml). The radioimmunoassay reported here may be useful for identifying factors which stimulate cellular release of 15-HETE and other 15-hydroxy-eicosanoids from endogenous arachidonic acid.     

Nitric oxide as a mediator of gastrointestinal mucosal injury?—Say it ain't so

Nitric oxide has been suggested as a contributor to tissue injury in various experimental models of gastrointestinal inflammation. However, there is overwhelming evidence that nitric oxide is one of the most important mediators of mucosal defence, influencing such factors as mucus secretion, mucosal blood flow, ulcer repair and the activity of a variety of mucosal immunocytes. Nitric oxide has the capacity to down-regulate inflammatory responses in the gastrointestinal tract, to scavenge various free radical species and to protect the mucosa from injury induced by topical irritants. Moreover, questions can be raised regarding the evidence purported to support a role for nitric oxide in producing tissue injury. In this review, we provide an overview of the evidence supporting a role for nitric oxide in protecting the gastrointestinal tract from injury.     

12/15-Lipoxygenase Mediates High-fat Diet-induced Endothelial Tight Junction Disruption and Monocyte Transmigration: A NEW ROLE FOR 15(S)-HYDROXYEICOSATETRAENOIC ACID IN ENDOTHELIAL CELL DYSFUNCTION*

A convincing body of evidence suggests that 12/15-lipoxygenase (12/15-LO) plays a role in atherosclerosis. However, the mechanisms of its involvement in the pathogenesis of this disease are not clear. Therefore, the purpose of this study is to understand the mechanisms by which 12/15-LO mediates endothelial dysfunction. 15(S)-Hydroxyeicosatetraenoic acid (15(S)-HETE), the major 12/15-LO metabolite of arachidonic acid (AA), induced endothelial barrier permeability via Src and Pyk2-dependent zonula occluden (ZO)-2 tyrosine phosphorylation and its dissociation from the tight junction complexes. 15(S)-HETE also stimulated macrophage adhesion to the endothelial monolayer in Src and Pyk2-dependent manner. Ex vivo studies revealed that exposure of arteries from WT mice to AA or 15(S)-HETE led to Src-Pyk2-dependent ZO-2 tyrosine phosphorylation, tight junction disruption, and macrophage adhesion, whereas the arteries from 12/15-LO knock-out mice are protected from these effects of AA. Feeding WT mice with a high-fat diet induced the expression of 12/15-LO in the arteries leading to tight junction disruption and macrophage adhesion and deletion of the 12/15-LO gene disallowed these effects. Thus, the findings of this study provide the first evidence of the role of 12/15-LO and its AA metabolite, 15(S)-HETE, in high-fat diet-induced endothelial tight junction disruption and macrophage adhesion, the crucial events underlying the pathogenesis of atherosclerosis.