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[PubMed] [Google Scholar] 8. one of these amino acid residues prevented its dissociation from occludin and restored TJ integrity and barrier function. In response to high-fat diet (HFD) feeding, WT, but not 12/15-lipoxygenase (LO)?/?, mice showed enhanced XO expression and its activity in the artery, which was correlated with increased aortic TJ disruption and barrier permeability with enhanced leukocyte adhesion and these responses were inhibited by allopurinol. These observations provide novel insights on the role of XO in 12/15-LO-induced JamA tyrosine phosphorylation and TJ disruption leading to increased vascular permeability in response to HFD. for 10 min, the white cell pellet was resuspended in PBS. Then, an equal volume of Lymphoprep solution was Lathyrol laid over the cell suspension and centrifuged at 800 for 23 min. The leukocyte-enriched layer was collected and resuspended in PBS followed by labeling with BCECF-AM. Aortas from WT and 12/15-LO?/? mice were dissected out, cleaned from connective and fat tissue, and treated with and without 15-(S)-HETE (0.5 M) in the presence and absence of the indicated inhibitors for 30 min. The aortas were then opened longitudinally and the BCECF-AM-labeled leukocytes (5.6 105 cells /ml) were added in serum-free medium and incubated for SLIT1 1 h at room temperature. The aortas were fixed with 3% paraformaldehyde for 30 min followed by 0.2% picric acid for 1 h, permeabilized in TBS [10 mM Tris-HCl and 150 mM NaCl (pH 8.0)] containing 3% BSA and 0.2% Triton X-100 for 10 min, blocked in 3% BSA for 1 h at room temperature, and probed with rabbit anti-JamA antibodies followed by Alexa Fluor 568-conjugated goat anti-rabbit secondary antibodies. In another set of experiments, aortas were isolated from both WT and 12/15-LO?/? mice that were on CD or HFD for 3 months, fixed and stained for JamA and Lathyrol leukocytes using rabbit anti-JamA and mouse anti-CD45 antibodies, followed by Alexa Fluor 568-conjugated goat anti-rabbit and Alexa Fluor 488-conjugated goat anti-mouse secondary antibodies. Fluorescence images of the luminal side of aortas were captured and quantified as described above in immunofluorescence staining of HUVECs. Miles assay The level of vascular permeability was determined by quantitative measurement of the Evans Blue dye diffusion into the aorta according to Miles test (24). WT and 12/15-LO?/? mice that were on either CD or HFD for 3 months were anesthetized, and 0.1 ml of 1% Evans Blue dye was injected into the inferior vena cava. After 30 min, blood vessels were perfused with PBS through the left ventriculum and aortas were isolated. Evans Blue dye was extracted from the arteries by incubating in formaldehyde at 55C for 24 h, cleared by centrifugation and the optical density was measured at 610 nm. Lathyrol Vascular permeability was expressed as the amount of Evans Blue extravasated per milligram of artery. Statistics All the experiments were performed three times and the data are presented as mean SD. The treatment effects were analyzed by one-way ANOVA followed by Students values 0.05 were considered to be statistically significant. RESULTS 15(S)-HETE induces tyrosine phosphorylation of JamA in the disruption of EC TJs Loss of barrier function is a hallmark of endothelial dysfunction (1, 2, 6). TJs play a crucial role in the maintenance of EC barrier function (3, 4). Previously, we have reported that 15(S)-HETE increases EC barrier permeability by promoting threonine and tyrosine phosphorylation of ZO1 and ZO2, respectively, and their dissociation from TJ complexes (20, 21). In the present study, we examined the role of transmembrane proteins, JamA and occludin, that are present in the TJ complexes in 15(S)-HETE-induced EC barrier disruption. First, we tested the effect of 15(S)-HETE on the steady state levels of JamA and occludin. 15(S)-HETE did not affect the steady state levels of either JamA or occludin (Fig. 1A). We then tested the effect of 15(S)-HETE on their tyrosine phosphorylation. 15(S)-HETE stimulated tyrosine phosphorylation of both JamA and occludin in a time-dependent manner with maximum increase between 5.