Effects of diclofenac and parecoxib treatment on collagen or U46619-induced platelet response.

<p>Diclofenac (1 mg.kg⁻¹), but not parecoxib (0.5 mg.kg⁻¹), significantly reduced the time to peak (panel A) and total peak area (panel C) of the thrombotic response to collagen. Neither diclofenac, nor parecoxib, significantly affected the thrombotic responses to U46619 (panels B and D). Data presented as mean ± SEM, n = 4–9 per treatment group, *p<0.05 by one-way ANOVA and Dunnett's <i>post-hoc</i> test.</p

Effects of diclofenac and parecoxib treatment on COX-1 and COX-2 activity.

<p>Diclofenac (1 mg.kg⁻¹, i.v.), but not parecoxib (0.5 mg.kg⁻¹, i.v.), reduced the formation of TXA₂ in Ca²⁺ ionophore-stimulated whole blood (panel A). Whole blood from mice treated with either diclofenac or parecoxib caused significant inhibition of PGE₂ release from LPS-induced J774 macrophages (panel B). Data presented as mean ± SEM, *p<0.001 by one-way ANOVA and Dunnett's <i>post-hoc</i> test, n = 3.</p

Representative pooled curves of platelet response.

<p>Radioactivity levels were recorded following administration of either collagen (50 µg.kg⁻¹, i.v.; n = 7; upper panel) or U46619 (210 µg.kg⁻¹, i.v.; n = 7; lower panel). Data is represented as % change from baseline (mean ± SEM) and plotted over time.</p

Effect of oral aspirin dosing on platelet response to collagen or U46619.

<p>From 6^th order polynomial regression analysis time to peak and total peak area were calculated for responses to collagen (50 µg.kg⁻¹, i.v.; panels A and C) and U46619 (210 µg.kg⁻¹, i.v.; panels B and D). In comparison to vehicle, aspirin (100 mg.kg⁻¹.day⁻¹ p.o. for 7 days) significantly reduced the time to peak (panel A) and total peak area (panel C). Aspirin had no effect upon responses to U46619 (panels B and D). Data presented as mean ± SEM, n = 6–7 per treatment group, *p<0.05 by one-way ANOVA and Dunnett's <i>post-hoc</i> test.</p

COX-2 Protects against Atherosclerosis Independently of Local Vascular Prostacyclin: Identification of COX-2 Associated Pathways Implicate Rgl1 and Lymphocyte Networks

<div><p>Cyxlo-oxygenase (COX)-2 inhibitors, including traditional nonsteroidal anti-inflammatory drugs (NSAIDs) are associated with increased cardiovascular side effects, including myocardial infarction. We and others have shown that COX-1 and not COX-2 drives vascular prostacyclin in the healthy cardiovascular system, re-opening the question of how COX-2 might regulate cardiovascular health. In diseased, atherosclerotic vessels, the relative contribution of COX-2 to prostacyclin formation is not clear. Here we have used apoE^−/−/COX-2^−/− mice to show that, whilst COX-2 profoundly limits atherosclerosis, this protection is independent of local prostacyclin release. These data further illustrate the need to look for new explanations, targets and pathways to define the COX/NSAID/cardiovascular risk axis. Gene expression profiles in tissues from apoE^−/−/COX-2^−/− mice showed increased lymphocyte pathways that were validated by showing increased T-lymphocytes in plaques and elevated plasma Th1-type cytokines. In addition, we identified a novel target gene, rgl1, whose expression was strongly reduced by COX-2 deletion across all examined tissues. This study is the first to demonstrate that COX-2 protects vessels against atherosclerotic lesions independently of local vascular prostacyclin and uses systems biology approaches to identify new mechanisms relevant to development of next generation NSAIDs.</p></div

COX-2 deletion does not alter prostacyclin production by atherosclerotic vessels.

<p>Production of Prostacyclin and PGE₂ by isolated rings of brachiocephalic artery (a) and aortic arch (b), and from plaque-bearing (c) and plaque-free regions of thoracic aorta (d) from fat-fed apoE^−/−/COX-2^+/+ and apoE^−/−/COX-2^−/− mice demonstrated little role for COX-2 in the production of either prostaglandin in atherosclerotic vessels. In agreement, <i>en face</i> confocal immunofluorescence imaging of the lesser curvature of the aortic arch (b) suggests that whilst both COX-1 and COX-2-like immunoreactivity was present in atherosclerotic vessels, the COX-1 isoform was the predominant isoform. Sequential (Z) scanning through <i>en face</i> vessel preparations suggested COX-2 was more abundant in the endothelial and intraplaque layers than the media. Images are merges of red (COX1 or COX2), green (CD31 – endothelial cells) and blue (DAPI – cell nuclei) channels. n = 4–6.</p

COX-2 deletion increases circulating levels of lymphocytes and lymphocyte-related cytokines in fat-fed apoE^−/− mice.

<p>Measurement of plasma levels of cytokines by multiplex immunoassay (a) demonstrated an increased in the lymphocyte-related cytokines IL-2 and IL-12 (total) and the neutrophil chemoattractant KC, but no change in levels of TNFα, IL-1β, IL-4, IL-10 or IFNγ. Cell counts in whole blood (b), measured in parallel, indicated an increase in circulating lymphocytes, but not monocytes, neutrophils or eosinophils. *; p<0.05 vs apoE^−/−/COX-2^+/+ by unpaired t-test; n = 10–25.</p

Deletion of COX-2 is associated with up-regulation of lymphocyte-related gene expression patterns, and down-regulation of rgl1 in tissue from fat-fed apoE^−/− mice.

<p>Microarray analysis indicated 82, 87 and 90 differentially expressed genes (DEGs), respectively, in thymus (a), liver (b) and lung (c) from fat-fed apoE^−/−/COX-2^+/+ and apoE^−/−/COX-2^−/− mice. Volcano plot summaries of these data sets illustrate the distribution of effect size and statistical significance. Gene expression differing between genotypes >1.2-fold with p<0.05 highlighted in red, were considered for pathway analysis. Differential gene expression patterns demonstrated only weak overlap between tissues, but one gene, rgl1, was altered (down-regulated) in all tissues studied (d). Rgl1 down-regulation was confirmed in each tissue of apoE^−/−/COX-2^−/− mice by qPCR (e). Ingenuity pathway analysis of this data showed that in the thymus (f), liver (g) and lung (h), biological pathways/processes related to T- and/or B-lymphocyte function were amongst the most consistently altered pathways in each tissue. Tables show the top 15 altered biological pathways/processes and implicated genes (green: increased expression; red: decreased expression). Statistical testing of microarray data was performed using a linear model for microarray data modified t-test. n = 4.</p

COX gene expression in tissue from atherosclerotic apoE^−/− mice.

<p>COX-2 (ptgs2) (a) and COX-1 (ptgs1) (b) gene expression levels measured by quantitative RT-PCR in a panel of tissues from atherosclerotic apoE^−/− mice indicated that COX-1 was expressed at a similar level and was the dominant isoform across all studied tissues. COX-2 expression showed more variation across tissues, with the highest levels present in the thymus, which also had the greatest COX-2:COX-1 expression ratio of any tissue examined (c). n = 4-6.</p

COX-2 deletion enhances T-lymphocyte infiltration into the adventitia of atherosclerotic vessels.

<p>Atherosclerotic lesions in the brachiocephalic artery of fat-fed apoE^−/−/COX-2^+/+ and apoE^−/−/COX-2^−/− mice were examined for B- and T-lymphocytes respectively by immunohistochemistry for B220 (a) and CD3 (b). B220+ cells were rarely observed in vessels of either genotype. By contrast, CD3+ cells were present as focal accumulations in the adventitial layer of apoE^−/− mice, and the number and density of CD3+ cells present here was increased by deletion of COX-2. *; p<0.05 vs apoE^−/−/COX-2^+/+ by unpaired t-test; n = 5–10.</p