COX-2 inhibitor treatment initiated 1 week after beginning AngII infusion effectively reduces AAA progression in ApoE-deficient mice.

<p>(A) AAA incidence was determined at necropsy following 1 (1 Wk AngII) or 6 weeks (Control and Celecoxib) of AngII infusion. (B) The maximal external diameter or (C) AAA classification of the suprarenal aorta following 1 (1 Wk AngII) or 6 weeks (Control and Celecoxib) of AngII infusion. (D) Incidence of aortic rupture in mice on control or celecoxib containing diet following 6 weeks of AngII infusion. (E) Survival of Control and Celecoxib-treaded mice during the AngII infusion. (F) Representative image of postmortem detection of hemorrhage in the abdominal cavity indicating aortic rupture. For aortic diameter, results depict mean ± SEM. AAA incidence and diameter: 1 Wk AngII <i>n</i> = 10, Control <i>n</i> = 11, Celecoxib <i>n</i> = 18; aortic rupture: Control <i>n</i> = 15, Celecoxib <i>n</i> = 18. <i>P</i> values were determined for incidence data using Fisher's exact test and an unpaired two-tailed <i>t</i>-test for aortic diameter. * indicates <i>P</i><0.05, ** indicates <i>P</i><0.01, *** indicates <i>P</i><0.001.</p

Immunohistochemical analysis of markers of smooth muscle cell differentiation and de-differentiation.

<p>(A) α-actin expressing smooth muscle cells localize to the side of the abdominal aorta involved in aneurysmal remodeling (100× magnification). (B) As compared to the non-involved aorta, α-actin expression is more diffuse (arrow) in the SMCs involved in aneurysmal remodeling (200× magnification). (C) α-actin expression is localized to SMCs within the medial layer and to SMCs outside of the external elastic lamina (400× magnification). (D) Hyaluronic acid expression is localized throughout the AAA and in cells within the medial side of the external elastic lamina. The boxed area is shown within the insert at 400× magnification. Brown staining from DAB indicates α-actin or hyaluronic acid detection and sections are counterstained with hematoxylin (blue).</p

COX-2 inhibitor treatment initiated 3 weeks after beginning AngII infusion effectively reduces AAA progression in ApoE-deficient mice.

<p>(A) AAA incidence was determined at necropsy following 3 (3 Wks AngII) or 8 weeks (Control and Celecoxib) of AngII infusion. (B) The maximal external diameter or (C) AAA classification of the suprarenal aorta following 3 (3 Wks AngII) or 8 weeks (Control and Celecoxib) of AngII infusion. For aortic diameter, results depict mean ± SEM. AAA incidence and diameter: 3 Wks AngII <i>n</i> = 12, Control <i>n</i> = 13, Celecoxib <i>n</i> = 14. <i>P</i> values were determined for incidence data using Fisher's exact test and an unpaired two-tailed <i>t</i>-test for aortic diameter. * indicates <i>P</i><0.05.</p

COX-2 inhibitor treatment maintains mRNA expression of markers of differentiated smooth muscle.

<p>Real-time PCR quantitation of (A) α-actin or (B) hyaluronic acid synthase-2 mRNA expression in the abdominal aorta. Mice were provided control or celecoxib containing diet beginning 1 week after initiating a 6-week AngII infusion. (C) Quantitation of α-actin mRNA expression in the abdominal aorta of mice provided control or celecoxib containing diet beginning 3 weeks after initiating an 8-week AngII infusion. Expression levels of the gene of interest were normalized to HPRT mRNA levels. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044369#s3" target="_blank">Results</a> indicate mean ± SEM and the number of mice in each group is provided within the graphs. <i>P</i> values were determined using an unpaired two-tailed <i>t</i>-test. * indicates <i>P</i><0.05.</p

COX-2 inhibitor treatment does not alter macrophage-dependent inflammation.

<p>Abdominal aorta was collected at necropsy for control or celecoxib-treated mice beginning 1 week after initiating a 6-week AngII infusion. Real-time PCR quantitation of mRNA expression of (A) the macrophage marker CD68, (B) the monocyte recruitment chemokine MCP-1, (C) the proinflammatory cytokine TNFα, (D–E) metalloproteinases (MMPs) −2 and −9, and (F) the mast cell marker CD34. Expression levels of the gene of interest were normalized to HPRT mRNA levels. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044369#s3" target="_blank">Results</a> indicate mean ± SEM and the number of mice in each group is provided within the graphs. <i>P</i> values were determined using an unpaired two-tailed <i>t</i>-test. * indicates <i>P</i><0.05.</p

Histological analysis of the abdominal aorta during aneurysm progression.

<p>(A) Analysis of abdominal aorta containing a hematoma following histochemical staining by H&E. (B) Immunohistochemical analysis of COX-2 expression in abdominal aorta adjacent to hematoma. COX-2 expression in cells of the outer layers of a well-defined tunica media. Arrows indicate medial cells with concentrated expression of COX-2 surrounding individual nuclei. (C) COX-2 expression in abdominal aorta with adventitial aneurysmal remodeling. COX-2 expression is concentrated on the side of the aorta with aneurysmal remodeling without detectable expression on the non-involved side of the aorta (100× magnification). (D) COX-2 expression in cells just outside of the medial layer into a region between the media and the remodeled adventitia (200× magnification). (E) Cells with the greatest COX-2 expression are localized on the outer side of the external elastic lamina of the tunica media (400× magnification). (F) Immunohistochemical analysis of COX-2 expression in abdominal aorta without aneurysmal pathology (400× magnification). Arrows indicate regions with concentrated COX-2 expression. Brown staining from DAB indicates COX-2 and sections are counterstained with hematoxylin (blue).</p

Chylomicron formation promotes intestinal absorption of full-length, antigenic OVA.

<p>Fasted mice were gavaged with 0.2 ml emulsions as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008442#pone-0008442-g001" target="_blank">Figure 1</a>, except that ¹²⁵I-OVA was replaced with 25 mg OVA. Blood samples were obtained from the submandibular vein at indicated time points and analyzed for OVA by Western blotting.</p

Chylomicron formation promotes intestinal OVA absorption.

<p>Fasted mice were gavaged with a dispersion of 0.05 ml ¹²⁵I-labeled OVA (black bars) in PBS plus 0.15 ml of either LCT, MCT, or LCT plus 6 µl of Pluronic L-81 (Pl-81). Radioactivity in the entire plasma per mouse (top panel) and in pooled MLN per mouse (bottom panel) was measured 90 minutes later. Another group of mice was gavaged with identical solutions, except that ¹²⁵I-OVA was replaced with [³H]-retinol (white bars). Shown are averages±S.D. of 4 mice per experimental group; * indicates statistically significant differences between feeding groups (P<0.05; ANOVA, Bonferroni's posthoc analysis). The figure shows a representative outcome of two repeats.</p

Chylomicron formation promotes systemic dissemination and antigen presentation of dietary antigen.

<p>Naïve BALB/C mice were injected with 2.5×10⁶ CFSE labeled T cells from DO11.10 TCR transgenic mice. After 24 h, the mice were fasted (4 h) and gavaged with OVA (25 mg) in 0.2 ml PBS or 25 mg OVA in 0.05 ml PBS+0.15 ml of either MCT, LCT, or LCT plus Pl-81. Mice were then fasted for an additional 6 h. Three days later, inguinal LN cells were isolated, stained with anti-CD4 and KJ1-26 (TCR clonotypic antibody), and analyzed by flow cytometry. Histograms show representative CFSE dilution profiles of gated CD4+, KJ1-26+ T cells as a measure of cell division. The % of cells under markers M1 and M2 represent cells which have not or have undergone cell division respectively. Each panel represents a typical result of three experimental repeats.</p

Plasma chylomicrons transport dietary OVA.

<p>Fasted mice were gavaged with 0.2 ml LCT-containing emulsions also containing 25 mg OVA. Plasma was isolated 1 h later, and 55 µl were fractionated via FPLC. The grey line of the chromatogram shows the elution profile of a mouse injected i.p. with Poloxamer P-407 1 h prior to gavage to inhibit chylomicron clearance, which caused a milky plasma appearance (inset) and a greatly increased first peak. The solid line shows the elution profile of a mouse not previously injected with Poloxamer P-407. The fractions of this mouse, indicated by the vertical separators, were subjected to immunoprecipitation for detection of OVA (lower panel).The experiment was repeated three times with similar outcomes.</p