Adhesion molecules expression analysis.

<p>(A) Relative mRNA levels of VCAM-1, ICAM-1, e-selectin and p-selectin in aortic arches from p55^+/+LDLR^−/− (n = 10) and p55^−/−LDLR^−/− (n = 8) mice. Values are represented relative to expression in p55^+/+LDLR^−/− arches. (B) Immunohistochemical staining of VCAM-1 expression on sections from aortic valve areas indicating a less intense endothelial staining in p55^−/−LDLR^−/− mice. Original magnification×200. (C) Staining quantification. * p = 0.01 by Student's t-test. Error bars indicate SEM.</p

Survival curves, aneurismal incidence and suprarenal aortic diameters.

<p>(A) Percent survival in p55^+/+LDLR^−/− (squares) and p55^−/−LDLR^−/− (triangles) mice following infusion with saline (n = 3 mice/group) or AngII (n = 13–14 mice/group). Comparison of survival curves between AngII-infused p55^+/+LDLR^−/− and p55^−/−LDLR^−/− mice gave a probability value of p = 0.054 by Pearson's chi-square test. (B) representative cross-sections of EVG stained suprarenal aortas from (a) saline infused and of advanced AAA in (b) p55^+/+LDLR^−/− and (c) p55^−/−LDLR^−/− mice indicating dissecting aneurysms and formation of thrombi. Original magnification×100 (a) and×50 (b,c). (C) Percent incidence of AAA in p55^+/+LDLR^−/− and p55^−/−LDLR^−/− mice (p = 0.2 by Fisher's exact test). (D) Suprarenal aortic diameters in p55^+/+LDLR^−/− (n = 12) and p55^−/−LDLR^−/− (n = 8) mice (p = 0.3 by Student's t-test).</p

Atherosclerosis quantification.

<p>(A) Atherosclerotic lesion area in the aortic sinuses of p55^+/+LDLR^−/− (squares, n = 18) and p55^−/−LDLR^−/− (triangles, n = 16) mice. Each symbol represents one animal; bars represent means. * p = 0.02 by Student's t-test. (B) Representative lesions from p55^+/+LDLR^−/− and p55^−/−LDLR^−/− mice are shown. Original magnification×40. (C) Lesion classification according to severity. (D) Gene expression analysis in p55^+/+LDLR^−/− (n = 10) and p55^−/−LDLR^−/− (n = 8) aortic arches. Values are represented relative to expression in p55^+/+LDLR^−/− arches. * p = 0.03 by Student's t-test. Error bars indicate SEM.</p

Cytokine and chemokine expression analysis.

<p>(A) Relative mRNA levels of IκBα, TNF, IL-6, IL-10 and (B) MCP-1, MIP-1α, MIP-1β, RANTES in aortic arches from p55^+/+LDLR^−/− (n = 10) and p55^−/−LDLR^−/− (n = 8) mice. Values are represented relative to expression in p55^+/+LDLR^−/− arches. (C) Plasma levels of pro-inflammatory cytokines and chemokines (n = 12–15 mice/group) after 8 weeks of high fat feeding. Error bars indicate SEM.</p

Body weight and plasma lipid levels.

<p>(A) Body weight (B) plasma cholesterol and (C) plasma triglyceride levels in p55^+/+LDLR^−/− (n = 18) and p55^−/−LDLR^−/− (n = 16) mice before and after 8 weeks of high fat feeding.</p

General characterization of AngII infused mice.

<p>(A) Body weight (B) plasma cholesterol and (C) plasma triglyceride levels in p55^+/+LDLR^−/− and p55^−/−LDLR^−/− mice before (n = 13–14 mice/group) and after 5 weeks of high fat feeding (4 weeks of AngII infusion; n = 8–12 mice/group). (D) Plasma levels of pro-inflammatory cytokines and chemokines (n = 7–9 mice/group) after 5 weeks of high fat feeding (4 weeks of AngII infusion). * p<0.05 by Student's t-test. Error bars indicate SEM.</p

CD40^fl/flLysM^cre mice have similar glucose tolerance and insulin resistance than their WT controls.

(A) Glucose tolerance test (n = 6–9 mice/group). (B) Insulin tolerance test (n = 6–16 mice/group). (C) Fasted glucose levels (n = 6–9 mice/group). (D) Fasted insulin levels (n = 5–8 mice/group). Data is presented as mean ± SEM.</p

Adipose tissue inflammation is slightly increased in macrophage CD40 deficient mice without resulting in altered cytokine release.

(A) Quantification of CD45 staining for leukocytes of EpAT (n = 16 for HFD and n = 7 for SFD). (B) Quantification of Mac3 staining for macrophages in EpAT (n = 16 for HFD and n = 7 for SFD). (C) Quantification of CD3+ staining for T cells in EpAT (n = 16 for HFD and n = 7 for SFD). (D) Flow cytometric analysis of leukocyte subsets in EpAT, as % of total CD45+ leukocytes (n = 7-9/group). (E) mRNA expression of inflammatory markers in EpAT (n = 7-9/group). Data is presented as mean ± SEM.</p

Adipose tissue function is not affected by macrophage CD40 deficiency.

(A) Adipocyte size after 13 weeks of dietary intervention. (B) Plasma triglycerides (TG). (C) Plasma total cholesterol levels. (D) plasma adiponectin levels are increased in lean macrophage CD40 deficient mice but are not different in obese mice. (E) Plasma leptin levels. (F) mRNA expression of adiponectin, leptin, PPARγ and insulin receptor (IR) in EpAT, (n = 6–9 mice/group). Data is presented as mean ± SEM. *p<0.05, as determined by non-parametric Mann Whitney test.</p

Body and adipose tissue weight of myeloid deficient mice.

(A) Body weight of the high fat diet (HFD) CD40fl/flLysMcre and WT mice (n = 16/group) and the standard fat diet mice (SFD, n = 7/group) during 13 weeks of diet. (B) Tissue weights after 13 weeks of diet. Data is presented as mean ± SEM.</p