Absence of p55 TNF Receptor Reduces Atherosclerosis, but Has No Major Effect on Angiotensin II Induced Aneurysms in LDL Receptor Deficient Mice

The aim of the current study was to investigate the role of p55 TNF Receptor (p55 TNFR), the main signaling receptor for the pro-inflammatory cytokine tumor necrosis factor (TNF), in the development of two vascular disorders: atherosclerosis and angiotensin (Ang) II-induced abdominal aortic aneurysms (AAA). p55 TNFR deficient mice were crossed to an LDL receptor deficient background and were induced for the development of either atherosclerosis or AngII-induced AAA, and compared to littermate controls, wild-type for p55 TNFR expression. p55 TNFR deficient mice developed 43% smaller atherosclerotic lesions in the aortic sinuses compared to controls. Moreover, expression of CD68, a macrophage specific marker, exhibited a 50% reduction in the aortic arches. Decreased atherosclerosis correlated with a strong down-regulation in the expression of adhesion molecules, such as VCAM-1 and ICAM-1, by p55 TNFR deficient endothelium. In addition, expression levels of the pro-inflammatory cytokines and chemokines TNF, IL-6, MCP-1 and RANTES were significantly reduced in aortas of p55 TNFR deficient mice. In contrast, in the AngII-induced model of AAA, p55 TNFR deficiency correlated with a slight trend towards increased aneurismal lethality, but the incidence of aortic rupture due to a dissecting aneurysm, and the expansion of the suprarenal aorta were not significantly different compared to controls. We found that p55 TNFR expression promotes atherosclerosis, among other mechanisms, by enhancing expression of endothelial adhesion molecules, while it seems to have no major role in the development of AngII-induced AA

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

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

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

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

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

Hematopoietic deficiency of miR155 promotes atherosclerotic lesion development in LDLR^−/− mice.

<p>Representative pictures of toluidine blue-stained sections of the aortic root of wildtype (A) and miR155^−/− (B) transplanted mice, magnification 40×. Hematopoietic miR155 deficiency increases plaque area (C) and promotes plaque progression towards more advanced lesions (D, Chi square test p<0.05). * p<0.05, n = 14 wildtypes and 19 miR155−/− mice.</p

Hematopoietic deficiency of miR155 enhances inflammation in atherosclerotic lesions.

<p>(A) Pathological examination of collagen, necrosis, foam cells, inflammatory cells, monocyte adhesion to the plaque and adventitial influx. AU: arbitrary units. (B) collagen content stained by Sirius red staining, (C) Monocyte/macrophage area stained by moma-2, (D) Neutrophil numbers stained by NIMP, (E) Newly recruited macrophages identified by ERMP58 and (F) T cell numbers stained by KT-3 antibody (directed against CD3). Arrowheads indicate positively stained cells. *p<0.05, n = 14 wildtypes and 19 miR155−/− mice.</p