Tumor Necrosis Factor Receptor Associated Factor 6 Is Not Required for Atherogenesis in Mice and Does Not Associate with Atherosclerosis in Humans

BACKGROUND: Tumor necrosis factor receptor-associated factors (TRAFs) are important signaling molecules for a variety of pro-atherogenic cytokines including CD40L, TNF alpha, and IL1beta. Several lines of evidence identified TRAF6 as a pro-inflammatory signaling molecule in vitro and we previously demonstrated overexpression of TRAF6 in human and Murine atherosclerotic plaques. This study investigated the role of TRAF6-deficiency in mice developing atherosclerosis, a chronic inflammatory disease. METHODOLOGY/PRINCIPAL FINDINGS: Lethally irradiated low density lipoprotein receptor-deficient mice (TRAF6(+/+)/LDLR(-/-)) were reconstituted with TRAF6-deficient fetal liver cells (FLC) and consumed high cholesterol diet for 18 weeks to assess the relevance of TRAF6 in hematopoietic cells for atherogenesis. Additionally, TRAF6(+/-)/LDLR(-/-) mice received TRAF6-deficient FLC to gain insight into the role of TRAF6 deficiency in resident cells. Surprisingly, atherosclerotic lesion size did not differ between the three groups in both aortic roots and abdominal aortas. Similarly, no significant differences in plaque composition could be observed as assessed by immunohistochemistry for macrophages, lipids, smooth muscle cells, T-cells, and collagen. In accord, in a small clinical study TRAF6/GAPDH total blood RNA ratios did not differ between groups of patients with stable coronary heart disease (0.034+/-0.0021, N = 178), acute coronary heart disease (0.029+/-0.0027, N = 70), and those without coronary heart disease (0.032+/-0.0016, N = 77) as assessed by angiography. CONCLUSION: Our study demonstrates that TRAF6 is not required for atherogenesis in mice and does not associate with clinical disease in humans. These data suggest that pro- and anti-inflammatory features of TRAF6 signaling outweigh each other in the context of atherosclerosis

CD40 Ligand Mediates Inflammation Independently of CD40 by Interaction With Mac-1

Background— Strong evidence supports a role for CD40 ligand (CD40L) as marker and mediator of inflammatory diseases such as atherosclerosis. Despite extensive characterization of CD40, the classic receptor of CD40L, its role in immune defense against inflammatory diseases remains uncertain. The present study aimed to characterize the contribution of CD40 signaling to atherogenesis. Methods and Results— Surprisingly, mice deficient in both CD40 and the low-density lipoprotein receptor did not develop smaller lesions in the aortic arch, root, and thoracoabdominal aorta compared with mice deficient only in the low-density lipoprotein receptor that consumed an atherogenic diet for 8 and 16 weeks. By flow cytometry, radioactive binding assays, and immunoprecipitation, we demonstrate that CD40L interacts with the integrin Mac-1, which results in Mac-1–dependent adhesion and migration of inflammatory cells as well as myeloperoxidase release in vitro. Furthermore, mice deficient in CD40L show significantly reduced thioglycolate-elicited invasion of inflammatory cells into the peritoneal cavity compared with mice deficient in CD40 and wild-type controls. Inhibition of Mac-1 in low-density lipoprotein receptor–deficient mice attenuates lesion development and reduces lesional macrophage accumulation. Conclusions— These observations identify the interaction of CD40L and Mac-1 as an alternative pathway for CD40L-mediated inflammation. This novel mechanism expands understanding of inflammatory signaling during atherogenesis

Successful reconstitution of peripheral blood cells by fetal liver cell transplantation.

<p>LDLR^−/− mice (CD45.2-positive/CD45.1-negative, A) were lethally irradiated (2×450 cGy) and reconstituted with fetal liver cells of 6–8 week old CD45.1-positive/CD45.2-negative mice (B). After an interval of 4 weeks, peripheral blood cells were immunostained with CD45.1-PE and CD45.2-FITC (exemplary donors are shown in C and D) or CD45.1-PE in combination with CD19-PECy (B-cell marker, E), CD3-APC (T-cell marker, F), and CD11b-FITC (monocytic marker, G) antibodies and analyzed by FACS.</p

TRAF6 deficiency does not alter lipid deposition in the abdominal aorta.

<p>Lethally irradiated 6 week old TRAF6^+/+/LDLR^−/− mice received TRAF6-deficient (hatched bars, N = 10) or competent fetal liver cells (white bars, N = 10), TRAF6^+/−/LDLR^−/− mice received TRAF6-deficient fetal liver cells (black bars, N = 10) only. Subsequently, all groups consumed high cholesterol diet (HCD) for 18 weeks. Abdominal aortas were fixed in formalin, pinned, and stained with oil red O to detect lipid deposition. Oil red O-positive staining in per cent of total area is shown as mean±SEM in the upper panel (A), representative images are shown below (B).</p

Subtypes of blood leukocytes.

<p>Subtypes of blood leukocytes.</p

TRAF6 expression in blood does not associate with acute or chronic coronary heart disease.

<p>325 patients undergoing coronary angiography were divided into three groups: no coronary heart disease (No CHD, N = 77), stable coronary heart disease (CHD, N = 178), and acute coronary syndromes (ACS, N = 70). TRAF6 and GAPDH mRNA was analyzed by quantitative real-time PCR in total blood RNA. Results are presented as mean±SD computed from the average measurements obtained from each group.</p

Combined non-invasive assessment of endothelial shear stress and molecular imaging of inflammation for the prediction of inflamed plaque in hyperlipidaemic rabbit aortas.

To evaluate the incremental value of low endothelial shear stress (ESS) combined with high-resolution magnetic resonance imaging (MRI)- and computed tomography angiography (CTA)-based imaging for the prediction of inflamed plaque. Twelve hereditary hyperlipidaemic rabbits underwent quantitative analysis of plaque in the thoracic aorta with 256-slice CTA and USPIO-enhanced (ultra-small superparamagnetic nanoparticles, P904) 1.5-T MRI at baseline and at 6-month follow-up. Computational fluid dynamics using CTA-based 3D reconstruction of thoracic aortas identified the ESS patterns in the convex and concave curvature subsegments of interest. Subsegments with low baseline ESS exhibited significant increase in wall thickness and plaque inflammation by MRI, in non-calcified plaque burden by CTA, and developed increased plaque size, lipid and inflammatory cell accumulation (high-risk plaque features) at follow-up by histopathology. Multiple regression analysis identified baseline ESS and inflammation by MRI to be independent predictors of plaque progression, while receiver operating curve analysis revealed baseline ESS alone or in combination with inflammation by MRI as the strongest predictor for augmented plaque burden and inflammation (low ESS at baseline: AUC = 0.84, P &lt; 0.001; low ESS and inflammation by molecular MRI at baseline: AUC = 0.89, P &lt; 0.001). Low ESS predicts progression of plaque burden and inflammation as assessed by non-invasive USPIO-enhanced MRI. Combined non-invasive assessment of ESS and imaging of inflammation may serve to predict plaque with high-risk features