TRAF5 deficiency accelerates atherogenesis in mice by increasing inflammatory cell recruitment and foam cell formation.

RATIONALE: Tumor necrosis factor receptor-associated factors (TRAFs) are cytoplasmic adaptor proteins for the TNF/interleukin-1/Toll-like receptor superfamily. Ligands of this family comprise multiple important cytokines such as TNFα, CD40L, and interleukin-1ß that promote chronic inflammatory diseases such as atherosclerosis. We recently reported overexpression of TRAF5 in murine and human atheromata and that TRAF5 promotes inflammatory functions of cultured endothelial cells and macrophages. OBJECTIVE: This study tested the hypothesis that TRAF5 modulates atherogenesis in vivo. METHODS AND RESULTS: Surprisingly, TRAF5(-/-)/LDLR(-/-) mice consuming a high-cholesterol diet for 18 weeks developed significantly larger atherosclerotic lesions than did TRAF5(+/+)/LDLR(-/-) controls. Plaques of TRAF5-deficient animals contained more lipids and macrophages, whereas smooth muscle cells and collagen remained unchanged. Deficiency of TRAF5 in endothelial cells or in leukocytes enhanced adhesion of inflammatory cells to the endothelium in dynamic adhesion assays in vitro and in murine vessels imaged by intravital microscopy in vivo. TRAF5 deficiency also increased expression of adhesion molecules and chemokines and potentiated macrophage lipid uptake and foam cell formation. These findings coincided with increased activation of JNK and appeared to be independent of TRAF2. Finally, patients with stable or acute coronary heart disease had significantly lower amounts of TRAF5 mRNA in blood compared with healthy controls. CONCLUSIONS: Unexpectedly, TRAF5 deficiency accelerates atherogenesis in mice, an effect likely mediated by increased inflammatory cell recruitment to the vessel wall and enhanced foam cell formation.

Tumor necrosis factor receptor-associated factor 1 (TRAF1) deficiency attenuates atherosclerosis in mice by impairing monocyte recruitment to the vessel wall.

BACKGROUND: Members of the tumor necrosis factor superfamily, such as tumor necrosis factor-alpha, potently promote atherogenesis in mice and humans. Tumor necrosis factor receptor-associated factors (TRAFs) are cytoplasmic adaptor proteins for this group of cytokines. METHODS AND RESULTS: This study tested the hypothesis that TRAF1 modulates atherogenesis in vivo. TRAF1(-/-)/LDLR(-/-) mice that consumed a high-cholesterol diet for 18 weeks developed significantly smaller atherosclerotic lesions than LDLR(-/-) (LDL receptor-deficient) control animals. As the most prominent change in histological composition, plaques of TRAF1-deficient animals contained significantly fewer macrophages. Bone marrow transplantations revealed that TRAF1 deficiency in both hematopoietic and vascular resident cells contributed to the reduction in atherogenesis observed. Mechanistic studies showed that deficiency of TRAF1 in endothelial cells and monocytes reduced adhesion of inflammatory cells to the endothelium in static and dynamic assays. Impaired adhesion coincided with reduced cell spreading, actin polymerization, and CD29 expression in macrophages, as well as decreased expression of the adhesion molecules intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 in endothelial cells. Small interfering RNA studies in human cells verified these findings. Furthermore, TRAF1 messenger RNA levels were significantly elevated in the blood of patients with acute coronary syndrome. CONCLUSIONS: TRAF1 deficiency attenuates atherogenesis in mice, most likely owing to impaired monocyte recruitment to the vessel wall. These data identify TRAF1 as a potential treatment target for atherosclerosis.

Multimodality cardiac computed tomography angiography and magnetic resonance with clinical-grade scanners provide robust assessment of cardiac morphology and function in rabbits.

BACKGROUND: Non-invasive computer tomography (CT)- and magnetic resonance (MR)-based cardiac imaging still remains challenging in rodents. To investigate the robustness of non-invasive multimodality cardiac imaging in rabbits using clinical-grade CT and MR scanners. METHODS: A total of 16 rabbits (2.7-4.0 kg) serially underwent cardiac-gated imaging using a clinical-grade 256-row CT and a 1.5 Tesla MR-scanner at baseline and at 4-month follow-up (16±1 weeks). Image analysis included image quality (5-grade scale), left ventricular (LV) volumes, LV stroke volume, LV diameters, LV wall thickness and ejection fraction (LVEF). RESULTS: Cardiac MR (CMR) and CT angiography (CTA) provide images with an overall good image quality (excellent or good quality: CMR 82% vs. CTA 78%, P=0.68). Linear regression analysis demonstrated a good correlation of all diameters (diam.) and volumes (vol.) as assessed by CTA and CMR (diam.: r=0.9, 95% CI: 0.8-0.9; vol.: r=0.8, 95% CI: 0.6-0.9; P<0.0001 for both). CTA-based volumetric analysis revealed slightly higher LVEF values as compared to CMR (CTA: 64%±1%, CMR: 59%±1%, P=0.002). Analysis of inter-/intra-observer agreement demonstrated excellent agreements for diameters (CMR: 98.5%/98.7%; CTA: 98.2%/97.4%) and volumes (CMR: 99.9%/98.8%; CTA 98.7%/98.7%). Finally, serial CMR- and CTA-based assessment of cardiac diameters and volumes delivered excellent intersession agreements of baseline versus follow-up data (diam.: CMR: r=0.89; CTA: r=0.92; vol.: CMR: r=0.87; CTA: r=0.96, P<0.0001 for all). CONCLUSIONS: Multimodality non-invasive assessment of cardiac function and aortic hemodynamics is feasible and robust in rabbits using clinical-grade and MR and CT scanners. These imaging modalities could improve serial cardiac assessment for disease monitoring in preclinical settings.

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.

Unexpected abundance of HLA class II presented peptides in primary renal cell carcinomas.

PURPOSE: To elicit a long-lasting antitumor immune response, CD8+ and CD4+ T cells should be activated. We attempted to isolate HLA-DR-presented peptides directly from dissected solid tumors, in particular from renal cell carcinoma, to identify MHC class II ligands from tumor-associated antigens (TAA) for their use in peptide-based immunotherapy. EXPERIMENTAL DESIGN: Tumor specimens were analyzed by immunohistochemical staining for their HLA class II expression. HLA class II peptides were subsequently isolated and identified by mass spectrometry. Gene expression analysis was done to detect genes overexpressed in tumor tissue. Peptides from identified TAAs were used to induce peptide-specific CD4+ T-cell responses in healthy donors and in tumor patients. RESULTS: In the absence of inflammation, expression of MHC class II molecules is mainly restricted to cells of the immune system. To our surprise, we were able to isolate and characterize hundreds of class II peptides directly from primary dissected solid tumors, especially from renal cell carcinomas, and from colorectal carcinomas and transitional cell carcinomas. Infiltrating leukocytes expressed MHC class II molecules and tumor cells, very likely under the influence of IFNgamma. Our list of identified peptides contains ligands from several TAAs, including insulin-like growth factor binding protein 3 and matrix metalloproteinase 7. The latter bound promiscuously to HLA-DR molecules and were able to elicit CD4+ T-cell responses. CONCLUSIONS: Thus, our direct approach will rapidly expand the limited number of T-helper epitopes from TAAs for their use in clinical vaccination protocols.

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

AIMS: 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. METHODS AND RESULTS: 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 < 0.001; low ESS and inflammation by molecular MRI at baseline: AUC = 0.89, P < 0.001). CONCLUSION: 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.

Off-resonance magnetic resonance angiography improves visualization of in-stent lumen in peripheral nitinol stents compared to conventional T1-weighted acquisitions: an in vitro comparison study.

To compare the value of inversion recovery with on-resonant water suppression (IRON) to conventional T1-weighted (T1w) MRA and computed tomography angiography (CTA) for visualization of peripheral nitinol stents. We visualized 14 different peripheral nitinol stents in vitro both using Gadolinium (Gd) and ultrasmall superparamagnetic iron nanoparticles (USPIOs) for conventional T1w and IRON-MRA using clinical grade 1.5T MR scanner and iodinated contrast material for CTA using a 256-slice CT scanner. Parameter assessment included signal- and contrast-to-noise ratio (S/CNR), relative in-stent signal and artificial lumen narrowing. X-ray angiography served as gold standard for diameter assessment. Gd-enhanced IRON-MRA exhibited highest in-stent SNR and CNR values compared to conventional T1w MRA (IRON (Gd/USPIO): SNR = 30 ± 3/21 ± 2, CNR = 23 ± 2/14 ± 1; T1w: SNR = 16 ± 1/14 ± 2, CNR = 12 ± 1/10 ± 1, all p < 0.05). Furthermore, IRON-MRA achieved highest relative in-stent signal both using Gd and USPIO (IRON (Gd/USPIO): 121 ± 8 %/103 ± 6 %; T1w: 73 ± 2 %/66 ± 4 %; CTA: 84 ± 6 %, all p < 0.05). However, artificial lumen narrowing appeared similar in all imaging protocols (IRON (Gd/USPIO): 21 ± 3 %/21 ± 2 %; T1w: 16 ± 4 %/17 ± 3 %; CTA: 19 ± 2 %, all p = NS). Finally, IRON-MRA provided improvement of the in-stent lumen visualization with an 'open-close-open' design, which revealed a complete in-stent signal loss in T1w MRA. IRON-MRA improves in-stent visualization in vitro compared to conventional T1w MRA and CTA. In light of the in vitro results with Gd-enhanced IRON-MRA, the clinical implementation of such an approach appears promising.

Epicardial Adipose Tissue Is Associated with Plaque Burden and Composition and Provides Incremental Value for the Prediction of Cardiac Outcome. A Clinical Cardiac Computed Tomography Angiography Study.

OBJECTIVES: We sought to investigate the association of epicardial adipose tissue (eCAT) volume with plaque burden, circulating biomarkers and cardiac outcomes in patients with intermediate risk for coronary artery disease (CAD). METHODS AND RESULTS: 177 consecutive outpatients at intermediate risk for CAD and completed biomarker analysis including high-sensitive Troponin T (hs-TnT) and hs-CRP underwent 256-slice cardiac computed tomography angiography (CCTA) between June 2008 and October 2011. Patients with lumen narrowing ≥50% exhibited significantly higher eCAT volume than patients without any CAD or lumen narrowing <50% (median (interquartile range, IQR): 108 (73-167) cm3 vs. 119 (82-196) cm3, p = 0.04). Multivariate regression analysis demonstrated an independent association eCAT volume with plaque burden by number of lesions (R2 = 0.22, rpartial = 0.29, p = 0.026) and CAD severity by lumen narrowing (R2 = 0.22, rpartial = 0.23, p = 0.038) after adjustment for age, diabetes mellitus, hyperlidipemia, body-mass-index (BMI), hs-CRP and hs-TnT. Univariate Cox proportional hazards regression analysis identified a significant association for both increased eCAT volume and maximal lumen narrowing with all cardiac events. Multivariate Cox proportional hazards regression analysis revealed an independent association of increased eCAT volume with all cardiac events after adjustment for age, >3 risk factors, presence of CAD, hs-CRP and hs-TnT. CONCLUSION: Epicardial adipose tissue volume is independently associated with plaque burden and maximum luminal narrowing by CCTA and may serve as an independent predictor for cardiac outcomes in patients at intermediate risk for CAD.

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.

CD40L induces inflammation and adipogenesis in adipose cells--a potential link between metabolic and cardiovascular disease.

CD40L figures prominently in atherogenesis. Recent data demonstrate elevated levels of sCD40L in the serum of patients with the metabolic syndrome (MS). This study investigated the role of CD40L in pro-inflammatory gene expression and cellular differentiation in adipose tissue to obtain insight into mechanisms linking the MS with atherosclerosis. Human adipocytes and preadipocytes expressed CD40 but not CD40L. Stimulation with recombinant CD40L or membranes over-expressing CD40L induced a time- and dose-dependent expression of IL-6, MCP-1, IL-8, and PAI-1. Supernatants of CD40L-stimulated adipose cells activated endothelial cells, suggesting a systemic functional relevance of our findings. Neutralising antibodies against CD40L attenuated these effects substantially. Signalling studies revealed the involvement of mitogen-activated protein kinases and NFkB. Furthermore, stimulation with CD40L resulted in enhanced activation of C/EBPa and PPARg and promoted adipogenesis of preadipose cells in the presence and absence of standard adipogenic conditions. Finally, patients suffering from the metabolic syndrome with high levels of sCD40L also displayed high levels of IL-6, in line with the concept that CD40L may induce the expression of inflammatory cytokines in vivo in this population. Our data reveal potent metabolic functions of CD40L aside from its known pivotal pro-inflammatory role within plaques. Our data suggest that CD40L may mediate risk at the interface of metabolic and atherothrombotic disease.

Human CD8+ T cells store CXCR1 in a distinct intracellular compartment and up-regulate it rapidly to the cell surface upon activation.

Activation and subsequent differentiation of naive CD8+ T cells lead to the development of memory subsets with distinct homing and effector capacities. On nonlymphoid homing subsets, expression of "inflammatory" chemokine receptors (such as CXCR3, CCR5, CX3CR1, and CXCR1) is believed to promote migration into sites of infection/inflammation. Here we show that CXCR1 can be up-regulated to the cell surface within minutes of activating human CD8+ T cells. No concurrent up-regulation of other inflammatory chemokine receptors was observed. Up-regulation of CXCR1 preferentially occurred on central memory CD8+ T cells-that is, cells with a lymph node homing phenotype-and was functionally relevant. Immunofluorescence microscopy showed CXCR1 to be present in intracellular vesicles that do not significantly colocalize with perforin, RANTES (regulated upon activation normal T cell expressed and secreted), or the lysosomal marker CD63. By contrast, partial colocalization with the Golgi marker GM130, the constitutive secretory pathway marker beta2-microglobulin, and the early endosome marker EEA1 was observed. Up-regulation of CXCR1 did not occur after T-cell receptor cross-linking. By contrast, supernatants from activated neutrophils, but not from monocytes or dendritic cells, induced its up-regulation. These results suggest that CD8+ T cells can rapidly adapt their homing properties by mobilizing CXCR1 from a distinct intracellular compartment.