1. TRAF5 Deficiency Accelerates Atherogenesis in Mice by Increasing Inflammatory Cell Recruitment and Foam Cell FormationNovelty and Significance
by Anna Missiou, Philipp Rudolf, Peter Stachon, Dennis Wolf, Nerea Varo, Peter Aichele, Christian Colberg, Natalie Hoppe, Sandra Ernst, Christian Münkel, Carina Walter, Benjamin Sommer, Ingo Hilgendorf, Hiroyasu Nakano, Christoph Bode, and Andreas Zirlik
Circulation Research
Volume 107(6):
September 17, 2010
Copyright © American Heart Association, Inc. All rights reserved.

2. TRAF5 deficiency accelerates murine atherosclerosis.
TRAF5 deficiency accelerates murine atherosclerosis. A and B, TRAF5−/−/LDLR−/− and TRAF5+/+/LDLR−/− mice consumed HCD for 18 weeks and intimal lesion size in the aortic root (A) and arch (B), as well as lipid deposition (oil red O) in the abdominal aortas (C) were quantified. Left, Pooled data±SEM (N≥12, 8, and 16 per group, respectively). Right, Images of representative sections stained for lipid deposition (oil red O).
Anna Missiou et al. Circ Res. 2010;107:
Copyright © American Heart Association, Inc. All rights reserved.

3. TRAF5-deficient plaques contain more macrophages and lipids.
TRAF5-deficient plaques contain more macrophages and lipids. A through D, Sections of the aortic arches and roots of mice treated as described above were subjected to analysis of macrophage-, smooth muscle cell–, lipid-, and collagen-specific staining. Mac-3–, α-actin–, oil red O–, and picrosirius red–positive staining in relation to total wall area is presented as means±SEM.
Anna Missiou et al. Circ Res. 2010;107:
Copyright © American Heart Association, Inc. All rights reserved.

4. TRAF5 deficiency promotes inflammatory cell recruitment to the endothelium.
TRAF5 deficiency promotes inflammatory cell recruitment to the endothelium. A, Adhesion and rolling of PMA-activated thioglycollate-elicited peritoneal leukocytes obtained from TRAF5−/−/LDLR−/− and TRAF5+/+/LDLR−/− mice were quantified on TNFα-activated ECs isolated from TRAF5−/−/LDLR−/− and TRAF5+/+/LDLR−/− mice under flow conditions (0.5 dyn/cm2, N≥3 each). Adherent and rolling leukocytes were counted under the microscope. Pooled data represent means±SEM. B, Venules (30 to 50 μm) of the cremaster muscle were evaluated for adhesion and rolling of leukocytes in TRAF5−/−/LDLR−/− and TRAF5+/+/LDLR−/− mice 4 hours after IP administration of 200 ng of TNFα (N≥3). Leukocyte numbers were counted manually and are presented at left. Representative images are displayed at right. Data represent the means±SEM.
Anna Missiou et al. Circ Res. 2010;107:
Copyright © American Heart Association, Inc. All rights reserved.

5. TRAF5 deficiency enhances adhesion molecule expression in ECs and macrophages.
TRAF5 deficiency enhances adhesion molecule expression in ECs and macrophages. A, TRAF5-deficient and -competent murine ECs were stimulated with or without 20 ng/mL TNFα and 10 ng/mL IL-1β. Cell lysates were analyzed for ICAM-1 protein expression by Western blotting. Pooled densitometric data adjusted for GAPDH expression are given as means±SEM (top), and representative blots are shown (bottom) (N=9). B, TRAF5−/−/LDLR−/− and TRAF5+/+/LDLR−/− mice consumed HCD for 8 weeks, total RNA was extracted from aortas, and ICAM-1 and VCAM-1 transcripts were quantified by RT-PCR. Data are presented as mean ratios of target/GAPDH±SEM (N≥3). C, Murine monocytes were obtained from TRAF5−/−/LDLR−/− and TRAF5+/+/LDLR−/− mice, injected with 200 ng of TNFα or vehicle IP 4 hours before isolation and analyzed for CD49d expression by FACS. Pooled data as means±SEM are shown (n≥3).
Anna Missiou et al. Circ Res. 2010;107:
Copyright © American Heart Association, Inc. All rights reserved.

6. TRAF5 deficiency promotes migration and chemokine expression.
TRAF5 deficiency promotes migration and chemokine expression. A, Sterile peritonitis was induced by instillation of 4% thioglycollate in TRAF5−/−/LDLR−/− and TRAF5+/+/LDLR−/− mice. After 72 hours, cells were counted under the microscope (N=3). Data represent means±SEM. B and C, Serum (B) and arterial tissue (C) of TRAF5−/−/LDLR−/− and TRAF5+/+/LDLR−/− mice 4 hours after IP administration of 200 ng of TNFα were assayed for MCP-1 expression by ELISA and cytometric bead array, respectively (N≥5 each). Data represent means±SEM. D, Sections of the aortic roots from TRAF5−/−/LDLR−/− and TRAF5+/+/LDLR−/− mice consuming HCD for 18 weeks were stained for MCP-1 and quantified by image analysis software. Data represent means±SEM. E, TRAF5−/−/ LDLR−/− and TRAF5+/+/LDLR−/− mice consumed HCD for 8 weeks, total RNA was extracted from aortas, and KC transcripts were quantified by RT-PCR. Data are presented as mean ratios of KC/GAPDH±SEM (N≥3).
Anna Missiou et al. Circ Res. 2010;107:
Copyright © American Heart Association, Inc. All rights reserved.

7. TRAF5 deficiency promotes lipid uptake.
TRAF5 deficiency promotes lipid uptake. A, BMMs obtained from TRAF5−/−/LDLR−/− and TRAF5+/+/LDLR−/− mice were incubated with fluorescent-labeled acetylated LDL (acLDL). Lipid uptake was quantified by FACS. Data represent means±SEM (N=4). B through D, Murine TRAF5-deficient and -competent BMMs were stimulated with TNFα 20 ng/mL or vehicle IP and analyzed for CD36 (B), TLR4 (C), and scavenger receptor A (SR-A) (D) expression by FACS and Western blotting. Data represent means±SEM (N≥4). E, In blood from TRAF5−/−/LDLR−/− and TRAF5+/+/LDLR−/− mice, CD115-positive cells were tested for ABCA1 expression by FACS. Data represent mean fluorescent intensity±SEM (N≥4).
Anna Missiou et al. Circ Res. 2010;107:
Copyright © American Heart Association, Inc. All rights reserved.

8. TRAF5 deficiency promotes atherogenesis independently of TRAF2.
TRAF5 deficiency promotes atherogenesis independently of TRAF2. A, Following lethal body irradiation, TRAF2+/+/LDLR−/− received TRAF2-deficient or -competent FLCs and consumed HCD for 18 weeks. Additionally, TRAF2+/−/LDLR−/− mice received TRAF2-deficient FLCs. Intimal lesion size was quantified in sections of the aortic root. Left, Pooled data are presented as means±SEM. Right, Representative images are shown. B, Sections from the aortic roots of animals treated as described for A were stained with oil red O and staining was quantified with image analysis software. Data represent means±SEM. C, TRAF5−/−/LDLR−/− and TRAF5+/+/LDLR−/− mice consumed HCD for 8 weeks, total RNA was extracted from aortas and spleens, and TRAF transcripts were quantified by RT-PCR. Data are presented as mean ratios of target/GAPDH±SEM (N≥3).
Anna Missiou et al. Circ Res. 2010;107:
Copyright © American Heart Association, Inc. All rights reserved.

9. TRAF5/GAPDH mRNA ratios decrease in patients with acute and chronic coronary heart disease.
TRAF5/GAPDH mRNA ratios decrease in patients with acute and chronic coronary heart disease. A, Patients (n=325) undergoing coronary angiography were divided into the 3 groups: no coronary heart disease (No CHD), stable coronary heart disease (CHD), and acute coronary syndromes (ACS). Blood was taken before angiography for RNA isolation, and TRAF5/GAPDH mRNA ratios were analyzed by quantitative real-time PCR. Differences across groups were compared by ANOVA, followed by the Bonferroni post hoc test. Results are presented as means±SEM, computed from the average measurements obtained from each group. B, Twelve patients with lowest TRAF5/GAPDH ratios were retested after a mean follow up of 30.8±1.8 months. Mean TRAF5/GAPDH ratios±SEM are displayed. C, Monocytes, lymphocytes, and neutrophils were isolated from buffy coats and analyzed from TRAF5 expression by RT-PCR (N=4). Data represent means±SEM.
Anna Missiou et al. Circ Res. 2010;107:
Copyright © American Heart Association, Inc. All rights reserved.