Tumor necrosis factor receptor-associated factor 1 (TRAF1) deficiency attenuates atherosclerosis in mice by impairing monocyte recruitment to the vessel wall - PubMed (original) (raw)
. 2010 May 11;121(18):2033-44.
doi: 10.1161/CIRCULATIONAHA.109.895037. Epub 2010 Apr 26.
Natascha Köstlin, Nerea Varo, Philipp Rudolf, Peter Aichele, Sandra Ernst, Christian Münkel, Carina Walter, Peter Stachon, Benjamin Sommer, Dietmar Pfeifer, Katja Zirlik, Lindsey MacFarlane, Dennis Wolf, Erdyni Tsitsikov, Christoph Bode, Peter Libby, Andreas Zirlik
Affiliations
- PMID: 20421522
- PMCID: PMC2995263
- DOI: 10.1161/CIRCULATIONAHA.109.895037
Tumor necrosis factor receptor-associated factor 1 (TRAF1) deficiency attenuates atherosclerosis in mice by impairing monocyte recruitment to the vessel wall
Anna Missiou et al. Circulation. 2010.
Abstract
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.
Conflict of interest statement
Conflict of Interest Disclosures:
Anna Missiou - Nothing to disclose
Natascha Köstlin - Nothing to disclose
Nerea Varo - Nothing to disclose
Philipp Rudolf - Nothing to disclose
Christian Münkel - Nothing to disclose
Sandra Ernst - Nothing to disclose
Carina Walter - Nothing to disclose
Peter Stachon - Nothing to disclose
Benjamin Sommer - Nothing to disclose
Dietmar Pfeifer - Nothing to disclose
Katja Zirlik - Nothing to disclose
Lindsey MacFarlane - Nothing to disclose
Dennis Wolf - Nothing to disclose
Erdyni Tsitsikov - Nothing to disclose
Christoph Bode - Nothing to disclose
Peter Libby - Nothing to disclose
Andreas Zirlik - Nothing to disclose
Figures
Figure 1. TRAF1 deficiency attenuates atherosclerosis in mice
A and B. TRAF1−/−/LDLR−/− and TRAF1+/+/LDLR−/− mice consumed a high cholesterol diet for 18 weeks and 8 weeks underwent analysis of intimal lesion size in the aortic root (A) and arch (B) (N=8 and 13). Pooled data±SEM are shown on the left; images of representative sections stained for lipid deposition (Oil-red-O) are displayed on the right. C. Sections of the aortic arches of mice treated as described above were analyzed for macrophage-, smooth muscle cell-, lipid-, and collagen-specific staining. Mac-3-, α-actin-, oil-red-O-, and picosirius red-positive staining in relation to total wall area is displayed as mean±SEM (N=8 and 13).
Figure 2. TRAF1 deficiency on bone marrow-derived and resident cells attenuates atherogenesis in mice
Bone marrow transplantations were conducted between TRAF1−/−/LDLR−/− and TRAF1+/+/LDLR−/− mice. Chimers consuming high cholesterol diet for 18 weeks were subjected to analysis of intimal lesion size in the aortic root. Pooled data represent mean±SEM. Mac-3- (macrophage), α-actin- (smooth muscle cell), CD4- (T cell), oil-red-O- (lipid), and picosirius red (collagen)-positive staining in relation to total wall area is displayed as mean ± SEM (N=8 and 5).
Figure 3. TRAF1 deficiency impairs adhesion of monocytes to endothelial cells and attenuates spreading of Murine macrophages
A. Murine monocytes and PBMCs of TRAF1-deficient and wild-type mice were stained with CFDA and allowed to interact with Murine endothelial cells isolated from TRAF1-deficient and wild-type mice (N=3). Adherent cells were counted under the microscope. Each symbol indicates an individual experiment and donor. B. Adhesion of PMA-activated thioglycollate-elicited peritoneal leukocytes obtained from TRAF1-deficient and wild-type mice was analyzed on TNFα-activated endothelial cells isolated from TRAF1-deficient and wild-type mice under flow conditions (0.5 dyne/cm2, N=5). Adherent leukocytes were quantified under the microscope. Pooled data represent mean±SEM. C. Mice were treated intraperitoneally with 200ng TNFα 4h prior to intravital microscopy. Venules (30–50µm) of the cremaster muscle were screened for adhesion of leucocytes. The number of adherent leucocytes was counted manually. Data represent the mean±SEM. D. Macrophages from wild-type and TRAF1-deficient mice were plated on serum-coated glass cover slips and incubated at 37°C. Cells were stained with Alexa Fluor 594-conjugated phalloidin and confocal microscope performed. Spreading was quantified and expressed as mean±SEM of spreading cells on the left (N=5 each); representative pictures are shown on the right.
Figure 4. TRAF1 deficiency attenuates expression of adhesion molecules and integrins on Murine endothelial cells and macrophages
A. Murine endothelial cells isolated from TRAF1-deficient and wild-type mice were stimulated with or without TNFα (20ng/ml), CD40L (10µg/ml), and IL-1β (10ng/ml), and cell lysates were analyzed for VCAM-1 (N=6) and ICAM-1 (N=7) by Western blotting. Pooled densitometric data adjusted for GAPDH given as mean±SEM are shown on top, representative blots below. B. Arterial tissue of TRAF1−/−/LDLR−/− and TRAF1+/+/LDLR−/− mice was isolated and lysates were examined for VCAM-1 and ICAM-1 protein expression by Western blotting. Data adjusted for GAPDH expression. Data are shown as grouped scatter plot. Representative blots are shown below (N=3). C. Sections of the aortic roots of TRAF1−/−/LDLR−/− and TRAF1+/+/LDLR−/− mice consuming a high cholesterol diet for 18 weeks underwent immunohistochemical analysis for VCAM-1- and ICAM-1 expression. VCAM-1- (N=6 and 10) and ICAM-1- (N=6 and 11)-positive staining in relation to total wall area is displayed as grouped scatter plot on the left, representative pictures are shown on the right. D. Murine bone marrow-derived macrophages (BMM) obtained from TRAF1-deficient and wild-type mice were stimulated with or without TNFα (20ng/ml) for 24h, and cell lysates were analyzed for CD29 (N=4) and CD11b (N=3) by Western blotting. Pooled densitometric data adjusted for GAPDH given as grouped scatter plot, representative blots below.
Figure 5. TRAF1 deficiency differentially regulates chemokine and chemokine receptor expression but does not alter inflammatory cell migration
A. Murine endothelial cells isolated from TRAF1-deficient and –competent mice were stimulated with or without TNFα (20ng/ml)´ for 24h. Kc (CXCL-1) and MIP-2 (CXCL 2) protein was assayed in the supernatant by ELISA (N=8 and 7). Data are given as mean±SEM and as a grouped scatter blot. B. Bone marrow-derived macrophages (BMM) of TRAF1-deficient and -competent mice were stimulated with or without TNFα (20ng/ml). CXCL-1 protein was measured in the supernatants by ELISA and CXCR-1 expression in the cell lysates by Western blotting (N=6 and 3). Data are given as mean±SEM and densitometric mean adjusted for GAPDH ±SEM, and as grouped scatter plot, a representative Western blot is shown below where appropriate. C. Murine monocytes of TRAF1−/−/LDLR−/− and TRAF1+/+/LDLR−/− mice consuming a high cholesterol diet were analyzed for CXCR2, CCR5, CXCR3, and CCR7 expression by FACS (N=3 each). Data represent percentage ±SEM in a grouped scatter blot. D. PBMCs isolated from TRAF1-deficient and –competent mice were assayed for their migratory capacity towards indicated concentrations of CXCL-1, MCP-1, and CXCL-2 in a modified Boyden chamber (N=3 each). Migrated PBMCs were quantified microscopically and given as percentage of loaded cells.
Figure 6. TRAF1 deficiency downregulates inflammatory cytokines but does not affect macrophage differentiation and cellular apoptosis
A. Serum from TRAF1−/−/LDLR−/− and TRAF1+/+/LDLR−/− mice was analyzed for TNFα, IL-1β, IL-6, and IL-1α at baseline and after consumption of high cholesterol diet for 18 weeks. Data represent mean±SEM (N=10 and 16 per group) B. Bone marrow derived macrophages (BMM) fixed on cover slips and sections of spleens of TRAF1−/−/LDLR−/− and TRAF1+/+/LDLR−/− mice were stained with the differentiation markers FA-11 and F4/80 (N=3 each). Similarly, sections of atherosclerotic plaques in the aortic arches of TRAF1−/−/LDLR−/− and TRAF1+/+/LDLR−/− mice fed a high cholesterol diet for 18 weeks were labeled with the proliferation marker Ki-67 (N=6 and 13). Representative images are shown on top. FA-11, F4/80, and Ki-67-positive cells per 100 cells counted were quantified, pooled and are given as grouped scatter plots. C. TRAF1−/−/LDLR−/− and TRAF1+/+/LDLR−/− mice consumed a high cholesterol diet for 18 weeks. Sections of the aortic root were analyzed by TUNEL assay. TUNEL-positive staining in relation to total wall area is displayed as mean±SEM (N=5 and 13).
Figure 7. TRAF1 deficiency does not alter general immune responses
(A) Splenocytes were analyzed ex vivo for CD4 and C8 T cells and their activation (CD44hi, CD62lo) and proliferation marker (KLRG-1hi). (B) Splenocytes were activated with αCD3/αCD28 for 3 days and intracellular TNFα and IFNγ determined. Supernatants were assayed for IL-6, IL12p70, and IL-10. (C) Serum of mice consumed a high cholesterol diet was examined for IFNγ, IL-2, IL12p70, IL-4, IL-5, IL-10 and IL-13. In addition, IFNγ, IL12p70 and IL-10 was assessed in arterial tissue of atherosclerotic and TNFα-stimulated mice as well as in the serum of TNFα -stimulated mice. Data represents mean±SEM.
Figure 8. TRAF1-silencing in EC and monocytes limits the expression of adhesion molecules and integrines and TRAF1 mRNA expression increases in acute coronary syndromes in humans
A. Human umbilical endothelial cells and human monocytes were transfected with TRAF1-specific- or scrambled siRNA. Cells were stimulated with TNFα (20ng/ml) for 24h and expression levels of VCAM-1 and ICAM-1 (N=4 each) in endothelial cells, and Integrin beta 1(CD29) and CXCR1 (N=3 each) in human monocytes analyzed by Western blotting. Densitometric results adjusted for GAPDH are presented as mean±SEM on top and as grouped scatter blot, representative blots below. B. 325 patients undergoing coronary angiography were divided into the three groups: no coronary heart disease (No CHD), stable coronary heart disease (CHD), and acute coronary syndromes (ACS). TRAF1 and GAPDH mRNA was analyzed by quantitative real-time PCR in total blood RNA. Spearman correlation coefficients for continuous variables were used to assess univariate correlations of TRAF1 levels with all variables. Results are presented as mean±standard error computed from the average measurements obtained from each group.
Similar articles
- TRAF5 deficiency accelerates atherogenesis in mice by increasing inflammatory cell recruitment and foam cell formation.
Missiou A, Rudolf P, Stachon P, Wolf D, Varo N, Aichele P, Colberg C, Hoppe N, Ernst S, Münkel C, Walter C, Sommer B, Hilgendorf I, Nakano H, Bode C, Zirlik A. Missiou A, et al. Circ Res. 2010 Sep 17;107(6):757-66. doi: 10.1161/CIRCRESAHA.110.219295. Epub 2010 Jul 22. Circ Res. 2010. PMID: 20651286 Clinical Trial. - P2Y6 deficiency limits vascular inflammation and atherosclerosis in mice.
Stachon P, Peikert A, Michel NA, Hergeth S, Marchini T, Wolf D, Dufner B, Hoppe N, Ayata CK, Grimm M, Cicko S, Schulte L, Reinöhl J, von zur Muhlen C, Bode C, Idzko M, Zirlik A. Stachon P, et al. Arterioscler Thromb Vasc Biol. 2014 Oct;34(10):2237-45. doi: 10.1161/ATVBAHA.114.303585. Epub 2014 Aug 7. Arterioscler Thromb Vasc Biol. 2014. PMID: 25104800 - Discoidin domain receptor 1 (ddr1) deletion decreases atherosclerosis by accelerating matrix accumulation and reducing inflammation in low-density lipoprotein receptor-deficient mice.
Franco C, Hou G, Ahmad PJ, Fu EY, Koh L, Vogel WF, Bendeck MP. Franco C, et al. Circ Res. 2008 May 23;102(10):1202-11. doi: 10.1161/CIRCRESAHA.107.170662. Epub 2008 May 1. Circ Res. 2008. PMID: 18451340 - Monocyte and macrophage dynamics during atherogenesis.
Ley K, Miller YI, Hedrick CC. Ley K, et al. Arterioscler Thromb Vasc Biol. 2011 Jul;31(7):1506-16. doi: 10.1161/ATVBAHA.110.221127. Arterioscler Thromb Vasc Biol. 2011. PMID: 21677293 Free PMC article. Review. - Monocyte trafficking in acute and chronic inflammation.
Ingersoll MA, Platt AM, Potteaux S, Randolph GJ. Ingersoll MA, et al. Trends Immunol. 2011 Oct;32(10):470-7. doi: 10.1016/j.it.2011.05.001. Epub 2011 Jun 12. Trends Immunol. 2011. PMID: 21664185 Free PMC article. Review.
Cited by
- Tumor necrosis factor receptor-associated factor 5 protects against intimal hyperplasia by regulation of macrophage polarization via directly targeting PPARγ.
Cheng WL, Chao SP, Zhao F, Cai HH, Zeng Z, Cao JL, Jin Z, Deng KQ, Hu X, Wang H, Lu Z. Cheng WL, et al. Inflamm Res. 2024 Jun;73(6):929-943. doi: 10.1007/s00011-024-01875-8. Epub 2024 Apr 20. Inflamm Res. 2024. PMID: 38642079 - Role of CD40(L)-TRAF signaling in inflammation and resolution-a double-edged sword.
Strohm L, Ubbens H, Münzel T, Daiber A, Daub S. Strohm L, et al. Front Pharmacol. 2022 Oct 4;13:995061. doi: 10.3389/fphar.2022.995061. eCollection 2022. Front Pharmacol. 2022. PMID: 36267276 Free PMC article. Review. - Scavenger receptor-targeted plaque delivery of microRNA-coated nanoparticles for alleviating atherosclerosis.
Bai Q, Xiao Y, Hong H, Cao X, Zhang L, Han R, Lee LKC, Xue EY, Tian XY, Choi CHJ. Bai Q, et al. Proc Natl Acad Sci U S A. 2022 Sep 27;119(39):e2201443119. doi: 10.1073/pnas.2201443119. Epub 2022 Sep 19. Proc Natl Acad Sci U S A. 2022. PMID: 36122215 Free PMC article. - Cellular Heterogeneity of the Heart.
Anto Michel N, Ljubojevic-Holzer S, Bugger H, Zirlik A. Anto Michel N, et al. Front Cardiovasc Med. 2022 Apr 25;9:868466. doi: 10.3389/fcvm.2022.868466. eCollection 2022. Front Cardiovasc Med. 2022. PMID: 35548426 Free PMC article. Review. - Circulating Autoantibodies Recognizing Immunodominant Epitopes From Human Apolipoprotein B Associate With Cardiometabolic Risk Factors, but Not With Atherosclerotic Disease.
Marchini T, Malchow S, Caceres L, El Rabih AAH, Hansen S, Mwinyella T, Spiga L, Piepenburg S, Horstmann H, Olawale T, Li X, Mitre LS, Gissler MC, Bugger H, Zirlik A, Heidt T, Hilgendorf I, Stachon P, von Zur Muehlen C, Bode C, Wolf D. Marchini T, et al. Front Cardiovasc Med. 2022 Apr 11;9:826729. doi: 10.3389/fcvm.2022.826729. eCollection 2022. Front Cardiovasc Med. 2022. PMID: 35479271 Free PMC article.
References
- Hansson GK, Libby P. The immune response in atherosclerosis: a double-edged sword. Nat Rev Immunol. 2006;6(7):508–519. - PubMed
- Libby P. Inflammation in atherosclerosis. Nature. 2002;420(6917):868–874. - PubMed
- Branen L, Hovgaard L, Nitulescu M, Bengtsson E, Nilsson J, Jovinge S. Inhibition of tumor necrosis factor-alpha reduces atherosclerosis in apolipoprotein E knockout mice. Arterioscler Thromb Vasc Biol. 2004;24(11):2137–2142. - PubMed
- Chi H, Messas E, Levine RA, Graves DT, Amar S. Interleukin-1 receptor signaling mediates atherosclerosis associated with bacterial exposure and/or a high-fat diet in a murine apolipoprotein E heterozygote model: pharmacotherapeutic implications. Circulation. 2004;110(12):1678–1685. - PubMed
- Mach F, Schonbeck U, Sukhova GK, Atkinson E, Libby P. Reduction of atherosclerosis in mice by inhibition of CD40 signalling. Nature. 1998;394(6689):200–203. - PubMed
Publication types
MeSH terms
Substances
Grants and funding
- R01 HL034636/HL/NHLBI NIH HHS/United States
- R01 HL034636-23/HL/NHLBI NIH HHS/United States
- R37 HL034636/HL/NHLBI NIH HHS/United States
- HL34636/HL/NHLBI NIH HHS/United States
LinkOut - more resources
Full Text Sources
Medical
Molecular Biology Databases
Research Materials