Dickkopf-3 Ablation Attenuates the Development of Atherosclerosis in ApoE-Deficient Mice

Abstract

Background: Dickkopf-3 (DKK3) is a negative regulator of the Wnt/β-catenin signaling pathway, which is involved in inflammation. However, little is known about the relationship between DKK3 expression and the progression of atherosclerosis. The aim of the present study was to define the role of DKK3 and its potential mechanism in the development of atherosclerosis.

Methods and results: Immunofluorescence analysis showed that DKK3 was strongly expressed in macrophages of atherosclerotic plaques from patients with coronary heart disease and in hyperlipidemic mice. The expression level was significantly increased in atherogenesis. DKK3-/-ApoE-/- mice exhibited a significant decrease in atherosclerotic lesions in the entire aorta, aortic sinus, and brachiocephalic arteries. Transplantation of bone marrow from DKK3-/-ApoE-/- mice into lethally irradiated ApoE-/- recipients resulted in a reduction of atherosclerotic lesions, compared with the lesions in recipients transplanted with ApoE-/- donor cells, suggesting that the effect of DKK3 deficiency was largely mediated by bone marrow-derived cells. A reduction in the necrotic core size, accompanied by increased collagen content and smooth muscle cells and decreased accumulation of macrophages and lipids, contributed to the stability of plaques in DKK3-/-ApoE-/- mice. Furthermore, multiple proinflammatory cytokines exhibited marked decreases in DKK3-/-ApoE-/- mice. Finally, we observed that DKK3 ablation increased β-catenin expression in the nuclei of macrophages both in vivo and in vitro.

Conclusions: DKK3 expression in macrophages is involved in the pathogenesis of atherosclerosis through modulation of inflammation and inactivation of the Wnt/β-catenin pathway.

Keywords: atherosclerosis; dickkopf‐3; inflammation; macrophage; β‐catenin.

© 2017 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.

Figures

Figure 1

Expression of DKK3 in atheromatous lesions of mice. A, Western blotting analysis of DKK3 protein levels in aorta of mice. The expression levels were normalized to GAPDH and quantified. n=4. *P<0.05. B, Representative images showing double immunofluorescence staining of mouse aortic sinus for DKK3 (green) and macrophages (red). Scale bar=50 μm. C, Western blot analysis of DKK3 expression in BMDMs on oxidized‐LDL treatment. *P<0.05. BMDMs indicates bone marrow‐derived macrophages; DKK3, Dickkopf‐3; LDL, low‐density lipoprotein.

Figure 2

Development of atherosclerosis resulted from DKK3 deficiency. A, En face Oil Red O staining of aortas from DKK3−/−ApoE−/− mice and control mice fed NC or a HFD. Lesion occupation was quantified and shown in the right and bottom panel. n=15 to 18. B, Hematoxylin and eosin staining of the lesions in aortic root sections. Scale bar=500 μm, n=8 to 10. C, Brachiocephalic artery sections were stained with hematoxylin and eosin. Scale bar=100 μm, n=11 to 12. D and E, Body weight, triglycerides, total cholesterol, and lipoprotein profiles from DKK3−/−ApoE−/− and ApoE−/− mice fed a HFD. n=20. *P<0.05 in A through C. DKK3 indicates Dickkopf‐3; HDL, high‐density lipoprotein; HFD, high‐fat diet; IDL, intermediate‐density lipoprotein; LDL, low‐density lipoprotein; NC, normal chow; TC, total cholesterol; TG, triglyceride; VLDL, very‐low‐density lipoprotein.

Figure 3

The necrotic area and plaque stability characteristics in DKK3‐deficient mice. A and B, Representative aortic sinus sections (A) and brachiocephalic arteries (B) from DKK3−/−ApoE−/− and ApoE−/− mice were stained with hematoxylin and eosin. The circular region indicates necrotic areas. Below panel: quantification of anuclear, afibrotic, and eosin‐negative necrotic areas. n=8 to 12. C and D, Cross sections of the aortic sinus plaques were stained with picrosirius red for collagen, α‐smooth muscle actin for smooth muscle cells, CD68 for macrophages, and Oil Red O for lipids. The assessment of plaque stability score in DKK3−/−ApoE−/− and ApoE−/− mice. n=5 to 10, Scale bar=100 μm. *P<0.05 in A, B, and D. DKK3 indicates Dickkopf‐3.

Figure 4

Atherogenesis of ApoE−/− mice transplanted with DKK3 deficient bone marrow cells. A and B, The genotype of ApoE (A) and DKK3 (B) in genomic DNA isolated from circulating white blood cells of recipient animals transplanted with DKK3−/−ApoE−/− or ApoE−/− bone marrow. n=4. C, Representative aorta stained with Oil Red O (n=10) (left panel) and quantification of plaque occupation in mice that received bone marrow cells from DKK3−/−ApoE−/− or ApoE−/− mice. D, Aortic root sections stained with hematoxylin and eosin (n=10) and quantification of plaque area. *P<0.05 in (C and D). DKK3 indicates Dickkopf‐3.

Figure 5

Inflammatory cytokine production and activity of the NF‐κB signaling pathway in DKK3 knockout mice. A, mRNA levels of proinflammatory markers in the aortas of ApoE−/− and DKK3−/−ApoE−/− mice were measured by real‐time PCR. n=6. B, Serum TNF‐α, MCP‐1, IL‐6 and IL‐1β levels were measured by ELISA in ApoE−/− and DKK3−/−ApoE−/− mice. n=10. C, Immunofluorescence staining for ICAM‐1, IL‐6, and p65 phosphorylation in the aortic sinuses of ApoE−/− and DKK3−/−ApoE−/− mice. n=5. D, Western blotting analysis of the NF‐κB signaling pathway in the aortas of ApoE−/− and IRF3−/−ApoE−/− mice, as assessed by the levels of IKKβ, IκBα, and p65 phosphorylation. Protein expression levels were normalized to GAPDH. *P<0.05 in A, B, and D. DKK3 indicates Dickkopf‐3; IL‐6, interleukin‐6; IL‐1β, interleukin‐1β; MCP‐1, monocyte chemoattractant protein‐1; TNF‐α, tumor necrosis factor‐α;

Figure 6

Activation of the Wnt/β‐catenin pathway in DKK3 deficiency. A, The total protein levels of β‐catenin in the nucleus and the phosphorylated and total protein levels of β‐catenin in the cytoplasm of DKK3−/−ApoE−/− and ApoE−/− mice. *P<0.05. B, Immunofluorescence costaining of atherosclerotic plaques with β‐catenin (red) and CD68 (green). n=3. Scale bar=20 μm. C, Western blot analysis of β‐catenin in the nucleus and the phosphorylated and total protein levels of β‐catenin in the cytoplasm of peritoneal macrophages isolated from ApoE−/− and DKK3−/− ApoE−/− upon oxidized‐LDL stimulation. *P<0.05. D, mRNA levels of proinflammatory markers in peritoneal macrophages isolated from ApoE−/− and DKK3−/− ApoE−/− treatment with oxidized LDL. *P<0.05. DKK3 indicates Dickkopf‐3; LDL, low‐density lipoprotein.

Figure 7

Expression of DKK3 in human atheromatous lesions. A, Western blotting analysis of DKK3 protein levels in the right coronary artery in humans. The expression levels were normalized to GAPDH and quantified. n=4. *P<0.05. B, Representative images showing double‐immunofluorescence staining of human coronary arteries for DKK3 (green) and macrophages (red). Scale bar=50 μm. DKK3 indicates Dickkopf‐3.