LRP5 deficiency down-regulates Wnt signalling and promotes aortic lipid infiltration in hypercholesterolaemic mice

Abstract

Low-density lipoprotein receptor-related protein 5 (LRP5) is a member of the LDLR family that orchestrates cholesterol homoeostasis. The role of LRP5 and the canonical Wnt pathway in the vascular wall of dyslipidaemic animals remains unknown. In this study, we analysed the role of LRP5 and the Wnt signalling pathway in mice fed a hypercholesterolaemic diet (HC) to trigger dyslipidaemia. We show that Lrp5(-/-) mice had larger aortic lipid infiltrations than wild-type mice, indicating a protective role for LRP5 in the vascular wall. Three members of the LDLR family, Lrp1, Vldlr and Lrp6, showed up-regulated gene expression levels in aortas of Lrp5(-/-) mice fed a hypercholesterolaemic diet. HC feeding in Lrp5(-/-) mice induced higher macrophage infiltration in the aortas and accumulation of inflammatory cytokines in blood. Wnt/β-CATENIN signalling proteins were down-regulated in HC Lrp5(-/-) mice indicating that LRP5 regulates the activation of Wnt signalling in the vascular wall. In conclusion, our findings show that LRP5 and the canonical Wnt pathway down-regulation regulate the dyslipidaemic profile by promoting lipid and macrophage retention in the vessel wall and increasing leucocyte-driven systemic inflammation.

Keywords: LRP5; atherosclerosis; canonical Wnt signalling; macrophages; plasma cholesterol.

Fig 1

Hypercholesterolaemic (HC) Lrp5^−/− mice model characterization. (A) Agarose gel showing Wt, Lrp5^−/− and Lrp5^−/+ alleles. (B) Serum cholesterol levels in Wt and Lrp5^−/− mice fed a normocholesterolaemic (NC) or a HC diet. (C) Non-HDL-C in Wt and Lrp5^−/− mice fed a NC or a HC diet. (D) Representative images of mouse thoracic aortas stained with ORO. (E) Quantification of lipid area in mice aortas. (F) Cholesteryl ester measurement by thin layer chromatography and bar graph showing CE quantification. *P < 0.05; ***P < 0.005.

Fig 2

Regression analyses between total cholesterol and aortic lipid coverage in Wt (A) and Lrp5^−/− mice (B) and between non-HDL cholesterol and aortic lipid coverage in Wt (C) and Lrp5^−/− mice (D) with their statistical significances (p), correlation coefficients (r) and slopes (b).

Fig 3

Hypercholesterolaemic (HC) Lrp5^−/− mice show increased aortic macrophage infiltration. (A) Lrp5 expression levels in blood leucocytes in normocholesterolaemic (NC) or HC Wt and Lrp5^−/− mice. (B) HC Wt and HC Lrp5^−/− mice aortas labelled with HAM56, L, lumen. (C) Quantitative analysis of B expressed as number of macrophages/field of vision. (D) White blood cells gene expression of pro-inflammatory cytokines in HC Wt and HC Lrp5^−/− mice. *P < 0.05; ***P < 0.005.

Fig 4

Wnt pathway modulation in aortas from Wt and Lrp5^−/− mice. (A) Representative images of aortas sections immunostained with β-CATENIN in normocholesterolaemic (NC) or hypercholesterolaemic (HC) Wt and Lrp5^−/− mice. (B) % area stained by β-CATENIN. (C) Representative images of aortas sections immunostained with MMP-7 in NC or a HC Wt and Lrp5^−/− mice. (D) Quantitative analysis of aortas in C. **P < 0.01.

Fig 5

Receptors expression in mice aortas. mRNA expression levels of Lrp2 (A), Lrp8 (B), Ldlr (C) and Cd36 (D) in aortas from Wt and Lrp5^−/− mice fed a normocholesterolaemic (NC) or a hypercholesterolaemic (HC) diet. *P < 0.05; ***P < 0.005.

Fig 6

Receptors expression in mice aortas. mRNA expression levels of Lrp1 (A), Vldlr (B) and Lrp6 (C) in aortas from Wt and Lrp5^−/− mice fed a normocholesterolaemic (NC) or a hypercholesterolaemic (HC) diet. *P < 0.05; ***P < 0.005. (D) Regression analyses between aortic lipid coverage and Lrp6 or Lrp1 gene expression levels in aortas of Lrp5^−/− mice with their statistical significances (p), correlation coefficients (r) and slopes (b).