Low-density lipoprotein receptor-related protein 1 prevents early atherosclerosis by limiting lesional apoptosis and inflammatory Ly-6Chigh monocytosis: evidence that the effects are not apolipoprotein E dependent

Abstract

Background: We previously demonstrated that macrophage low-density lipoprotein receptor (LDLR)-related protein 1 (LRP1) deficiency increases atherosclerosis despite antiatherogenic changes including decreased uptake of remnants and increased secretion of apolipoprotein E (apoE). Thus, our objective was to determine whether the atheroprotective effects of LRP1 require interaction with apoE, one of its ligands with multiple beneficial effects.

Methods and results: We examined atherosclerosis development in mice with specific deletion of macrophage LRP1 (apoE(-/-) MΦLRP1(-/-)) and in LDLR(-/-) mice reconstituted with apoE(-/-) MΦLRP1(-/-) bone marrow. The combined absence of apoE and LRP1 promoted atherogenesis more than did macrophage apoE deletion alone in both apoE-producing LDLR(-/-) mice (+88%) and apoE(-/-) mice (+163%). The lesions of both mouse models with apoE(-/-) LRP1(-/-) macrophages had increased macrophage content. In vitro, apoE and LRP1 additively inhibit macrophage apoptosis. Furthermore, there was excessive accumulation of apoptotic cells in lesions of both LDLR(-/-) mice (+110%) and apoE(-/-) MΦLRP1(-/-) mice (+252%). The apoptotic cell accumulation was partially due to decreased efferocytosis as the ratio of free to cell-associated apoptotic nuclei was 3.5-fold higher in lesions of apoE(-/-) MΦLRP1(-/-) versus apoE(-/-) mice. Lesion necrosis was also increased (6 fold) in apoE(-/-) MΦLRP1(-/-) versus apoE(-/-) mice. Compared with apoE(-/-) mice, the spleens of apoE(-/-) MΦLRP1(-/-) mice contained 1.6- and 2.4-fold more total and Ly6-C(high) monocytes. Finally, there were 3.6- and 2.4-fold increases in Ly6-C(high) and CC-chemokine receptor 2-positive cells in lesions of apoE(-/-) MΦLRP1(-/-) versus apoE(-/-) mice, suggesting that accumulation of apoptotic cells enhances lesion development and macrophage content by promoting the recruitment of inflammatory monocytes.

Conclusion: Low-density lipoprotein receptor protein 1 exerts antiatherogenic effects via pathways independent of apoE involving macrophage apoptosis and monocyte recruitment.

Figure 1. Atherosclerosis in LDLR^−/− mice transplanted with BM from apoE^−/− or apoE^−/−MΦLRP^−/− mice

Quantitation of the mean Oil-red-O (A) and MOMA-2 (B) stained cross-sectional area of proximal aortas from LDLR^−/− mice transplanted with either apoE^−/− (n=10) or apoE^−/−MΦLRP^−/− (n=10) BM. *p<0.05, Mann-Whitney test. C. Representative images show Oil-red-O and MOMA-2 stain in aortic root sections.

Figure 2. Atherosclerosis in apoE^−/− and apoE^−/−MΦLRP^−/− mice

Quantitation of the mean Oil-red-O (A) and MOMA-2 (B) stained cross-sectional area of proximal aortas from apoE^−/− and apoE^−/−MΦLRP^−/− mice (n=12 per group). *p<0.05, Mann-Whitney test. C. Images show Oil-redO and MOMA-2 stain in aortic root sections.

Figure 3. In vitro macrophage apoptosis and atherosclerotic lesion apoptosis

A and B. Images show TUNEL positive (red) and negatively stained macrophages after 16h with DMEM (A). Quantitation of the percent TUNEL positive cells in WT, apoE^−/−, LRP^−/−, and apoE^−/−LRP^−/−cultures (n=6 per group). (B). *, #, and & denotes statistically significances compared to WT, apoE^−/−, and LRP^−/− cells, respectively, p<0.05, ANOVA with Bonferroni’s post test. C. Quantitation of apoptotic cells in proximal aorta sections from apoE^−/− (n=6) or apoE^−/−MΦLRP^−/−(n=7) BM recipient LDLr^−/− mice. *p<0.05, Mann-Whitney test. D. Images show TUNEL staining (dark red) of nuclei in aortic root sections.

Figure 4. Macrophage apoptosis and efferocytosis in lesions of apoE^−/− and apoE^−/− MΦLRP^−/− mice

A Micrographs show nuclei (Hoechst, blue), nuclei + TUNEL positive staining (red), and merged images of macrophage cytoplasm (green), nuclei, and TUNEL in aortic root sections from apoE^−/− and apoE^−/−MΦLRP^−/− mice. Quantitation of the number of apoptotic cells (B) and of the ratio of free versus macrophage associated TUNEL positive cells (C) in proximal aorta sections of lesions from apoE^−/− and apoE^−/−MΦLRP^−/− mice (n=6 per group). *p<0.05, Mann-Whitney test.

Figure 5. Necrosis in lesions of apoE^−/− and apoE^−/−MΦLRP^−/− mice

A Images show hematoxylin and eosin staining of aortic root sections from apoE^−/− and apoE^−/−MΦLRP^−/− mice. NEC denotes necrotic area. B. Quantitation of the percent necrotic area in the aortic lesions from apoE^−/− and apoE^−/−MΦLRP^−/− mice (n=6 per group). *p<0.05, Mann-Whitney test.

Figure 6. Total and Ly-6C^high monocytes in blood and spleens of WT, MΦLRP^−/−, apoE^−/−, and apoE^−/−MΦLRP^−/− mice

A-D WT (n=10), MΦLRP^−/− (n=8), apoE^−/− (n=10), and apoE^−/−MΦLRP^−/− (n=10) mice were fed a western type diet for 8 weeks and then blood (A-B) and spleen (C-D) total (A,C) and Ly-6C^high monocytes (B,D) were measured by flow cytometry analysis. A-D. *, #, and & denotes statistically significances compared to WT, apoE^−/−, and MΦLRP^−/− mice, respectively, p<0.05, ANOVA with Bonferroni’s post test.

Figure 7. Ly-6C and CCR2 positive cells in lesions of apoE^−/− and apoE^−/−MΦLRP^−/− mice

A and D Representative images show nuclei (blue, Dapi) and merged images of Ly-6C (A, green) and CCR positive staining (D, red) and nuclei. B and C. Quantitation of the percent Ly-6C (B) and CCR2 (C) positive cells in aortic root sections from apoE^−/− and apoE^−/−MΦLRP^−/− mice (n=6 per group). *p<0.05, Mann-Whitney test.