Serum amyloid A directly accelerates the progression of atherosclerosis in apolipoprotein E-deficient mice

Abstract

Although serum amyloid A (SAA) is an excellent marker for coronary artery disease, its direct effect on atherogenesis in vivo is obscure. In this study we investigated the direct effect of SAA on promoting the formation of atherosclerosis in apolipoprotein E-deficient (ApoE⁻/⁻) mice. Murine SAA lentivirus was constructed and injected into ApoE⁻/⁻ mice intravenously. Then, experimental mice were fed a chow diet (5% fat and no added cholesterol) for 14 wks. The aortic atherosclerotic lesion area was larger with than without SAA treatment. With increased SAA levels, the plasma levels of interleukin-6 and tumor necrosis factor-α were significantly increased. Macrophage infiltration in atherosclerotic regions was enhanced with SAA treatment. A migration assay revealed prominent dose-dependent chemotaxis of SAA to macrophages. Furthermore, the expression of monocyte chemotactic protein-1 and vascular cell adhesion molecule-1 (VCAM-1) was upregulated significantly with SAA treatment. SAA-induced VCAM-1 production was detected in human aortic endothelial cells in vitro. Thus, an increase in plasma SAA directly accelerates the progression of atherosclerosis in ApoE⁻/⁻ mice. SAA is not only a risk marker for atherosclerosis but also an active participant in atherogenesis.

Figure 1

Quantification of atherosclerosis in mice fed a high-fat diet. (A) En face analysis of aortas. Atherosclerotic lesions were identified by Oil-Red-O staining. (B) Total atherosclerotic lesion area indicating level of atherogenesis. (C) Oil-Red-O staining of aortic sinus cryosections (bar = 500 μm). (D) Ratio of total atherosclerotic lesion area to aorta lumen area indicating mean size of atherosclerotic plaque. Data are mean ± SD. *P > 0.05 compared with lenti-null group (n = 10 for both groups).

Figure 2

Quantification of atherosclerosis in mice fed a chow diet. (A) En face analysis of aortas. Atherosclerotic lesions were identified by Oil-Red-O staining. (B) Total atherosclerotic lesion area indicating level of atherogenesis (n = 18 for both groups). (C) H&E and Oil-Red-O staining of aortic sinus cryosections (bar = 500 μm). (D) The ratio of total atherosclerotic lesion area to aorta lumen area indicating mean size of atherosclerotic plaque. Data are mean ± SD. *P < 0.01 compared with the lenti-null group (n = 25 for both groups).

Figure 3

Elevated plasma SAA level induces the accumulation of macrophages in atherosclerotic regions of chow-fed mice. (A) Representative images by immunohistochemistry staining for macrophages (brown) (bar = 100 μm). *P < 0.01 compared with the lenti-null group (n = 10 for both groups). (B) The colocalization of SAA (red) with macrophages (green) by immunofluorescence analysis (Magnification 100×). (C) Migration assay of chemotaxis of SAA to macrophages by dilution dose of SAA with DMEM. Data are mean ± SD from 5 separate fields in each sample from 3 independent experiments (bar = 50 μm). *P < 0.05 versus control group and ^#P < 0.05 versus 1:50 group.

Figure 4

SAA upregulates MCP-1 secretion in atherosclerotic plaque of chow-fed mice. (A, B) Immunohistochemistry staining and quantification of MCP-1 (brown, bar = 100 μm; n = 10 for both groups). (C) Real-time PCR analysis of mRNA expression of MCP-1. Relative expression was normalized to that of reference genes β-actin, TBP and GAPDH. Geometric mean of reference gene expressions was used to confirm the robustness of experimental data. Data are mean ± SD. *P < 0.01 compared with the lenti-null group (n = 12 for both groups).

Figure 5

SAA induces VCAM-1 production in atherosclerotic lesions of chow-fed mice and in vitro. (A, B) Expression of VCAM-1 (green) by immunofluorescence analysis and quantification (magnification 200×; n = 10 for both groups). (C) Real-time PCR analysis of VCAM-1 mRNA expression in aortas (n = 12 for both groups). Relative expression was normalized to that of a reference gene group including β-actin, TBP and GAPDH. Geometric mean of reference gene expressions was used to confirm the robustness of experimental data. Data are mean ± SD. *P < 0.01 compared with lenti-null group. (D) and (E) HAECs were stimulated with recombinant human SAA (20 μg/mL) for various times. Western blot and real-time PCR analysis of VCAM-1 protein and mRNA expression. (F) Human aortic endothelial cells were treated with different concentrations of SAA for 24 h. Protein expression of VCAM-1 was investigated with Western blot analysis. Data are mean ± SD from 3 independent experiments performed in duplicate. *P < 0.05 compared with control.