Serum amyloid P component therapeutically attenuates atherosclerosis in mice via its effects on macrophages

Abstract

Background: A hallmark of atherosclerosis is the formation of macrophage-derived foam cells. Serum amyloid P component (SAP), a member of the pentraxin family of proteins, is known to affect macrophage activation. However, the role of SAP in atherosclerosis is still unclear. Methods: Apolipoprotein E-deficient (Apoe^-/-) mice fed a high-fat diet were given intraperitoneal injections of SAP (6 mg/kg) every other day for a total of 2 weeks to characterize atherosclerosis development. Results: We showed that intraperitoneal injection of SAP attenuated atherosclerosis in Apoe^-/- mice. Immunostaining of aortic roots indicated that SAP was up-taken by the lesion area. In SAP-treated mice, serum paraoxonase1 (PON1) activity was increased whereas high-density lipoprotein inflammatory index (HII) was reduced. The cholesterol efflux rate in macrophages was elevated along with the expression of cholesterol efflux proteins. Through bioinformatics analysis followed by experimental validation, we found that proline/serine-rich coiled-coil protein 1 (Psrc1) was an important downstream effector of SAP in macrophages. Conclusions: Our findings reveal an anti-atherosclerotic role of SAP and extend the current knowledge regarding this molecule as a marker for atherosclerosis.

Keywords: atherosclerosis; cholesterol; macrophage; serum amyloid P component.

Figure 1

The effect of SAP treatment on atherosclerotic development in Apoe^-/- mice. (A) A diagram of the experimental design. (B) Staining of the aortic tree (upper panel) and quantification of positive area (lower panel, mean ± SEM, n = 6). (C) Oil Red O staining of the aortic roots (upper panel) and quantification of the lesion area (lower panel). (D) CD68 and SAP immunostaining of aortic roots of Apoe^-/- mice treated with SAP. Quantification of CD68-positive staining area as well as SAP-positive staining area are shown in the right panel. Data are shown as mean ± SEM.

Figure 2

Altered HDL function in Apoe^-/- mice. (A) The serum lipid profiles. HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; TC, total cholesterol; TG, triglyceride. (B) The quantification results of lipoprotein cholesterol efflux capacity. (C) The quantification results of paraoxonase-1 (PON1) activity. (D) The quantification results of HDL inflammatory index (HII). All parameters were screened using 6 mice randomly selected in each group. for the analysis of cholesterol efflux capacity, all 12 mice in each group were used to ensure a statistical power of more than 0.8. (E) The expression of lipid transport-related proteins in RAW264.7 cells treated with serum from Apoe^-/- mice. Western blot was repeated 3 times and a representative image is shown. The mean values of quantified data are labeled on the right.

Figure 3

The role of SAP in macrophages. RAW264.7 cells were treated with different concentrations of SAP. (A) The effect of different concentration of SAP on cholesterol efflux rate in RAW264.7 cells after 24 h treatment (mean ± SEM, n = 4). (B) The effect of SAP on cholesterol efflux rate in RAW264.7 cells across various time points (mean ± SEM, n = 4). *, p < 0.05; **, p < 0.01. (C) The expression of lipid transport-related genes in RAW264.7 cells after SAP treatment for 24 h assessed by qRT-PCR analysis of. (D) Western blot analysis of lipid transport-related genes.

Figure 4

Identification of SAP downstream target genes. (A) Transcriptome analysis revealed 10 AS-related genes were differentially expressed upon SAP treatment. (B) Validation of gene expression by using qRT-PCR. (C) Western blot analysis of Psrc1 expression in vivo.

Figure 5

The involvement of Psrc1 in SAP signaling in RAW264.7 cells. (A) RAW264.7 cells were transfected with siPsrc1. The transfection efficacy was measured by qRT-PCR. (B) The change of cholesterol efflux rate after siPsrc1 transfection. (C) The expression of lipid transport-related genes was tested by qRT-PCR. (D) Western blot analysis of lipid transport-related genes.