The Mechanism of Simvastatin-Mediated M1 Macrophage Polarization Contributing to Osteogenesis and Angiogenesis

Abstract

Background: The immune response is essential for bone regeneration, and macrophages in the immune microenvironment contribute to bone metabolism and angiogenesis. Emerging evidence demonstrates that simvastatin is a promising candidate for bone repair and promotes bone formation both in vitro and in vivo. However, the effect of simvastatin on macrophages and the following outcomes are still unclear. Objectives: This study aimed to investigate the potential immunomodulatory effect of simvastatin on M1 macrophages and its subsequent impact on osteogenesis and angiogenesis. Methods: Cell viability was assessed by CCK-8. Osteogenic and angiogenic markers were evaluated by RT-qPCR, Western blotting, and immunofluorescence. M1 macrophage phenotype was analyzed by flow cytometry. Osteogenesis was examined by histological staining, and angiogenic capacity was assessed using functional assays. Results: The present study found that simvastatin decreased M1 macrophage markers (CD86) and stimulated M1 macrophages to express high levels of pro-regenerative cytokines (BMP-2 and VEGF). In addition, simvastatin promoted osteogenic differentiation in MC3T3-E1 cells and angiogenic gene expression in HUVECs. Importantly, simvastatin enhanced the osteogenic capacity of MC3T3-E1 and the angiogenic potential of HUVECs by inhibiting M1 macrophage polarization in vitro. Conclusions: We demonstrated that simvastatin could confer favorable bone immunomodulatory properties and influence the crosstalk behavior between immune cells and osteoblasts and vascular endothelial cells to promote bone healing.

Keywords: angiogenesis; bone immune; macrophage polarization; osteogenesis; simvastatin.

Figure 1

Simvastatin promotes MC3T3-E1 cells’ osteogenic differentiation ability. (A) Cell proliferation of MC3T3-E1 treated with simvastatin for 1, 2, and 3 days. (B) ALP activity of MC3T3-E1 cells treated with simvastatin for 7 days. (C) Expression of osteogenic-related genes of MC3T3-E1 cells treated with simvastatin for 7 and 14 days. (D) Alizarin red staining and quantitative analysis of MC3T3-E1 cells treated with simvastatin after 14 days of osteogenesis induction. * p < 0.05, ** p < 0.01, *** p < 0.001. SIM, simvastatin.

Figure 2

Simvastatin promotes HUVECs’ angiogenic capability. (A) Cell proliferation of HUVECs with simvastatin for 12 h, 24 h, 48 h, and 72 h. (B) Representative images and relative quantification of wound healing assays of HUVECs with simvastatin treatment (scale of 100 μm). (C) Representative images and relative quantification of node number, branch number, and total branching length of tube formation assay (scale of 100 μm). * p < 0.05, ** p < 0.01, *** p < 0.001. SIM, simvastatin.

Figure 3

Simvastatin increases the expression of angiogenic-related genes in HUVECs. (A) Detection of CD31, VEGF, and HIF-1α expression in HUVECs after 3 days of simvastatin treatment. (B) Representative images of immunofluorescence staining of HUVECs treated with simvastatin after 3 days for CD31, VEGF, and HIF-1α and quantification of expression (a scale of 100 μm) * p < 0.05, ** p < 0.01, *** p < 0.001. SIM, simvastatin; MFI, Mean Fluorescence Intensity.

Figure 4

Simvastatin inhibits M1 macrophage polarization in RAW264.7 cells. (A) Cell proliferation of RAW264.7 treated with simvastatin for 1, 2, and 3 days. (B) Detection of CD86 expression in RAW264.7 cells treated with simvastatin. (C) Immunofluorescence staining of CD86 on RAW264.7 cells treated with simvastatin (scale of 50 μm). (D) Flow cytometry analysis of the expression of CD86 treated with simvastatin. * p < 0.05, ** p < 0.01, *** p < 0.001. SIM, simvastatin.

Figure 5

Simvastatin modulates macrophage polarization to promote osteogenesis. (A) Detection of BMP2 and VEGF expression in RAW264.7 cells after 12 h of simvastatin treatment. (B) Western blotting assay and quantification of BMP2 expression in RAW264.7 cells treated with simvastatin. (C) Alizarin red staining and quantitative analysis of co-cultured MC3T3-E1 cells and RAW264.7 pretreated with simvastatin after 14 days of osteogenesis induction. (D) Detection of ALP and RUNX2 expression in the co-culture of MC3T3-E1 cells and RAW264.7 pretreated with simvastatin after 7 and 14 days of osteogenesis induction. * p < 0.05, ** p < 0.01, *** p < 0.001. SIM, simvastatin.

Figure 6

Simvastatin regulates macrophage polarization to promote angiogenesis. (A) Representative images and relative quantification of wound healing assays of co-cultured HUVECs and RAW264.7 with a simvastatin treatment (scale of 100 μm). (B) Representative images and relative quantification of node number, branch number, and total branching length of tube formation assay of co-cultured HUVECs and RAW264.7 with simvastatin treatment (scale of 100 μm). (C) Representative images of immunofluorescence staining of co-cultured HUVECs and RAW264.7 pre-treated with or without 0.5 μM simvastatin after 3 days for VEGF (green) and VEGF (red) and quantification of expression (scale of 200 μm). (D) Detection of CD3, VEGF, and HIF-1α expression in co-cultured HUVECs and RAW264.7 pretreated with simvastatin after 5 days. * p < 0.05, ** p < 0.01, *** p < 0.001. SIM, simvastatin.