Atorvastatin improves plaque stability in ApoE-knockout mice by regulating chemokines and chemokine receptors

Abstract

It is well documented that statins protect atherosclerotic patients from inflammatory changes and plaque instability in coronary arteries. However, the underlying mechanisms are not fully understood. Using a previously established mouse model for vulnerable atherosclerotic plaque, we investigated the effect of atorvastatin (10 mg/kg/day) on plaque morphology. Atorvastatin did not lower plasma total cholesterol levels or affect plaque progression at this dosage; however, vulnerable plaque numbers were significantly reduced in the atorvastatin-treated group compared to control. Detailed examinations revealed that atorvastatin significantly decreased macrophage infiltration and subendothelial lipid deposition, reduced intimal collagen content, and elevated collagenase activity and expression of matrix metalloproteinases (MMPs). Because vascular inflammation is largely driven by changes in monocyte/macrophage numbers in the vessel wall, we speculated that the anti-inflammatory effect of atorvastatin may partially result from decreased monocyte recruitment to the endothelium. Further experiments showed that atorvastatin downregulated expression of the chemokines monocyte chemoattractant protein (MCP)-1, chemokine (C-X3-C motif) ligand 1 (CX3CL1) and their receptors CCR2 and, CX3CR1, which are mainly responsible for monocyte recruitment. In addition, levels of the plasma inflammatory markers C-reactive protein (CRP) and tumor necrosis factor (TNF)-α were also significantly decrease in atorvastatin-treated mice. Collectively, our results demonstrate that atorvastatin can improve plaque stability in mice independent of plasma cholesterol levels. Given the profound inhibition of macrophage infiltration into atherosclerotic plaques, we propose that statins may partly exert protective effects by modulating levels of chemokines and their receptors. These findings elucidate yet another atheroprotective mechanism of statins.

Figure 1. Effect of atorvastatin (10 mg/kg/d) on atherosclerotic plaque morphology in ApoE-/- mice.

(A) Representative images of H&E staining (scale bar = 100 µm) and intimal surface/media ratio quantification in the control and atorvastatin-treated groups (p>0.05, n = 10). (B) Representative images of immunostaining for α-SMC actin (scale bar  = 100 µm) and quantification (p>0.05, n = 6). (C) Representative immunostaining for macrophages (scale bar  = 100 µm) and quantification (*p<0.05, n = 6). (D) Representative images of Oil Red O staining (scale bar  = 100 µm) and quantification (*p<0.05, n = 6). (E) Representative images of Sirius red staining (scale bar  = 100 µm) and quantification (*p<0.05, n = 6). Values are the mean ± SEM.

Figure 2. Effect of atorvastatin (10 mg/kg/d ) on collagenase activity and MMP-8 and MMP-13 expression in the neointima.

(A) Representative images of in situ collagenase activity (scale bar  = 100 µm) and quantification in the control and atorvastatin-treated groups (*p<0.05, n = 6). (B) Representative images of MMP-8 immunohistochemical labeling (scale bar  = 100 µm) and quantification in the control and atorvastatin-treated groups (*p<0.05, n = 6). (C) Representative images of MMP-13 immunohistochemical staining (scale bar  = 100 µm) and quantification in the control and atorvastatin-treated groups (*p<0.05, n = 6). Values are the mean ± SEM.

Figure 3. Effect of atorvastatin (10 mg/kg/d)on expression levels of intimal chemokines and their receptors e on peripheral blood monocytes in ApoE-/- mice.

(A) Representative images of immunostaining for MCP-1 and CX3CL1 (scale bar  = 100 µm) and quantification in the control and atorvastatin-treated groups (*p<0.05, n = 6). (B) Representative FACS analysis for monocytes with double-immunofluorescent labeling for CCR2 and CX3CR1. Data represent the mean ± SEM of the percentage of marker-positive cells from six experiments. *p<0.05 compared with control.

Figure 4. Effect of atorvastatin (10 mg/kg/d ) on plasma inflammatory markers TNF-α and CRP in ApoE-/- mice.

Blood was collected 8 weeks after isosmotic saline or atorvastatin administration. Data represent the mean ± SEM of six ELISA experiments. *p<0.05 compared with control.