All-trans-retinoic acid ameliorated high fat diet-induced atherosclerosis in rabbits by inhibiting platelet activation and inflammation

Abstract

Background: All-trans-retinoic acid (atRA) is effective for many proliferative diseases. We investigated the protective effects of atRA against atherosclerosis.

Methods: Rabbits were randomly allocated to receive basal diet or an HFD for 4 weeks. HFD group then received rosuvastatin (3 mg/day), atRA (5 mg/kg/day), or the same volume of vehicle, respectively, for next 8 weeks.

Results: HFD group showed increases in plasma lipids and aortic plaque formation. P-selectin expression and fibrinogen binding on platelets or deposition on the intima of the aorta also increased significantly as did the levels of TNF-α, IL-6, and fibrinogen in plasma. After 8 weeks of treatment with atRA, there was a significant decrease in plasma lipids and improvement in aortic lesions. AtRA also inhibited the expression of P-selectin and fibrinogen binding on platelets and deposition on the intima of the aorta.

Conclusion: AtRA can ameliorate HFD-induced AS in rabbits by inhibiting platelet activation and inflammation.

Figure 1

Characteristics of arterial lesions. Pathological sections of arterial lesions were examined by HE staining in different groups. In the control group (a), the vessel walls were thin and smooth with even thicknesses; in the HFD group (b), there were more foam cells and necrotic substances in the intima. Compared with the HFD group, there were less foam cells, necrotic substances in the intima from rosuvastatin group (c) and atRA group (d) (magnification ×200). Statistical results of aortic intimal thickness (e) and maximal vessel wall thickness (f) among different groups; *P < 0.05 compared to control group, ^▲P < 0.05 compared to HFD group.

Figure 2

Platelet fibrinogen binding in different groups. Platelets isolated from rabbits were incubated with FITC-labelled anti-fibrinogen antibody and analyzed with flow cytometry to examine the platelets surface change. Representative histograms are shown. (a) The binding of fibrinogen on the resting platelets surface from different groups. (b) The binding of fibrinogen on the thrombin-activated platelets from different groups. (c) The binding of fibrinogen on the ADP-activated platelets from different groups. (d) Mean fluorescence intensity (MFI) of fibrinogen binding on platelets surface in different groups. Data are mean ± standard deviation (SD). Comparisons between groups were carried out using one-way analysis of variance (ANOVA) and Student-Newman-Keuls (SNK) method (n = 6). **P < 0.01 compared to control group, ^▲P < 0.05, ^▲▲P < 0.01 compared to HFD group.

Figure 3

Platelet CD62-P expressions in different groups. Isolated platelets were incubated with FITC-labelled anti-CD62P antibody, and surface CD62-P changes were monitored by flow cytometry. Representative histograms from every group are shown. (a) The expression of CD62-P on the resting platelets surface from different groups. (b) The expression of CD62-P on the thrombin-activated platelets from different groups. (c) The expression of CD62-P on the ADP-activated platelets from different groups. (d) Mean fluorescence intensity (MFI) of CD62-P expression on platelets surface in different groups. Data are mean ± standard deviation (SD). Comparisons between groups were carried out using one-way analysis of variance (ANOVA) and Student-Newman-Keuls (SNK) method (n = 6). **P < 0.01 compared to control group, ^▲P < 0.05, ^▲▲P < 0.01 compared to HFD group.

Figure 4

Fibrinogen deposition in arteries from different groups with immunohistochemistry. Areas of positive fibrinogen deposition are shown in brown. The representative photographs of immunohistochemistry from different groups are shown. In the control group (a), the deposition of fibrinogen in the intima was minimum, while in the HFD group (b), there was much more fibrinogen deposition in the intima. In the rosuvastatin group (c), there was less fibrinogen deposition in the intima compared with HFD group. In the atRA group (d), the fibrinogen deposition was significantly less in comparison with either HFD or rosuvastatin group (magnification ×400).

Figure 5

CD62-P expressions in atherosclerotic lesion from different groups with immunohistochemistry. Representative photographs of immunohistochemistry from different groups are shown. Positive CD62-P expression is shown in brown. In the control group (a), the expression of CD62-P in the intima was minimum. In the HFD group (b), there was significantly increased CD62-P expression in the intima. In the rosuvastatin group (c), there was less but considerable CD62-P expression in the intima. Compared to the HFD group, there was significantly reduced CD62-P expression in the intima from atRA group (d) (magnification ×400).