Citreoviridin Enhances Atherogenesis in Hypercholesterolemic ApoE-Deficient Mice via Upregulating Inflammation and Endothelial Dysfunction

Abstract

Vascular endothelial dysfunction and inflammatory response are early events during initiation and progression of atherosclerosis. In vitro studies have described that CIT markedly upregulates expressions of ICAM-1 and VCAM-1 of endothelial cells, which result from NF-κB activation induced by CIT. In order to determine whether it plays a role in atherogenesis in vivo, we conducted the study to investigate the effects of CIT on atherosclerotic plaque development and inflammatory response in apolipoprotein E deficient (apoE-/-) mice. Five-week-old apoE-/- mice were fed high-fat diets and treated with CIT for 15 weeks, followed by assay of atherosclerotic lesions. Nitric oxide (NO), vascular endothelial growth factor (VEGF) and endothelin-1 (ET-1) were detected in serum. Levels of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), VEGF, and ET-1 in plaque areas of artery walls were examined. NF-κB p65 expression and NF-κB activation in aorta also were assessed. CIT treatment significantly augmented atherosclerotic plaques and increased expressions of ICAM-1, VCAM-1, VEGF and ET-1 in aorta. Mechanistic studies showed that activation of NF-κB was significantly elevated by CIT treatment, indicating the effect of CIT on atherosclerosis may be regulated by activation of NF-κB.

Fig 1. Body weight of apoE^-/- mice.

Data are presented as mean ± SEM (n = 10). The statistical analysis of one-way analysis of variance (ANOVA) was performed. There was no significant difference among the three groups.

Fig 2. Serum lipid levels in apoE^-/- mice.

Data are presented as mean ± SD (n = 10). The statistical analysis of one-way analysis of variance (ANOVA) was performed. There was no significant difference among the three groups.

Fig 3. Effect of CIT on atherosclerotic lesions in apoE^-/- mice.

(A) Representative image of en face Oil Red O staining of aorta. (B) Representative cross-sectional view of aortic root stained with oil red O (40×). (C) Ratio of plaque area stained with oil red O to total luminal surface area. Data are presented as mean ± SEM (n = 5). (D) Plaque area stained with oil red O in aortic root (n = 10). The statistical analyses of one-way analysis of variance (ANOVA) and Student-Newman-Keuls (SNK) multiple comparison were performed to determine the significant difference among the three groups. *P < 0.05, **P < 0.01 versus vehicle-treated group; ^# P < 0.05, ^## P < 0.01 versus low dosage CIT treatment group.

Fig 4. Serum NO concentration in apoE^-/- mice.

Data are presented as mean ± SEM (n = 10). The statistical analyses of one-way analysis of variance (ANOVA) and Student-Newman-Keuls (SNK) multiple comparison were performed to determine the significant difference among the three groups. *P < 0.05, **P < 0.01 versus vehicle-treated group; ^# P < 0.05, ^## P < 0.01 versus low dosage CIT treatment group.

Fig 5. ET-1 expression in aorta of apoE^-/- mice treated with CIT.

(A) Immunohistochemical staining of aortic sections (blue = nuclei, brown = target protein, 100×) (B) Integral optical density (IOD) values of ET-1. Data are presented as mean ± SEM (n = 10). (C) The concentration of ET-1 in the serum of apoE^-/- mice (n = 10). The statistical analyses of one-way analysis of variance (ANOVA) and Student-Newman-Keuls (SNK) multiple comparison were performed to determine the significant difference among the three groups. *P < 0.05, **P < 0.01 versus vehicle-treated group; ^# P < 0.05, ^## P < 0.01 versus low dosage CIT treatment group.

Fig 6. VEGF expression in aorta of apoE^-/- mice treated with CIT.

(A) Immunohistochemical staining of aortic sections (blue = nuclei, brown = target protein, 100×). (B) Integral optical density (IOD) values of VEGF. Data are presented as mean ± SEM (n = 10). (C) The concentrations of VEGF in serum of apoE^-/- mice(n = 10). The statistical analyses of one-way analysis of variance (ANOVA) and Student-Newman-Keuls (SNK) multiple comparison were performed to determine the significant difference among the three groups. *P < 0.05, **P < 0.01 versus vehicle-treated group; ^# P < 0.05, ^## P < 0.01 versus low dosage CIT treatment group.

Fig 7. ICAM-1 expression in aorta of apoE^-/- mice.

(A) Immunohistochemical staining of aortic sections (blue = nuclei, brown = target protein, 100×). (B) Integral optical density (IOD) value of ICAM-1. Data are presented as mean ± SEM (n = 10). (C) The relative ICAM-1mRNA level (n = 5). The values are shown as ratios compared to the levels of mRNA expression in control mice. The statistical analyses of one-way analysis of variance (ANOVA) and Student-Newman-Keuls (SNK) multiple comparison were performed to determine the significant difference among the three groups. *P < 0.05, **P < 0.01 versus vehicle-treated group; ^# P < 0.05, ^## P < 0.01 versus low dosage CIT treatment group.

Fig 8. VCAM-1 expression in aorta of apoE^-/- mice treated with CIT.

(A) Immunohistochemical staining of aortic sections (blue = nuclei, brown = target protein, 100×). (B) Integral optical density (IOD) value of VCAM-1. Data are presented as mean ± SEM (n = 10). (C) The relative VCAM-1mRNA level (n = 5). The values are shown as ratios compared to the levels of mRNA expression in control mice. The statistical analyses of one-way analysis of variance (ANOVA) and Student-Newman-Keuls (SNK) multiple comparison were performed to determine the significant difference among the three groups. *P < 0.05, **P < 0.01 versus vehicle-treated group; ^# P < 0.05, ^## P < 0.01 versus low dosage CIT treatment group.

Fig 9. CIT increased NF-κB activation in aorta.

(A) Expression of NF-κB p65, phospho-NF-κB p65, I-κB, and phospho-I-κB in aorta analyzed by Western blotting. (B) The levels of phospho-NF-κB p65/NF-κB p65 and phospho-I-κB/I-κB. Data are presented as mean ± SEM (n = 5). The statistical analyses of one-way analysis of variance (ANOVA) and Student-Newman-Keuls (SNK) multiple comparison were performed to determine the significant difference among the three groups. *P < 0.05, **P < 0.01 versus vehicle-treated group; ^# P < 0.05, ^## P < 0.01 versus low dosage CIT treatment group.