Vitamin D and macrophage polarization in epicardial adipose tissue of atherosclerotic swine

Figures

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  Fig 1. Evaluation of blood parameters and weight in different animal groups. (A) Serum 25(OH) D level in vitamin D-deficient (Vit.D.Def.), vitamin D-sufficient (Vit.D.Suf.) (p< 0.014) and vitamin D-supplemented swine (Vit.D.Sup.) (p< 0.0004). (B) Bar graph showing levels of serum glucose (mg/dL) at baseline and at time of euthanasia. (C) Bar graph showing weight (kg) at baseline and at time of euthanasia. (D) Bar graph showing levels of serum total cholesterol (mg/dL) at baseline and at time of euthanasia. (n = 3 in each experimental group).

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  Fig 2. Hematoxylin and Eosin (H&E) staining and expression of vitamin D receptor (VDR). (A-C) shows the H&E staining of post-balloon angioplasty site of the left circumflex coronary artery (LCx); (D-F) shows the effect of vitamin D on vitamin D receptor (VDR) expression in EAT of: vitamin D-deficient (Vit.D.Def.), vitamin D-sufficient (Vit.D.Suf.) and vitamin D-supplemented (Vit.D.Sup.) groups; (G) shows the percent stenosis. (n = 3 in each treatment group); ***p<0.0005, **p<0.003. (H) The mean fluorescent intensity (MFI) was measured for VDR+cells in each treatment group. (n = 3 in each experimental group); *p<0.04. (I) A scatter plot showing the MFI of VDR with change in the serum 25(OH) D level.

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  Fig 3. Effect of vitamin D on TNF-α and MCP1 expression. Comparison of Immunofluorescence staining for TNF-α (A-C) and MCP1 (D-F) cells in the EAT of: vitamin D-deficient (Vit.D.Def.), vitamin D-sufficient (Vit.D.Suf.) and vitamin D-supplemented (Vit.D.Sup.) groups. (G) The mean fluorescent intensity (MFI) values for TNF-α+cells were measured for each treatment group. (H) The MFI values for MCP1 cells were measured for each treatment group. Data show the MFI ± SEM (n = 3 in each treatment group); **p <0.0012. ****p <0.0001.

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  Fig 4. Effect of vitamin D on CD14/CCR7 expression. (A-I) Double immunofluorescence staining for CCR7 and CD14 in EAT of: vitamin D-deficient (Vit.D.Def.), vitamin D-sufficient (Vit.D.Suf.) and vitamin D-supplemented (Vit.D.Sup.) groups. (A-C) Cy-3 (red) images are CCR7+ Cells. (D-F) FITC (green) images are all CD14+ Cells. (J) Data reveal that more cells were expressing CCR7 in vitamin D-deficient swine and lower in vitamin D-supplemented swine than with vitamin D-sufficient swine. (n = 3 in each treatment group); ***p <0.0001.

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  Fig 5. Effect of vitamin D on CD14/CD206 expression. (A-I) Double immunofluorescence staining for CD206 and CD14 in EAT of: vitamin D-deficient (Vit.D.Def.), vitamin D-sufficient (Vit.D.Suf.) and vitamin D-supplemented (Vit.D.Sup.) groups. (A-C) Cy-3 (red) images are CD206+ Cells. (D-F) FITC (green) images are all CD14+ Cells. (J) Data reveals that few cells were expressing CD206 in vitamin D-deficient swine and more in vitamin D-supplemented swine than with vitamin D-sufficient swine. (n = 3 in each treatment group); *p <0.05, ****p <0.0001.

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  Fig 6. A scatter plot of the M1 and M2 macrophage. (A) A representation of the M1 and M2 macrophage density in the epicardial adipose tissue (EAT) of each animal against their serum 25 (OH)D levels. The solid circle (●) represents M1 macrophage number for the swine under 10 high power field (HPF). The solid square (■) represents M2 macrophage number for the swine under 10 HPF. (B) A scatter plot of the M1/M2 macrophage ratio against their serum 25 (OH)D level in each animal.

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  Fig 7. Pictorial representation of the effects of vitamin D in regulating pro-inflammatory signaling in the epicardial adipose tissue (EAT). Vascular smooth muscle cells (VSMC).