Vitamin D Deficiency Accelerates Coronary Artery Disease Progression in Swine

Abstract

Objective: The role of vitamin D deficiency in coronary artery disease (CAD) progression is uncertain. Chronic inflammation in epicardial adipose tissue (EAT) has been implicated in the pathogenesis of CAD. However, the molecular mechanism underlying vitamin D deficiency-enhanced inflammation in the EAT of diseased coronary arteries remains unknown. We examined a mechanistic link between 1,25-dihydroxyvitamin D-mediated suppression of nuclear factor-κB (NF-κB) transporter, karyopherin α4 (KPNA4) expression and NF-κB activation in preadipocytes. Furthermore, we determined whether vitamin D deficiency accelerates CAD progression by increasing KPNA4 and nuclear NF-κB levels in EAT.

Approach and results: Nuclear protein levels were detected by immunofluorescence and Western blot. Exogenous KPNA4 was transported into cells by a transfection approach and constituted lentiviral vector. Swine were administered vitamin D-deficient or vitamin D-sufficient hypercholesterolemic diet. After 1 year, the histopathology of coronary arteries and nuclear protein expression of EAT were assessed. 1,25-dihydroxyvitamin D inhibited NF-κB activation and reduced KPNA4 levels through increased vitamin D receptor expression. Exogenous KPNA4 rescued 1,25-dihydroxyvitamin D-dependent suppression of NF-κB nuclear translocation and activation. Vitamin D deficiency caused extensive CAD progression and advanced atherosclerotic plaques, which are linked to increased KPNA4 and nuclear NF-κB levels in the EAT.

Conclusions: 1,25-dihydroxyvitamin D attenuates NF-κB activation by targeting KPNA4. Vitamin D deficiency accelerates CAD progression at least, in part, through enhanced chronic inflammation of EAT by upregulation of KPNA4, which enhances NF-κB activation. These novel findings provide mechanistic evidence that vitamin D supplementation could be beneficial for the prevention and treatment of CAD.

Keywords: 1,25-dihydroxyvitamin D; KPNA4; atherosclerosis; coronary artery disease; vitamin D.

Figure 1

VD3-dependent suppression of TNF-α-induced p65 nuclear translocation not related to IκBα in the swine epicardial preadipocytes. (A) VD3 had no effect on basal p65 localization, but prevented TNF-α-induced nuclear translocation, as determined by immunofluorescent assay. (B) Western blot confirmed immunofluorescence results shown in panel A. (C) VD3 had no effect on IκBα expression and TNF-α-induced IκBα phosphorylation. **p<0.01 vs. individual control, ^##p<0.01 vs. TNF-α alone (n=3). C: control; TNF: TNF-α.

Figure 2

Effect of VD3 and TNF-α on KPNA4 signature and the role of VD3 in VDR expression. (A and B) VD3 reduced KPNA4 protein and mRNA levels. (C) VD3 increased VDR expression. (D and E) VDR siRNA significantly reduced VDR expression and VD3-induced VDR levels, and knockdown of VDR by the specific siRNA eliminated VD3 effect on KPNA4. (F) TNF-α did not affect either KPNA4 expression or VD3-mediated suppression of KPNA4. The experiments were repeated three to five times. **p<0.01, *p<0.05 vs. individual no treatment or control groups.

Figure 3

VD3 failed to prevent p65 nuclear translocation induced by TNF-α in the cells with overexpression of KPNA4. The experiments were repeated three times and the representative immunofluorescent images are shown.

Figure 4

Forced expression of KPNA4 significantly rescued VD3-dependent inhibition of NF-κB activity. The cells were transfected with NF-κB-Luc/Renilla-Luc in the presence of different doses of CMV6-KPNA4 expression vector or control vector. After 24 hours of transfection, the cells were treated with VD3 for 24 hours and TNF-α for the final 8 hours. **p<0.01, *p<0.05 vs. individual control groups. ^##p<0.01, ^#p<0.05 vs. individual TNF-α alone groups, ^§ p<0.05 vs. TNF-α with the control vector (n=4–6).

Figure 5

Vitamin D deficiency accelerated the progression of CAD induced by hypercholesterolemic diets. The representative images of HE staining from swine right coronary arteries (RCA) are shown and the pooled data in the graph display the quantification of stenosis area in the RCAs from the swine fed on vitamin D-deficient Diet-1(VD-Def, n=4) and sufficient control (VD-Suf, C-1, n=5) diet (A and B) for one year, and vitamin D-deficient Diet-2 (n=4) and sufficient control (C-2) diet (n=3) (C and D) for one year. The relationship between plasma 25(OH)D level versus % stenosis in each swine is shown in (E). **p<0.01 vs. individual control group. ADV: adventitia, IEL: internal elastic lamina, L: lumen, EEL: external elastic lamina, NI: neointima.

Figure 6

Quantitative immunofluorescence (IF) showing increased nuclear p65 and KPNA4 levels in the EAT of swine fed on vitamin D deficient hypercholesterolemic Diet-2 vs. sufficient hypercholesterolemic diet (C-2). The experiments were performed using the sections from right coronary arteries and the EAT of three different swine from each group. KPNA4 (A) and nuclear p65 (B) levels were determined by the overlap of the staining using the specific antibodies and DAPI. The representative immunofluorescent images are shown and the graphs display the intensity of immunofluorescence measured in relative units (RU) in approximately 150 nuclei in 5–6 random fields from three animals per group. **p<0.01 vs. individual control group.