Statin suppresses the development of excessive intimal proliferation in a Kawasaki disease mouse model

Abstract

Kawasaki disease (KD) causes vascular injury and lifelong remodeling. Excessive intimal proliferation has been observed, resulting in coronary artery lesions (CALs). However, the mechanisms underlying vascular remodeling in CAL and statin treatment have not been comprehensively elucidated. This study aimed to investigate the effects of statins on vascular remodeling using a KD mouse model. Candida albicans water-soluble substance (CAWS) was intraperitoneally injected in 5-week-old male apolipoprotein-E-deficient mice. They were categorized as follows (n = 4): control, CAWS, CAWS+statin, and late-statin groups. The mice were euthanized at 6 or 10 weeks after injection. Statins (atorvastatin) were initiated after CAWS injection, except for the late-statin group, for which statins were internally administered 6 weeks after injection. Elastica van Gieson staining and immunostaining were performed for evaluation. Statins substantially suppressed the marked neointimal hyperplasia induced by CAWS. Additionally, CAWS induced TGFβ receptor II and MAC-2 expression around the coronary arteries, which was suppressed by the statins. KD-like vasculitis might promote the formation of aneurysm by destroying elastic laminae and inducing vascular stenosis by neointimal proliferation. The anti-inflammatory effects of statins might inhibit neointimal proliferation. Therefore, statin therapy might be effective in adult patients with KD with CAL by inhibiting vascular remodeling.

Keywords: Candida albicans water‐soluble substance; Kawasaki disease; coronary artery lesions; statin; vascular remodeling; vasculitis.

FIGURE 1

(a) Experimental procedure. Candida albicans water‐soluble substance (CAWS; 4 mg for five consecutive days; the control group received phosphate‐buffered saline (PBS) instead) was intraperitoneally injected into 5‐week‐old Apo E−/− mice to induce Kawasaki disease‐like vasculitis (the control group received PBS instead). The diet of all mice was changed to a high‐fat diet to promote atherosclerosis after 2 weeks of CAWS administration. Mice were categorized into four groups: 1. control group, 2. CAWS group, 3. CAWS + statin group and 4. late‐statin group. Statins (10 mg/kg/day) were dissolved in 0.5 mL of 0.5% methylcellulose solution and subsequently orally administered with a sonde. The control group was administered a methylcellulose solution without statin. The CAWS and CAWS + statin groups daily received statins from 2 weeks after CAWS administration to the end of the experiment. The late‐statin group daily received statins from 6 weeks after CAWS administration to the end of the experiment. Mice were euthanized after 6 and 10 weeks of CAWS administration, and the samples were collected. (b) Histological findings at the coronary arteries. Histological findings at the aortic root after CAWS administration for 10 weeks were recorded. In one base specimen, two coronary arteries were identified, enlarged, and analyzed. Immunostaining for macrophage cells and Elastica van Gieson (EVG) staining for elastic fibers around the coronary arteries were performed. Anti‐MAC‐2 antibody staining. Right and left; scale bar, 100 μm. Middle; scale bar, 500 μm. (c) Definition of the percentage of stenosis at the distance between the internal elastic lamina (IEL). Coronary artery neointimal thickness was selected as an indicator of intima formation and defined as the thickened intima extending into the IEL. To reduce the bias owing to the presence or absence of aneurysms, the coronary percentage of stenosis between IELs was comparatively evaluated. Scale bar, 100 μm.

FIGURE 2

(a) Immunostaining findings for coronary arteries. The area ratio of MAC‐2 and transforming growth factor (TGF) βR II expression around coronary arteries was calculated. Candida albicans water‐soluble substance (CAWS) enhanced MAC‐2 and TGFβR II expression. Statin treatment suppressed macrophage cell infiltration and vascular remodeling at the coronary arteries; these effects of statins were also observed in the late‐statin group. Scale bar, 100 μm. (b) Macrophage cell invasion area ratio and TGFβ receptor II expression area ratio. Macrophage cell infiltration area and TGFβR II expression area ratios were calculated and compared between the groups. Area ratios were expressed as percentages. CAWS substantially promoted macrophage cell infiltration and TGFβR II expression at 6 and 10 weeks after CAWS administration. Additionally, statins considerably inhibited macrophage cell infiltration and TGFβR II expression 6 and 10 weeks after CAWS administration. The inhibitory effect of statins was also observed in the late‐statin group. Data were analyzed by the Kruskal–Wallis test. The Wilcoxon test was corrected with a post‐hoc test. The values are mean ± SD. n = 3–4 mice. All data were generated from one experiment in the same timeframe. (c) Correlations between macrophage cell, TGFβR II, and neointimal percentage. Correlation coefficient scatter diagrams are shown. The neointimal percentage at the distance between the internal elastic lamina was markedly associated with the macrophage cell invasion area ratio and TGFβR II expression area ratio in coronary arteries. Scatter plots showing Spearman correlations and correlation coefficients (ρ) between TGFβR II, macrophage cell invasion area ratio, and the neointimal percentage. The same sites were observed and analyzed for two coronary arteries observed in one individual specimen. n = 3–4 mice. All data were generated from one experiment in the same timeframe.

FIGURE 3

(a) Structural evaluation of the elastic fibers in the coronary arteries using Elastica van Gieson staining. These images show the histological findings after 10 weeks of Candida albicans water‐soluble substance (CAWS) administration. CAWS vasculitis disrupted the elastic fiber structure of the coronary arteries. No recovery was observed using statin medication once the elastic fiber structure was broken. Top: Elastica van Gieson (EVG) staining of the coronary artery; transverse section. Scale bar, 100–500 μm. Bottom: Close‐up view of the elastic fiber structure. Black arrow: Internal elastic lamina. Scale bar, 50 μm. (b) Elastin breaks in the elastic lumina. Elastin breaks in the elastic lumina were semi‐quantitatively evaluated to evaluate the effect of statins on CAWS‐induced elastin breaks; EVG staining of coronary arteries from mice treated with phosphate‐buffered saline (PBS), CAWS and CAWS + statin. CAWS‐induced vasculitis damaged the elastic fiber structure of the coronary arteries. Statin therapy did not improve them. Data were analyzed by the Kruskal–Wallis test. The Wilcoxon test was corrected with a post‐hoc test. The values are mean ± SD. n = 3–4 mice. All data were obtained from a single experiment in the same time frame. (c) Percentage of stenosis at the distance between the internal elastic lamina (IEL). The intimal formation was defined as the thickened intima afferently overhanging the inside of the IEL. As an indicator of intimal formation, we chose to use intimal thickness. Neointima in the coronary arteries was markedly increased by CAWS vasculitis. Statins did not suppress neointimal proliferation 6 weeks after CAWS administration. However, statin therapy considerably suppressed neointimal proliferation at 10 weeks. Notably, neointimal proliferation was also suppressed in the late‐statin group. Data were analyzed by the Kruskal–Wallis test. The Wilcoxon test was corrected with a post‐hoc test. The values are mean ± SD. n = 4 mice. All data were generated from one experiment in the same timeframe.