Small leucine-rich proteoglycans in atherosclerotic lesions: novel targets of chronic statin treatment?

Abstract

Small leucine-rich proteoglycans (SLRPs), such as decorin and biglycan, regulate the assembly and turnover of collagenous matrix. The aim of the study was to analyse the effect of chronic rosuvastatin treatment on decorin, biglycan and the collagen matrix in ApoE-deficient mice. Twenty-week-old male ApoE-deficient mice received normal chow or 20 mg rosuvastatin/kg × day for 32 weeks. Subsequently, matrix composition was analysed by histochemistry and immunostaining at the aortic root and in innominate arteries of ApoE deficient mice as well as in human carotid endarterectomy specimens. Immunoblotting of proteoglycans was performed from aortic extracts of ApoE-deficient mice. Immunohistochemistry and immunoblotting revealed strongly increased decorin and biglycan deposition in atherosclerotic plaques at the aortic root and in innominate arteries. In contrast, versican and perlecan expression was not changed by rosuvastatin. Furthermore, matrix metalloproteinase 2 and gelatinolytic activity were decreased in response to rosuvastatin and a condensed collagen-rich matrix was formed. In carotid endarterectomy specimens of statin-treated patients increased decorin and biglycan accumulation was detected as well. Drug treatment did not change low-density lipoprotein (LDL) plasma levels in ApoE-deficient mice and did not significantly affect lipid retention at the aortic root level as demonstrated by oil-red O staining and immunohistochemistry of LDL. Long-term treatment with rosuvastatin caused pronounced remodelling of atherosclerotic plaque matrix characterized specifically by enrichment with SLRPs and formation of a condensed collagen matrix. Therefore, decorin and biglycan might represent novel targets of statin treatment that contribute to a stable plaque phenotype.

Fig 1

Increased accumulation of collagen in aortic root lesions of ApoE^−/− mice in response to rosuvastatin. ApoE^−/− mice were chronically treated with rosuvastatin (20 mg/kg × day) from 20 to 52 weeks of age. Sirius red staining of collagen and plaque morphology at the aortic root. (A) control; (B) rosuvastatin treated ApoE^−/−; (C) quantitative image analysis. Tightly packed collagen matrix was visualized by birefringence analysis of picrosirus red staining under polarized light of (D) control; (E) rosuvastatin treated ApoE^−/−; (F) quantitative image analysis, *P < 0.05; control, n= 10, rosu-vastatin, n= 18.

Fig 2

Accumulation of decorin and biglycan was increased in aortic root plaques and in aortic extracts in response to rosuvastatin. (A, D) control and (B, E) rosuvastatin. (A, B) decorin immonostaining; (D, E) biglycan immunostaining; (C, F) quantitative image analysis of intimal SLRP staining; *, P < 0.05; control, n= 8, rosuvastatin, n= 10. (G–J) to verify the immunostaining Western blotting of both SLRPs was performed from aortic extracts normalized to mg dry weight of total aorta. (G, I) Western blots, (G) decorin and I, biglycan; (H, J) quantitative densitometry, n= 4; *, P < 0.05.

Fig 3

Condensed collagen matrix and increased accumulation of SLRPs in innominate arteries of rosuvastatin treated ApoE^−/− mice. Collagen and collagen matrix arrangement were analysed by picrosirius red staining and birefringence analysis, respectively. In addition decorin and biglycan were detected by immunohistochemistry in plaques of innominate arteries. (A, D, G, J) control and (B, E, H, K) rosuvastatin. (A–F) picrosirius red staining and quantitative image analysis. (D, E) picrosirius red staining viewed under polarized light. (G–I) biglycan immunostaining and quantitative image analysis. (J–L) decorin immunostaining and quantitative image analysis; control, n= 4, rosuvastatin, n= 11, *, P < 0.05.

Fig 4

Rosuvastatin inhibits collagen degradation and gelatinolytic activity in atherosclerotic plaque of ApoE^−/− mice. Collagen cleavage was demonstrated by staining of collagen neoepitopes originating from MMP mediated cleavage in aortic root plaques. (A) control and (B) rosuvastatin. (C) quantitative image analysis, n= 8; *P < 0.05. (D) ratio between collagen neoepitopes and total collagen as determined by picrosirius red staining (Fig. 1). (E, F) MMP activity as detected by in situ zymography on cryosections in control versus rosuvastatin. (G)quantitative image analysis. (H, I)expression of MMP-2 (H) and MMP-9 (I) was analysed by immunostaining, quantitative data from the image analysis is provided; n= 8–9, *, P < 0.05.

Fig 5

Three dimensional collagen gels cause increased bigly-can and decorin expression. To investigate whether rosuvastatin or three dimensional collagen type 1 matrix affect SLRP expression in vitro experiments were performed. (A)–(B) human coronary SMC were cultured on plastic and incubated with or without 1 μM rosuvastatin in normal growth medium (5% FCS) for 5 days. Subsequently mRNA expression of (A) biglycan, decorin and (B) human collagen type 1 and 3 were determined by real-time RT-PCR, n= 10. (C), human coronary SMC were cultured either on plastic under routine conditions or in a gel of polymeric collagen type 1 (20,000 cells/400 mm³), after 24 days mRNA expression of biglycan and decorin was determined by real-time RT-PCR and expressed as fold of the monolayer cultures on plastic; n= 4; *, P < 0.05. (D), to investigate whether the SLRP expression in collagen gels is further influenced by rosu-vastatin, the experiments shown in C were repeated in the presence or absence of 1 μM rosuvastatin. n= 8–11; *, P < 0.05 versus control.

Fig 6

(A–D) Retention of lipids was not affected by rosuvastatin in aortic roots of ApoE^−/− mice. Oil Red O staining and immunostaining of apoB48 of plaques at the aortic root was performed in order to analyse whether the changes in matrix composition were associated with effects on lipid retention. (A)(C) control; (B, D) rosuvastatin, (E) quantification of lipid staining by image analysis. (F) quantification of Apo B48 immunostaining; n= 7; *, P < 0.05.

Fig 7

Condensed collagen matrix and increased SLRPs in statin treated endarterectomies. Decorin, biglycan and collagen in human atherosclerosis after statin treatment. (A, D, G) control and (B, E, H) specimens derived from patients treated with statins for at least one month. (A–F) picrosirius red staining of human carotid endarterectomies. (A, B) viewed under polarized light. (C, F) quantitative image analysis. (G, H) co-immunostaining of bigly-can and decorin, (I) quantitative image analysis; n= 10; *, P < 0.05.