Cartilage oligomeric matrix protein (COMP) in murine brachiocephalic and carotid atherosclerotic lesions

Abstract

Objective: To investigate the hypothesis that COMP can influence the morphology, stability and size of murine atherosclerotic lesions.

Methods: ApoE- and ApoE/COMP-knockout mice were fed a high-fat diet to develop atherosclerotic plaques at lesion sites of three different types; inflammatory and fibrous plaques induced in the carotid artery by low or oscillatory shear stress, respectively, and spontaneously developing plaques in the brachiocephalic artery. The localization of COMP in the plaques and the effect of COMP deficiency on plaque development were evaluated.

Results: COMP immunoreactivity was observed in about half of the investigated plaques from the ApoE null mice, mainly located along the intima-medial border. There were no significant differences in the size of inflammatory and fibrous carotid plaques between the genotypes. Plaques in the brachiocephalic artery from ApoE mice lacking COMP were increased in size with 54%. In these plaques the collagen content was also increased by 48%. There were no differences in relative collagen content in inflammatory and fibrous carotid plaques between genotypes. Polarized light microscopy showed that the increase in total collagen in brachiocephalic plaques was more than proportionally accounted for by an increase in thicker collagen fibrils.

Conclusion: We have shown that COMP deficiency has a significant impact on atherosclerotic plaque morphology and size. Our data also suggest that an altered collagen metabolism may be an important mechanism in this finding.

Keywords: ApoE-KO mouse; Atherosclerosis; COMP; Collagen fibre; Plaque morphology.

Fig. 1

COMP, PCNA, collagen and smooth muscle α-actin in carotid plaques. A–H: Consecutive sections from a proximal (A–D) and a distal (E–H) carotid plaque stained for COMP (A,E), PCNA (B,F), collagen (C,G) and α-SMA (D,H). COMP and α-SMA are represented by red immunofluorescence, while green colour represents autofluorescence in the respective images. PCNA positive cells, individually revealed by evaluation of the DAB-developed original slide under the microscope, are indicated by red dots in the morphometrically-processed images. Collagen appears blue in the masson-trichrome (MTC) processed images. Scale bar: 100 μm. I + J: Analysis of proximal and of distal carotid plaques of either ApoE-deficient (ApoE^−/−) or ApoE and COMP deficient (ApoE/COMP^−/−) animals. I: Collagen contents in % of the total plaque area as determined by masson-trichrome. (ApoE^−/−: n = 19; proximal ApoE/COMP^−/−: n = 15; distal ApoE/COMP^−/−: n = 16). J: Number of PCNA positive cells per mm². (ApoE^−/−: n = 17; proximal ApoE/COMP^−/−: n = 14; distal ApoE/COMP^−/−: n = 15). K: Analysis of PCNA positive cells per mm² in plaques showing either clear COMP immunoreactivity (clear COMP pos., n = 14) or very weak and diffuse or no COMP immunoreactivity (diff./COMP neg., n = 20). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Morphometric analysis of carotid and brachiocephalic plaques. A: Plaque/media ratios of inflammatory (proximal) and fibrous (distal) carotid (CA) and brachiocephalic (BCA) lesions of either ApoE-deficient (ApoE^−/−) or ApoE and COMP deficient (ApoE/COMP^−/−) animals. BCA ratios show significant differences between genotypes (proximal and distal CA ApoE^−/−: n = 19; proximal CA ApoE/COMP^−/−: n = 15; distal CA ApoE/COMP^−/−: n = 16; BCA ApoE^−/−: n = 20; BCA ApoE/COMP^−/−: n = 23). B: Outer circumferences of inflammatory (proximal) and fibrous (distal) carotid lesions and contra lateral carotid arteries of either ApoE-deficient (ApoE^−/−) or ApoE and COMP deficient (ApoE/COMP^−/−) animals. Circumferences are expressed in arbitrary length units calculated by the program used to analyze the scanned images. Around inflammatory lesions outer circumferences show significant differences between genotypes (proximal and distal CA ApoE^−/−: n = 19; proximal and distal CA ApoE/COMP^−/−: n = 15; contra lateral CA: n = 4).

Fig. 3

COMP, GEP, ADAMTS7 and PCNA immunofluorescence in brachiocephalic plaques. A–E: Brachiocephalic sections stained fluorescence-immunohistochemically for COMP (A), GEP (B), ADAMTS7 (C), and PCNA (D,E), all represented by red colour. Green colour represents autofluorescence, except for particularly bright green fluorescence in (B), which indicates α-SMA. Sections A-D are from ApoE-deficient mice, while section E is from an ApoE and COMP deficient mouse. Arrowheads point to PCNA positive cells. Scale bar: 200 μm. F: Number of PCNA positive cells per mm² (ApoE^−/−: n = 15; ApoE/COMP^−/−: n = 15). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Collagen content and fibre size and circumference of picrosirius red stained brachiocephalic lesions. A-D Examples of picrosirius red stained brachiocephalic plaques observed under normal (A,C) and polarized (B,D) light. Scale bar: 200 μm. E: Total collagen content and fibre size associated collagen content in plaque and media. Values showing significant differences (p < 0.05) between ApoE single KO's (striped bars) and ApoE/COMP double-KO's (filled bars) are indicated by stars. F: Outer elastic circumferences of the brachiocephalic lesions of either ApoE-deficient (ApoE^−/−; n = 20) or ApoE and COMP deficient (ApoE/COMP^−/−; n = 23) animals.