Levels of Circulating MMCN-151, a Degradation Product of Mimecan, Reflect Pathological Extracellular Matrix Remodeling in Apolipoprotein E Knockout Mice

Abstract

Aim: Arterial extracellular matrix (ECM) remodeling by matrix metalloproteinases (MMPs) is one of the major hallmarks of atherosclerosis. Mimecan, also known as osteoglycin has been implicated in the integrity of the ECM. This study assessed the validity of an enzyme-linked immunosorbent assay (ELISA) developed to measure a specific MMP12-derived fragment of mimecan, MMCN-151, in apolipoprotein-E knockout (ApoE-KO) mice.

Methods and results: A mouse monoclonal antibody raised against MMCN-151 was used to develop a competitive ELISA. The assay was validated using samples from 20 ApoE-KO and 20 wild type [C57 BL/6] male mice fed a normal or high-fat diet (HFD) for up to 20 weeks. The technical reliability of the assay was established with intra-assay variability <2% and inter-assay variability <10%. The lowest limit of quantification of MMCN-151 was 0.5 ng/ml. ApoE-KO mice fed a HFD for 20 weeks had four-fold increased circulating levels of MMCN-151 compared to baseline, whereas MMCN-151 levels in control mice on HFD increased two-fold compared with baseline. After 10 weeks of a HFD, a significant difference in MMCN-151 levels was observed between ApoE-KO and control mice (P = 0.005) and became more significant at 20 weeks (P = 0.002).

Conclusions: The newly developed assay is a reliable detector of MMCN-151 levels which ultimately may be useful indicators of arterial remodeling in patients affected by atherosclerotic disease.

Keywords: MMCN-151; atherosclerosis; biomarker; extracellular matrix; matrix metalloproteinases; mimecan; proteoglycans.

Figure 1

(A) Example of a 4-parametric fitted standard curve in a competitive ELISA setting with peptide concentrations of 0, 3.125, 6.25, 12.5, 25, 50, 100 and 200 ng/ml. (B) Competitive ELISA test of MMCN-151 antibody reaction towards free peptide, elongated peptide, mimecan cleaved with MMP12 and intact mimecan. The x-axis is representative of the concentrations of free and elongated peptide. Concentrations of samples containing mimecan and mimecan cleaved with MMP12 are 43 ug/ml as the highest concentration followed by 2-fold dilutions of samples.

Figure 2

Plasma levels of MMCN-151 in control (ctrl) and ApoE-KO mice, at baseline (BL), after 10 weeks and after 20 weeks of feeding either standard chow (A) or high fat diet (B), and (C) effect of diet on circulating levels of MMCN-151 neoepitope after 20 weeks of feeding. Notes: *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 3

Cholesterol levels measured in mice plasma at baseline (BL) and after 20 weeks of feeding normal diet (ND) or high fat diet (HFD) in either control mice (ctrl) or in apolipoportein-E knock-out (ApoE-KO) mice.

Figure 4

Linear correlation between (A) plasma cholesterol of control mice fed HFD and their plasma levels of MMCN-151 and (B) plasma cholesterol of ApoE KO mice fed HFD and the levels of MMCN-151.

Figure 5

Linear correlation between plasma levels of MMCN-151 and average pixel intensity (%) corresponding to the total proteoglycan area of each mouse aorta. The average pixel intensity is reflected as the intensity of Alcian blue, which is a standard proteoglycan staining. Alcian blue binds to glycosaminoglycan chains on proteoglycans.