Development and validation of an enzyme-linked immunosorbent assay for the quantification of a specific MMP-9 mediated degradation fragment of type III collagen-A novel biomarker of atherosclerotic plaque remodeling
University of Southern Denmark, Odense, Denmark. Clinical biochemistry
(Impact Factor: 2.28).
07/2011; 44(10-11):900-6. DOI: 10.1016/j.clinbiochem.2011.04.004
Degradation of collagen in the arterial wall by matrix metalloproteinases is the hallmark of atherosclerosis. We have developed an ELISA for the quantification of type III collagen degradation mediated by MMP-9 in urine.
A monoclonal antibody targeting a specific MMP-9 generated fragment of collagen III was used in a competitive ELISA. The assay was validated in urine and arterial tissue of Apolipoprotein-E knockout (ApoE-KO) mice.
The lower limit of detection was 0.5ng/mL, intra- and inter-assay coefficients of variation were below 10%. By the end of 20weeks of the study, urine levels of the novel CO3-610 biomarker in ApoE-KO mice increased by two-fold (p<0.0001) and were three-fold higher than in control mice. Western blots confirmed high expression of CO3-610 in arterial extracts of ApoE-KO mice.
We have developed a novel competitive ELISA, capable of measuring a urine biomarker indicative of pathological extracellular matrix remodeling in a mouse model of atherosclerosis.
Available from: Susanne Hosbond
- "MMP-degradation of proteins generates specific cleavage sites on fragments which in turn enable the development of new epitopes. Our group previously discussed neoepitopes that may have potential utility as biomarkers of unbalanced ECM remodeling in a number of different pathologies and can be measured in biological fluids such as serum, plasma and urine [22-27]. Key benefits of measuring biomarkers in body fluids are that this process is non-invasive and, because the specific neoepitopes represent a unique ‘fingerprint’ of the proteolytic cleavage of the protein, they identify the specific tissue being turned over and also detect whether the tissue is diseased or healthy. "
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Titin is a muscle-specific protein found in cardiac and skeletal muscles which is responsible for restoring passive tension. Levels and functioning of titin have been shown to be affected by cardiac damage. Due to the inherent difficulty of measuring titin levels in vivo in a clinical setting, we aimed to develop an assay that could reliably measure fragments of degraded titin in serum and potentially be used in the assessment of cardiac muscle damage.
A competitive ELISA was developed to specifically measure levels of the titin sequence 12670’ NVTVEARLIK 12679’, derived by the degradation of titin by matrix metalloproteinase (MMP)-12. Serum samples from 90 individuals were divided into 3 equally sized groups. One group had been diagnosed with acute myocardial infarction (AMI) while the remaining two were asymptomatic individuals either with CT-scan signs of coronary calcium (CT-plusCa) or without coronary calcium (CT-noCa).
Mean geometric levels of the titin fragment in the CT-noCa group were 506.5 ng/ml (±43.88). The CT-plusCa group showed 50.6% higher levels of the marker [763 ng/ml (±90.14)] (P < 0.05). AMI patients showed 56.3% higher levels [792 ng/ml (±149)] (P < 0.05).
The titin-12670 fragment is present in both individuals with undiagnosed and diagnosed CVD. The statistically significant increase in level of the marker in the AMI group is indicative that this neoepitope biomarker may be a useful serological marker in AMI.
Available from: Efstathios Vassiliadis
- "However, we saw that the increase in MMCN-151 is not mediated by a high cholesterol diet, since only ApoE KO mice had elevated plasma biomarker levels. Moreover, it is interesting that we found no difference in MMCN-151 plasma levels in ApoE KO and control mice which received normal diet as we have seen previously with our collagen III, CO3-610 urine biomarker where significant biomarker increase was demonstrated in ApoE KO mice fed standard rodent chow.33 However, we have seen that the increase in MMCN-151 in ApoE KO mice seems to be mediated by a high cholesterol diet, whereas no significant increase in the circulating levels of MMCN-151 were observed in control mice when comparing the two diet regimens. "
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ABSTRACT: 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.
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).
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.
Available from: Massimo Coletta
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ABSTRACT: Human matrix metalloproteinases (MMPs) belong to the M10 family of the MA clan of endopeptidases. They are ubiquitarian enzymes, structurally characterized by an active site where a Zn(2+) atom, coordinated by three histidines, plays the catalytic role, assisted by a glutamic acid as a general base. Various MMPs display different domain composition, which is very important for macromolecular substrates recognition. Substrate specificity is very different among MMPs, being often associated to their cellular compartmentalization and/or cellular type where they are expressed. An extensive review of the different MMPs structural and functional features is integrated with their pathological role in several types of diseases, spanning from cancer to cardiovascular diseases and to neurodegeneration. It emerges a very complex and crucial role played by these enzymes in many physiological and pathological processes.
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