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: 2.02). 07/2011; 44(10-11):900-6. DOI: 10.1016/j.clinbiochem.2011.04.004
Source: PubMed

ABSTRACT 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.

  • [Show abstract] [Hide abstract]
    ABSTRACT: The hallmark of a variety of fibrotic diseases such as liver fibrosis, lung fibrosis, skin fibrosis and atherosclerosis is extensive extracellular matrix remodeling (ECMr) of the disease affected tissue. Inflammation often leads to tissue disruption and destruction, upon which locally released battery of proteases such as matrix metalloproteinases and cysteine proteases degrade the surrounding matrix. The degradation products of ECM proteins, the co-called neoepitopes, are released into the systemic circulation. By recent development of Enzyme-Linked Immunosorbent Assays (ELISAs) detecting the pathological tissue turnover in atherosclerosis and liver fibrosis, we have introduced a novel class of biomarkers into the field of fibrotic diseases, which have been proved efficient in the early diagnosis. This work has resulted in identification of common mechanisms involving specific cell types, proteins and proteases as well as pathways shared among the fibrotic diseases. In this analysis we seek to answer following questions: a) Are there common disease mechanisms and cell types involved in both atherosclerosis and fibrosis? b) Can the lessons learned in developing fibrosis biomarkers be used for the development of atherosclerosis biomarkers? Our hypothesis is that by answering the above questions, we may be able to improve general understanding of the early-stage disease initiation and progression of fibrotic diseases, which in turn may aid in early diagnosis, prognosis and ultimately patient management.
    American Journal of Translational Research 01/2013; 5(1):1-14. · 3.23 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The multi-targeted therapy for liver cancer has been considered as a novel strategy to fight hepatocellular carcinoma. In this study, we first found that sprengerinin C, a naturally derived compound strongly suppressed tumor angiogenesis on human umbilical vein endothelial cells. Mechanism study revealed that sprengerinin C blocked vascular endothelial growth factor receptor 2-dependent phosphoinositide 3-kinase/Akt/mTOR/matrix metalloproteinase and p38 MAPK/matrix metalloproteinase pathways, two major pathways for tumor angiogenesis. Moreover, sprengerinin C inhibited vascular endothelial growth factor release, a vital event for early angiogenesis response, from hypoxic HepG-2/BEL7402 cells by suppressing hypoxia-inducible factor-1α transcriptional activity. Furthermore, sprengerinin C induced HepG-2/BEL7402 cell apoptosis by activating NADPH oxidase/reactive oxygen species-dependent caspase apoptosis pathway and suppressed HepG-2/BEL7402 cell growth through p53-mediated G2/M-phase arrest. Sprengerinin C also showed significant anti-tumor effect in the nude mouse xenograft model of human hepatocellular carcinoma. These results provide new insights into development of potent candidate compounds for liver cancer through affecting multiple tumor progression steps of angiogenesis, apoptosis and proliferation.
    European journal of pharmacology 05/2013; · 2.59 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Osteoarthritis (OA) is a degenerative disease with a subset of patients experiencing joint inflammation, but C-reactive protein (CRP) has shown limited use in OA as a diagnostic marker. The aim was to identify subpopulations of patients with high or low levels of acute (hsCRP) and/or matrix metalloproteinase (MMP) derived inflammation (CRPM) and investigate the subpopulations' association with biomarkers of collagen degradation and Kellgren-Lawrence score (KL). hsCRP, CRPM and MMP-degraded type I, II and III collagen (C1M, C2M and C3M) were quantified by ELISA in serum of 342 patients with symptomatic knee OA of which 60 underwent total knee replacement (TKR). KL was obtained. Patients were divided into quartiles by hsCRP and CRPM levels, where Q1 and Q4 were low or high in both. The biomarker levels of healthy adults provided in the ELISA kits were used as reference level. hsCRP was elevated in TKR (5.9(3.6-8.2 95% CI)μg/mL) compared to reference level (3μg/mL), while CRPM was highly elevated with OA independent of KL (10-14ng/mL) compared to reference level (5ng/mL). Q4 had higher KL than Q1 (P<0.001), Q2 (P=0.017) and Q3 (P<0.001). C1M, C2M and C3M were lowest in Q1. C1M was elevated in Q3 compared to Q2 (P<0.001), whereas C3M was lower (P=0.019). A bigger proportion of patients were elevated in CRPM compared to hsCRP, indicating MMP-derived inflammation as a component of OA. Moreover, the levels of MMP-degraded collagens differed between the subgroups segregated by inflammation, indicating distinctively different subpopulation selected by inflammation.
    Osteoarthritis and Cartilage 11/2013; · 4.26 Impact Factor