A novel marker for assessment of liver matrix remodeling: An enzyme-linked immunosorbent assay (ELISA) detecting a MMP generated type I collagen neo-epitope (C1M) (1)
Liver fibrosis is one of the leading causes of death world- wide mostly due to viral or alcohol-induced injury. The most well studied extracellular matrix (ECM) biochemical marker for liver fibrosis is the propeptide of type III collagen (PIIINP), which has shown good potential as a fibrogenesis marker, however still with limitation. Endopeptidases such as MMPs play major roles in the degradation of extracellular macromolecules such as collagens and proteoglycans, resulting in neo-epitopes. A neo-epitope is a protease-generated post-translational modification (PTM) of a protein, and has potential as a biochemical marker of ECM degradation.
Since bone tissue mainly consists of type I collagen it was worthwhile to aim for elimination the potential background coming from bone, which may be the case if the main protease of the bone resorbing cells, osteoclasts, cleaved type I collagen inside the selected epitope as described for cathepsin K (Cat K). The sequence 755′GSPGKDGVRG⇓‘764 (CO1-764) in the alpha 1 chain of type I collagen generated by MMP-2, -9, -13 (⇓) was identified by LC-MS and selected for immunizations since it is unique to type I collagen and it is further cleaved by Cat K (#) on the second position from the C-terminus of the peptide (GSPGKDGVR#G⇓). This sequence is 100% homologous to human, rat, mouse and bovine type I collagen.
The native reactivity of the hybridoma clone was high against both human serum and urine; rat serum and urine; and mouse serum. The typical standard curve show a 4-parametric fit for the assay.
The lower limit of detection (LDL) for the assay was 0.83 ng/mL. Dilution recovery was within 100 ± 15% except for mouse urine (mean 118%) and mouse plasma. The inter- and intra-assay variation was around or below 10% for assessments in both assays.
Analyte stability in human serum samples were acceptable; percent recovery was between 100 ± 20% for samples values after storage 2–48 h at 4- and 20°C. Finally, the analyte stability was acceptable for 2–7x freeze/thaw cycles: all were within 100 ± 20% in human- and rat serum.
From the ELISA characterization it was observed that MMP-2, -9 and -13 were able to generate the CO1-764 fragment. The test of whether Cat K destroys the CO1-764 fragment showed that a MMP-9 cleaved sample lost approximately 50% of its reactivity following subsequent cleavage by Cat K. No cross reactivity was seen to the collagen type V, VII and elongated synthetic peptide sequences that have high homology with the immunization sequence of type I collagen.
At the 4 week termination point CO1-764 levels were significantly elevated in BDL rats compared with baseline levels (+75% increased) and compared with sham termination levels (+97% increased). From the qPCR data it was observed that BDL provoked a massive 100-fold increase of type I collagen gene expression in the liver.
In the CCL4 rat model it was seen that the levels of CO1- 764 were statistically elevated at week 12, 16 and 20 in CCL4 treated rats. The correlation between CO1-764 and total collagen was highly significant in CCL4 treated rats.
The mean values of CO1-764 in patients stratified according to cancer type and presence or absence of BM.
There was no correlation between the CO1-764 marker and Col1a1 mRNA in the livers. Since type I collagen is considered an index of fibrogenesis activation, these results suggest that CO1- 764, contrary to other degradation fragments of liver collagens such as CO3-610, is less dependent of fibrogenesis, and potentially reflects tissue destruction caused by pathological events other than fibrogenesis. The serum CO1-764 marker is a true degradation marker and not a formation marker since there is a time displacement prior to its release from the liver.