MMP Mediated Degradation of Type VI Collagen Is Highly Associated with Liver Fibrosis – Identification and Validation of a Novel Biochemical Marker Assay

Abstract

Background and AimsDuring fibrogenesis, in which excessive remodeling of the extracellular matrix occurs, both the quantity of type VI collagen and levels of matrix metalloproteinases, including MMP-2 and MMP-9, increase significantly. Proteolytic degradation of type VI collagen into small fragments, so-called neo-epitopes, may be specific biochemical marker of liver fibrosis. The aim of this study was to develop an ELISA detecting a fragment of type VI collagen generated by MMP-2 and MMP-9, and evaluate this assay in two preclinical models of liver fibrosis.MethodsMass spectrometric analysis of cleaved type VI collagen revealed a large number of protease-generated neo-epitopes. A fragment unique to type VI collagen generated by MMP-2 and MMP-9 was selected for ELISA development. The CO6-MMP assay was evaluated in two rat models of liver fibrosis: bile duct ligation (BDL) and carbon tetrachloride (CCl4)-treated rats.ResultsIntra- and inter-assay variation was 4.1% and 10.1% respectively. CO6-MMP levels were significantly elevated in CCl4-treated rats compared to vehicle-treated rats at weeks 12 (mean 30.9 ng/mL vs. 12.8 ng/mL, p = 0.002); week 16 (mean 34.0 ng/mL vs. 13.7 ng/mL, p = 0.0018); and week 20 (mean 35.3 ng/mL vs. 13.3 ng/mL, p = 0.0033) with a tight correlation between hepatic collagen content and serum levels of CO6-MMP (R2 = 0.58, p<0.0001) in CCl4- treated rats. In BDL rats, serum levels of CO6-MMP were significantly elevated compared to the levels in sham-operated animals both at 2 weeks (mean 29.5 ng/mL vs. 14.2 ng/mL, p = 0.0001) and 4 weeks (mean 33.0 ng/mLvs. 11.8 ng/mL, p = 0.0003).ConclusionsThis novel ELISA is the first assay enabling assessment of MMP degraded type VI collagen, allowing quantification of type VI collagen degradation, which would be relevant for different pathologies. The marker was highly associated with liver fibrosis in two liver fibrosis animal models, suggesting type VI turnover to be a central player in fibrogenesis.

Serum endotrophin identifies optimal responders to PPARγ agonists in type 2 diabetes

“…In support of a role for type VI collagen in NASH-related fibrosis, studies have demonstrated its prominent expression in areas of active scar formation [35,36], and elevated serum levels of the collagen VI core structure (which lacks the endotrophin domain) have been shown to be associated with advanced liver fibrosis in rodents [37], humans [38][39][40][41], and with elevated portal pressure [42]. The expression of procollagen α3(VI) is regulated by PPARγ and our findings are in alignment with this.…”

Extracellular Matrix Remodeling: The Common Denominator in Connective Tissue DiseasesPossibilities for Evaluation and Current Understanding of the Matrix as More Than a Passive Architecture, but a Key Player in Tissue Failure

“…Promising candidates have been reported, including those derived from type IV collagen, 196,197 type I collagen, 198 type V collagen, 199 and type VI collagen. 200 Systemic approaches, such as global profiling of serum glycoproteins, have also been utilized, 201 and this technique is now being validated in rodent models (e.g., Fang et al, 202 Blomme et al,…”

“…A range of serological assessments have been developed to investigate some of the key structural proteins of the ECM (Table 3). 56,60,129,144,[196][197][198][199][200]217,274,276,278,311,314,[331][332][333][334][335][336][337][338][339][340][341][342][343][344][345][346][347] Measurement of these proteins may provide key information in clinical settings on the tissue turnover profile, and thereby assists in patient diagnosis, in identification of those patients in most need of treatment, and finally, in monitoring of clinical efficacy of interventions. These technologies may also be used in preclinical settings, in ex vivo and in vitro cultures, to determine the molecular mode of action in the assembly and maintenance of the matrix.…”

“…atherosclerosis (type I and III collagens, titin and versican) 217 . various fibrotic diseases including liver (type I, III, IV, V, VI collagens and biglycan), [59][60][61]144,196,197,199,200,218 lung (elastin, type I, III, and V collagen), 74,[220][221][222][223][224][225][226][227][228][229][230][231][232] and kidney. 233 Key lessons on the importance of the structural components of the matrix may be harvested from the genetic mutations that lead to pathologies.…”

Cyanidin-3-O-β-glucoside Purified from Black Rice Protects Mice against Hepatic Fibrosis Induced by Carbon Tetrachloride via Inhibiting Hepatic Stellate Cell Activation

“…On the other hand, MMPs are promptly expressed by activated HSCs in response to diverse hepatic injury and are considered to promote disease progression. 38 Thus, MMPs play a dual role in liver fibrosis, depending on the timing of their production. The early temporary high expression of MMPs may destroy the surrounding tissue in order to deposit newly synthesized ECM.…”