Citrullination and MMP degradation of vimentin, a key event in fibrosis and tissue destruction

Abstract

Introduction: Inflammation is a common denominator in both autoimmune disorders and fibrotic processes. We hypothesized that citrullinated peptides, known to be present in autoimmune diseases, may be present in liver fibrosis. We aimed to evaluate whether a citrullinated and degraded fragment of vimentin, produced by proteolytic activity of MMP-2 and -8, was associated with liver fibrosis. Materials and methods: A vimentin peptide identified by mass spectrometry in fibrotic tissue extracts, and proteolytic degradation. A monoclonal antibody against sequence RLRSSVPGV-Citrulline (VICM) was developed, which was specific for citrullinated and MMP-degraded vimentin with no reactivity to intact vimentin. This was evaluated in a carbon tetrachloride (CCl4) rat model of liver fibrosis and a cohort of 88 hepatitis C (HCV) patients and 68 non-alcoholic fatty liver disease (NAFLD) patients, aged 18 to 75 years, and compared to healthy controls (n=19). Hepatitis C patients were stratified according the METAVIR fibrosis score (F0-F4 with F0: no fibrosis and F4: cirrhosis) and NAFLD patients according to the NAFLD fibrosis and activity score (NAS). Results: Systemic VICM was increased by 31% in 12 weeks (176 ng/mL, p < 0.001); 41.7% in 16 weeks (190 ng/mL, p < 0.001) and 49.2% at 20 weeks (200 ng/ml, p < 0.001) (control group 134 ng/ml) in CCl 4-treated rats. Serum VICM correlated to total amount of hepatic collagen determined by Sirius red staining of CCL 4-treated rat livers (r=0.7570, p < 0.05). In the HCV cohort, VICM levels were compared to controls 63% increased in F0 specimens (632 ± 251 ng/mL, p < 0.05), 54% in F1 (597 ± 274 ng/mL, p < 0.05) and 62.6% in F2 (628 ± 308 ng/mL, p < 0.05). In the NAFLD cohort, VICM levels were 20.6% increased in F0 specimens (339 ± 53 ng/mL, p < 0.05), 23.8% in F1 (348 ± 59 ng/mL, p < 0.05) and 28.8% in F2 (362 ±80 ng/mL, p < 0.05). Discussion and conclusion: For the first time we demonstrated that increased serological levels of VICM were associated with the extent of liver fibrosis in the CCl 4-induced rat liver fibrosis model. We also showed that serum VICM was elevated in early fibrosis in human HCV and NAFLD patients. This novel marker may in combination with other extracellular matrix remodeling markers provide value information in patient assessment, individualized therapy and treatment evaluation.