Background Transforming growth factor-β-inducible gene-h3 (βig-h3) is an ECM protein, which consists of integrin-interacting structures including four homologous fas-1 domains and an Arg-Gly-Asp (RGD) motif, is abundantly found within synovial tissues and fluid of rheumatoid arthritis (RA). In the pathogenesis of RA, βig-h3 intensifies inflammatory processes by facilitating adhesion and migration of fibroblast-like synoviocyte (FLS) via binding to αvβ3 integrin. Based on these functions, fragmented peptides of βig-h3 have been developed as therapeutic agents for chronic inflammatory arthritis. Expression of MMP-2 is highly upregulated within the invasive pannus and associated with radiographic erosions in RA. Therefore, therapeutic agents that are transformable via introducing cleavable linkage by locally enriched MMP-2 within the pannus tissue would enhance targetability and therapeutic efficacy on chronic inflammatory arthritis.
Objectives To investigate whether MMP-2 cleavable peptide complex consisted of fas-1 domain and RGD peptide blocks the interaction between βig-h3 and resident cells and leads to the amelioration of inflammatory arthritis.
Methods We designed βig-h3-derivatives, including the fourth fas-1 domain truncated for H1 and H2 sequences of mouse (MFK00) and MMP-2-cleavable peptide complex (MFK902). MMP-2 selectivity was examined by treatment with a series of proteases. MFK902 efficacy was determined by the adhesion and migration assay with NIH3T3 cells in vitro and collagen-induced arthritis (CIA) model in vivo. The mice were treated intraperitoneally with MFK902 at different dosages (1, 10, or 30 mg/kg).
Results MFK902 was specifically cleaved by active MMP-2 in a concentration-dependent manner. Both MFK00 and MFK902 revealed a dose-dependent inhibition of βig-h3-mediated adhesion of fibroblasts and were similarly effective at a lower concentration (1 μM), compared with RGD peptide. In the migration assay, a very high concentration of RGD peptide (500 μM) reduced fibroblast migration by less than 50%. The lowest effective concentration of MFK00 for inhibition was 1 μM with the IC50 of 13.56 ± 1.28 μM. The IC50 of cell migration for MFK902 was 4.56 ± 0.25 μM with the degree of inhibition higher than 2 fold at 1 μM and higher than 5 fold at 20 μM. In in vivo assay with a murine CIA model, treatment with all dosages of MFK902 resulted in a slower onset of arthritis, followed by less steep increase of clinical arthritis index up to day 50 with significantly lower scores, compared with controls. On day 50, the incidence of arthritic paws for the control group was 94%, whereas those for 1, 10, and 30 mg/kg groups with MFK902 treatment were 54, 41, and 38%, respectively. MFK902 ameliorated histopathologic deterioration and reduced the expression of inflammatory mediators in parallel with improvement of clinical features. In addition, a favorable safety profile of MFK902 was demonstrated in vivo.
Conclusions The present study reveals that the MMP-2-cleavable peptide complex, based on βig-h3 structure, was a potent and safe therapeutic agent for chronic inflammatory arthritis and provides a reliable evidence for the MMP-2-cleavable mechanism as a tissue-targeted strategy to treat rheumatoid arthritis.
Disclosure of Interest None declared