Fig 1.
Identification of matrix metalloproteinase-2 (MMP-2)-specific cleavage of MMP-2-cleavable peptide complex, MFK902.
(A) Sequence of recombinant murine MFK902 consisting of MFK00 (murine 4th fas-1 peptide truncated for the H1 and H2 sequences) and an RGD motif linked by a MMP-2 substrate (GPLGVRG). (B) Schematic representation of the pET-29b(+) vector containing the MFK902 sequence. (C) Immunoblot of purified recombinant MFK902. (D) MMP-2–specific cleavage of MFK902 was performed as detailed in the Methods section.
Fig 2.
MFK902 inhibition of NIH3T3 cell adhesion and migration.
βig-h3-derivatives, including RGD (GGRGDSP), MFK00, and MFK902 peptides, were prepared as described in the Methods section. Inhibition of adhesion by (A) the RGD and RGE, (B) MFK00, and (C) MFK902 peptides. Inhibition of migration by (D) the RGD and RGE, (E) MFK00, and (F) MFK902 peptides. *p < 0.05. Values are presented as the mean ± SEM.
Fig 3.
Modulation of clinical arthritis in mice with collagen-induced arthritis (CIA) after MFK902 treatment.
(A) Distribution of Cy5.5-labeled MFK902 in the mice with active CIA. MFK902-Cy5.5 or free Cy5.5 were intravenously injected into mice and then near-infrared fluorescence (NIRF) images were captured using an eXplore Optix system. Representative color-coded images and total photon counts (mean at ± SD) indicated times are shown. (B-C) Mice with CIA were treated intraperitoneally with MFK902 daily at indicated doses, methotrexate (MTX, 1 mg/kg twice weekly), or phosphate-buffered saline (Control) beginning on day 23 after the first immunization. Arthritis scores are shown for 7–8 mice per group. (B) Clinical arthritis index. (C) Incidence of paw involvement. *p < 0.05. Data are expressed as the mean ± SEM.
Fig 4.
Amelioration of histopathologic severity in mice with CIA after MFK902 treatment.
(A) Representative histopathologic findings of ankle joints from control and MFK902-treated mice on day 50. Upper panel, H&E-stained joints. Lower panels, Immunohistochemical staining for CD3, CD31, and ICAM-1 (brown). (B) Histologic scoring of arthritis severity in control and MFK902-treated mice (mg/kg). Histologic scores for synovial hyperplasia, pannus formation, cartilage destruction, and bone erosion (range: 0–3 for each parameter) were quantified as described in the Methods section. *p < 0.05 vs. controls. Data are expressed as the mean ± SEM. ICAM-1, intercellular adhesion molecule-1. Scale bar: 50 μm
Fig 5.
Downregulation of inflammatory mediators after MFK902 treatment in the arthritis tissues from CIA mice.
(A) Semi-quantitative measurement of inflammatory mediators from joint tissues using reverse transcription-polymerase chain reaction (RT-PCR). The comparison of transcript levels was performed using 18S ribosomal RNA as the denominator and normalized according to the relative expression level of inflammatory mediators from non-arthritic mice. (B) Immunoblot analysis of ICAM-1 and RANKL expression. *p < 0.05 vs. controls. Values are expressed as the mean ± SEM. CCL2, chemokine (C-C motif) ligand 2; ICAM-1, intercellular adhesion molecule-1; RANKL, Receptor activator of nuclear factor kappa-B ligand; VCAM-1, vascular cell adhesion molecule-1.
Table 1.
In vivo safety profile in CIA mice treated with MFK902.