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Jang, Kang, Sa, Han, Seo, Kim, Nam, Kim, and Kang: Therapeutic Effect of a Recombinant βig-h3 Fragment-RGD Peptide for Chronic Inflammatory Arthritis
Original Article
Journal of Rheumatic Diseases

Journal of Rheumatic Diseases 2012; 19(2): 73-81.

Published online: 30 April 2012

DOI: https://doi.org/10.4078/jrd.2012.19.2.73

Therapeutic Effect of a Recombinant βig-h3 Fragment-RGD Peptide for Chronic Inflammatory Arthritis

Ji Ae Jang1, 2, Jin Hee Kang1, 2, Keum Hee Sa1, 2, Seung Woo Han1, Jae Seok Seo1, Kyung Hoon Kim1, Eon Jeong Nam1, In San Kim2, 3, Young Mo Kang1, 2, 3

1Division of Rheumatology, Department of Internal Medicine, Kyungpook National University School of Medicine, Daegu, Korea.

2Department of Biochemistry and Cellular Biology, Kyungpook National University School of Medicine, Daegu, Korea.

3Cell and Matrix Research Institute, Kyungpook National University School of Medicine, Daegu, Korea.

Corresponding to: Young Mo Kang, Division of Rheumatology, Kyungpook National University School of Medicine, 50, Samdeok-dong, Jung-gu, Daegu 700-706, Korea. ymkang@knu.ac.kr

Received 2 November 2011       Revised 21 November 2011       Accepted 22 November 2011

Copyright © 2012 by The Korean College of Rheumatology

(free-access):

This is a Free Access article, which permits unrestricted non-commerical use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Objective

βig-h3 is a 68kDa extracellular matrix protein which is overexpressed in synovial tissues of rheumatoid arthritis (RA). Previous results proved that βig-h3 fragments are relevant to adhesion and migration of synovial fibroblast and angiogenesis through interaction with αvβ 3 integrin. We designed a recombinant βig-h3 protein consisting of a fas-1 domain and RGD motif and evaluated the therapeutic efficacy in RA.

Methods

Inhibitory effect of adhesion and migration of NIH3T3 cell line was evaluated in 96 well microtiter and transwell plates coated with βig-h3. Clinical arthritis index was evaluated after treating CIA mice with MFK12. Immunohistochemical staining in synovial tissues were performed. Expression of transcripts and proteins of inflammatory mediators were analyzed by semi-quantitative RT-PCR and immunoblotting.

Results

Recombinant protein consisted of 4th fas-1 domain truncated for H1 and H2 sequences and RGD peptide (MFK12), had M.W. of 10.4kDa. βig-h3 mediated adhesion and migration of NIH3T3 cell line were significantly inhibited in a dose-dependent manner. Arthritis severity and incidence were efficiently reduced when CIA mice were treated with MFK12 at 30 mg/kg/day compared with the control. Immunohistochemical staining of joint tissues in MFK12 treated mice exhibited reduced angiogenesis. In treated mice, expression of transcripts regarding inflammatory mediators was markedly suppressed and immunoblotting of ICAM-1 and RANKL from whole extract of hind paws also showed a significant reduction.

Conclusion

This study shows that MFK12 is effective in treating RA, although further study is warranted to improve the therapeutic efficacy.

Keywords: Rheumatoid arthritis, Inflammation, βig-h3, collagen-induced arthritis, Fas-1

Figures and Tables

Figure 1
Design of the recombinant MFK12 peptide. (A) A schematic representation of pET-29b(+) containing MFK12 sequence. (B) MFK12 peptide was produced by combination of dhfas-1 (amino acid 502-632) with RGD motif. (C) Western blot analysis of purified recombinant MFK12.
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Figure 2
Effective inhibition of βig-h3-mediated adhesion and migration by MFK12. To elucidate the efficiency of MFK12 in the regulation of cell function, MFK12 was incubated with NIH3T3 cell line in different concentrations before performing adhesion (A) and migration assay (B). Data demonstrates the relative percentage of inhibition by MFK12 compared with that by the control. Results are the mean and SEM of 3 independent experiments (*p<0.05 versus control).
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Figure 3
MFK12 induces amelioration of collagen-induced arthritis (CIA) where βig-h3 is unregulated. (A) Immunohistochemical staining of F4/80 and βig-h3 in joint tissues from normal and CIA mice. Expression of F4/80-expressing macrophages and βig-h3 in serial sections of joint tissues (×200). (B&C) Development of arthritis in MFK12-treated mice. From day 22 following the first immunization, mice were treated with MFK12 (0, 10, and 30mg/kg/day, intraperitoneally). (B) Clinical arthritis index was recorded daily. Values are the mean and SEM. (C) Incidence of arthritis among the four paws was recorded at day 30, 40, and 50 after the first immunization. Values are the mean and SEM. *p<0.05 versus the control.
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Figure 4
Histologies and immunohistologies of joints tissues from control and MFK-12 treated (10 and 30 mg/kg) CIA mice. Expression of CD31 and ICAM-1 was detected in CIA mice. (Hematoxylin counter stained for immunohistochemical stain, ×200).
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Figure 5
Effect of MFK12 treatment on the expression of inflammatory mediators in CIA mice. (A) Semi-quantitative measurement of transcripts of inflammatory mediators from joint tissues. Transcripts were calculated as target gene/reference gene (18s ribosome RNA) ratios and normalized to the relative expression level of non-arthritis mice. (B) Immunoblotting analysis of ICAM-1 and RANKL for proteins extracted from joint tissues of representative paws. Graphs show the mean and SD optical density (O.D.) from at least 3 independent experiments. The results are means±SD (*p<0.05 versus 0 mg/kg).
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