J Rheum Dis 2012; 19(2): 73-81
Published online April 30, 2012
© Korean College of Rheumatology
장지애1,2ㆍ강진희1,2ㆍ사금희1,2ㆍ한승우1ㆍ서재석1ㆍ김경훈1ㆍ남언정1ㆍ김인산2,3ㆍ강영모1,2,3
경북대학교 의학전문대학원 류마티스내과학교실1, 생화학세포생물학교실2, 경북대학교 세포기질연구소3
Correspondence to : Young Mo Kang
Objective. Ղig-h3 is a 68kDa extracellular matrix protein which is overexpressed in synovial tissues of rheumatoid arthritis (RA). Previous results proved that Ղig-h3 fragments are relevant to adhesion and migration of synovial fibroblast and angiogenesis through interaction with ՁvՂ3 integrin. We designed a recombinant Ղig-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 Ղig-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. Ղig-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, Ղig-h3, collagen-induced arthritis, Fas-1
J Rheum Dis 2012; 19(2): 73-81
Published online April 30, 2012
Copyright © Korean College of Rheumatology.
장지애1,2ㆍ강진희1,2ㆍ사금희1,2ㆍ한승우1ㆍ서재석1ㆍ김경훈1ㆍ남언정1ㆍ김인산2,3ㆍ강영모1,2,3
경북대학교 의학전문대학원 류마티스내과학교실1, 생화학세포생물학교실2, 경북대학교 세포기질연구소3
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
Division of Rheumatology, Departments of Internal Medicine1, Biochemistry and Cellular Biology2, Cell and Matrix Research Institute3, Kyungpook National University School of Medicine, Daegu, Korea
Correspondence to:Young Mo Kang
Objective. Ղig-h3 is a 68kDa extracellular matrix protein which is overexpressed in synovial tissues of rheumatoid arthritis (RA). Previous results proved that Ղig-h3 fragments are relevant to adhesion and migration of synovial fibroblast and angiogenesis through interaction with ՁvՂ3 integrin. We designed a recombinant Ղig-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 Ղig-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. Ղig-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, Ղ,ig-h3, collagen-induced arthritis, Fas-1
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