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A novel single-chain Fv antibody for connective tissue growth factor against the differentiation of fibroblast into myofibroblast

  • Applied genetics and molecular biotechnology
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Abstract

This study was aimed to investigate the effect of a single-chain fragment variable antibody of connective tissue growth factor (anti-CTGF scFv) against the differentiation of fibroblast into myofibroblast. The scFv antibody was firstly expressed in Escherichia coli cells and was then purified by affinity chromatography. The yield scFv protein reached a purity over 95% after purification. Immunoreactivity assay demonstrated that scFv possessed a special affinity toward CTGF. RT-PCR, western blot, and immunofluorescence experiments showed that increased expression of α-smooth muscle actin induced by TGF-β1 could be suppressed by this scFv antibody through inhibiting the phosphorylation of Akt.

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References

  • Abraham D (2008) Connective tissue growth factor: growth factor, matricellular organizer, fibrotic biomarker or molecular target for anti-fibrotic therapy in SSc? Rheumatology (Oxford) 47(Suppl 5):v8–v9

    Article  CAS  Google Scholar 

  • Ball DK, Rachfal AW, Kemper SA, Brigstock DR (2003) The heparin-binding 10 kDa fragment of connective tissue growth factor (CTGF) containing module 4 alone stimulates cell adhesion. J Endocrinol 176(2):R1–R7

    Article  CAS  Google Scholar 

  • Bottinger EP, Letterio JJ, Roberts AB (1997) Biology of TGF-[beta] in knockout and transgenic mouse models. Kidney Int 51(5):1355–1360

    Article  CAS  Google Scholar 

  • De Marco A (2011) Biotechnological applications of recombinant single-domain antibody fragments. Microb Cell Fact 10(1):44–57

    Article  Google Scholar 

  • Duncan MR, Frazier KS, Abramson S, Williams S, Klapper H, Huang X, Grotendorst GR (1999) Connective tissue growth factor mediates transforming growth factor beta-induced collagen synthesis: down-regulation by cAMP. FASEB J 13(13):1774–1786

    CAS  Google Scholar 

  • Grotendorst GR (1997) Connective tissue growth factor: a mediator of TGF-beta action on fibroblasts. Cytokine Growth Factor Rev 8(3):171–179

    Article  CAS  Google Scholar 

  • Grotendorst GR, Duncan MR (2005) Individual domains of connective tissue growth factor regulate fibroblast proliferation and myofibroblast differentiation. FASEB J 19(7):729–738

    Article  CAS  Google Scholar 

  • Guha M, Xu ZG, Tung D, Lanting L, Natarajan R (2007) Specific down-regulation of connective tissue growth factor attenuates progression of nephropathy in mouse models of type 1 and type 2 diabetes. FASEB J 21(12):3355–3368

    Article  CAS  Google Scholar 

  • Gurujeyalakshmi G, Giri SN (1995) Molecular mechanisms of antifibrotic effect of interferon gamma in bleomycin-mouse model of lung fibrosis: downregulation of TGF-beta and procollagen I and III gene expression. Exp Lung Res 21(5):791–808

    Article  CAS  Google Scholar 

  • Hautmann MB, Adam PJ, Owens GK (1999) Similarities and differences in smooth muscle alpha-actin induction by TGF-beta in smooth muscle versus non-smooth muscle cells. Arterioscler Thromb Vasc Biol 19(9):2049–2058

    Article  CAS  Google Scholar 

  • Horowitz JC, Thannickal VJ (2006) Idiopathic pulmonary fibrosis : new concepts in pathogenesis and implications for drug therapy. Treat Respir Med 5 (5):325–342

    Article  Google Scholar 

  • Hu B, Wu Z, Liu TJ, Ullenbruch MR, Jin H, Phan SH (2007) Gut-enriched Kruppel-like factor interaction with Smad3 inhibits myofibroblast differentiation. Am J Respir Cell Mol Biol 36(1):78–84

    Article  CAS  Google Scholar 

  • Huber PE, Bickelhaupt S, Peschke P, Tietz A, Wirkner U, Lipson KE (2010) Reversal of established fibrosis by treatment with the anti-CTGF monoclonal antibody FG-3019 in a murine model of radiation-induced pulmonary fibrosis. Am J Respir Crit Care Med 181:A1054, 1_MeetingAbstracts

    Google Scholar 

  • Ikawa Y, Ng PS, Endo K, Kondo M, Chujo S, Ishida W, Shirasaki F, Fujimoto M, Takehara K (2008) Neutralizing monoclonal antibody to human connective tissue growth factor ameliorates transforming growth factor-beta-induced mouse fibrosis. J Cell Physiol 216(3):680–687

    Article  CAS  Google Scholar 

  • Kolb M, Margetts PJ, Galt T, Sime PJ, Xing Z, Schmidt M, Gauldie J (2001) Transient transgene expression of decorin in the lung reduces the fibrotic response to bleomycin. Am J Respir Crit Care Med 163(3 Pt 1):770–777

    CAS  Google Scholar 

  • Kolb M, Bonniaud P, Galt T, Sime PJ, Kelly MM, Margetts PJ, Gauldie J (2002) Differences in the fibrogenic response after transfer of active transforming growth factor-beta1 gene to lungs of “fibrosis-prone” and “fibrosis-resistant” mouse strains. Am J Respir Cell Mol Biol 27(2):141–150

    CAS  Google Scholar 

  • Kulkarni AB, Huh CG, Becker D, Geiser A, Lyght M, Flanders KC, Roberts AB, Sporn MB, Ward JM, Karlsson S (1993) Transforming growth factor beta 1 null mutation in mice causes excessive inflammatory response and early death. Proc Natl Acad Sci U S A 90(2):770–774

    Article  CAS  Google Scholar 

  • Liu BC, Zhang JD, Zhang XL, Wu GQ, Li MX (2006) Role of connective tissue growth factor (CTGF) module 4 in regulating epithelial mesenchymal transition (EMT) in HK-2 cells. Clin Chim Acta 373(1–2):144–150

    Article  CAS  Google Scholar 

  • Liu NF, Wu GQ, Li H, Li LX, Xing HL, Zhang C, Lu HX (2009) A novel peptide isolated from phage display peptides library recognized by an antibody against connective tissue growth factor (CTGF). Int Immunopharmacol 9(3):291–297

    Article  CAS  Google Scholar 

  • Luo GH, Lu YP, Song J, Yang L, Shi YJ, Li YP (2008) Inhibition of connective tissue growth factor by small interfering RNA prevents renal fibrosis in rats undergoing chronic allograft nephropathy. Transplant Proc 40(7):2365–2369

    Article  CAS  Google Scholar 

  • Mageto Y, Flaherty K, Brown K, Fong A, Raghu G (2004) Safety and tolerability of human monoclonal antibody FG-3019, anti-connective tissue growth factor, in patients with idiopathic pulmonary fibrosis. Chest 126(4):773S-a-

    Google Scholar 

  • Minato M, Kubota S, Kawaki H, Nishida T, Miyauchi A, Hanagata H, Nakanishi T, Takano-Yamamoto T, Takigawa M (2004) Module-specific antibodies against human connective tissue growth factor: utility for structural and functional analysis of the factor as related to chondrocytes. J Biochem 135(3):347–354

    Article  CAS  Google Scholar 

  • Nakanishi T, Nishida T, Shimo T, Kobayashi K, Kubo T, Tamatani T, Tezuka K, Takigawa M (2000) Effects of CTGF/Hcs24, a product of a hypertrophic chondrocyte-specific gene, on the proliferation and differentiation of chondrocytes in culture. Endocrinology 141(1):264–273

    Article  CAS  Google Scholar 

  • Perbal B (2004) CCN proteins: multifunctional signalling regulators. Lancet 363(9402):62–64

    Article  CAS  Google Scholar 

  • Phanish MK, Winn SK, Dockrell ME (2010) Connective tissue growth factor-(CTGF, CCN2)—a marker, mediator and therapeutic target for renal fibrosis. Nephron Exp Nephrol 114(3):e83–e92

    Article  CAS  Google Scholar 

  • Qi W, Chen X, Poronnik P, Pollock CA (2008) Transforming growth factor-beta/connective tissue growth factor axis in the kidney. Int J Biochem Cell Biol 40(1):9–13

    Article  CAS  Google Scholar 

  • Scotton CJ, Chambers RC (2007) Molecular targets in pulmonary fibrosis: the myofibroblast in focus. Chest 132(4):1311–1321

    Article  Google Scholar 

  • Shull MM, Ormsby I, Kier AB, Pawlowski S, Diebold RJ, Yin M, Allen R, Sidman C, Proetzel G, Calvin D, Annunziata N, Doetschman T (1992) Targeted disruption of the mouse transforming growth factor-beta 1 gene results in multifocal inflammatory disease. Nature 359(6397):693–699

    Article  CAS  Google Scholar 

  • Steffen CL, Ball-Mirth DK, Harding PA, Bhattacharyya N, Pillai S, Brigstock DR (1998) Characterization of cell-associated and soluble forms of connective tissue growth factor (CTGF) produced by fibroblast cells in vitro. Growth Factors 15(3):199–213

    Article  CAS  Google Scholar 

  • Takehara K (2003) Hypothesis: pathogenesis of systemic sclerosis. J Rheumatol 30(4):755–759

    Google Scholar 

  • Wang XH, Wu GQ, Gou LX, Liu ZZ, Wang XY, Fan XB, Wu LY, Liu NF (2011) A novel single-chain-Fv antibody against connective tissue growth factor attenuates bleomycin-induced pulmonary fibrosis in mice. Respirology 16(3):500–507

    Article  Google Scholar 

  • Weisser NE, Hall JC (2009) Applications of single-chain variable fragment antibodies in therapeutics and diagnostics. Biotechnol Adv 27(4):502–520

    Article  CAS  Google Scholar 

  • Wu GQ, Fan XB, Wu HB, Liu NF, Li XF, Gou LX, Nie Y, Zhao R, Xi T (2010) Bioscreening of phage display antibody library and expression of a humanized single-chain variable fragment antibody against human connective tissue growth factor (CTGF/CCN2). Biotechnol Appl Biochem 56(3):95–102

    Article  CAS  Google Scholar 

  • Yokoi H, Mukoyama M, Nagae T, Mori K, Suganami T, Sawai K, Yoshioka T, Koshikawa M, Nishida T, Takigawa M, Sugawara A, Nakao K (2004) Reduction in connective tissue growth factor by antisense treatment ameliorates renal tubulointerstitial fibrosis. J Am Soc Nephrol 15(6):1430–1440

    Article  CAS  Google Scholar 

Download references

Acknowledgments

We thanked the Natural Science Foundation of Jiangsu Province, China (no. BK2009274), the Science Foundation of Southeast University (no. 3290000102), and the National Science Foundation of China (grant numbers 30970809 and 30840042) for the financial support to this work. Meanwhile, we thanked Dr. Xiaobo Fan and Dr. Jing Xiong for the careful revision to this paper.

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Authors and Affiliations

  1. Center of Clinical Laboratory Medicine of Zhongda Hospital, Southeast University, Nanjing, 210009, China

    Guoqiu Wu

  2. Medical School, Southeast University, Nanjing, 210009, China

    Xiyong Wang, Xiulei Xue & Xuejiao Yan

  3. Biotechnology Center, Department of Life Science and Biotechnology, China Pharmaceutical University, Nanjing, 210009, China

    Xuepeng Deng & Pengpeng Wu

  4. Department of Respiratory, Zhongda Hospital, Southeast University, Nanjing, 210009, China

    Xihua Wang

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  1. Guoqiu Wu
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  2. Xiyong Wang
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  3. Xuepeng Deng
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  4. Pengpeng Wu
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  5. Xiulei Xue
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  6. Xuejiao Yan
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  7. Xihua Wang
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Correspondence to Guoqiu Wu or Xihua Wang.

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Guoqiu Wu and Xiyong Wang contributed equally to this article.

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Wu, G., Wang, X., Deng, X. et al. A novel single-chain Fv antibody for connective tissue growth factor against the differentiation of fibroblast into myofibroblast. Appl Microbiol Biotechnol 93, 2475–2482 (2012). https://doi.org/10.1007/s00253-011-3755-8

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  • DOI: https://doi.org/10.1007/s00253-011-3755-8

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