Yonsei Med J.  2020 Jan;61(1):85-93. 10.3349/ymj.2020.61.1.85.

Follistatin Mitigates Myofibroblast Differentiation and Collagen Synthesis of Fibroblasts from Scar Tissue around Injured Flexor Tendons

Affiliations
  • 1BK21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul, Korea.
  • 2Department of Orthopaedic Surgery, Yonsei University College of Medicine, Seoul, Korea. YRCHOI@yuhs.ac

Abstract

PURPOSE
The aim of this study was to investigate the effect of FST gene on the inhibition of fibrosis in fibroblastic cells from scar tissue around repaired zone II flexor tendons.
MATERIALS AND METHODS
Immunohistochemistry was conducted on fibroblast cells transfected with adenovirus-LacZ (Ad-LacZ) as a marker gene (control), or with adenovirus-FST (Ad-FST) as a therapeutic gene. Fibroblast cultures without adenoviral exposure served as controls.
RESULTS
Fibroblastic cells transfected with Ad-FST demonstrated significant decrease in collagen type I, MMP-1, MMP2, and α-SMA mRNA expressions compared to those transfected with Ad-LacZ. In addition, fibroblastic cells transfected with Ad-FST exhibited significant decrease in MMP-1, TIMP-1, fibronectin, PAI-1, TRPV4, α-SMA, desmin, and PAX7 protein expressions.
CONCLUSION
Based on these findings, we conclude that FST may be a novel therapeutic strategy for preventing scar adhesions around repaired tendons by inhibiting fibroblasts from differentiating into myofibroblasts, in addition to producing type I collagen and regulating extracellular matrix turnover via the downregulation of MMP-1 and TIMP-1. FST may also decrease contracture of the scar by inhibiting Ca²âº-dependent cell contraction.

Keyword

Follistatin; tendon injuries; tendon adhesion; fibrosis; gene therapy

MeSH Terms

Cicatrix*
Collagen Type I
Collagen*
Contracture
Desmin
Down-Regulation
Extracellular Matrix
Fibroblasts*
Fibronectins
Fibrosis
Follistatin*
Genetic Therapy
Immunohistochemistry
Myofibroblasts*
Plasminogen Activator Inhibitor 1
RNA, Messenger
Tendon Injuries
Tendons*
Tissue Inhibitor of Metalloproteinase-1
Collagen
Collagen Type I
Desmin
Fibronectins
Follistatin
Plasminogen Activator Inhibitor 1
RNA, Messenger
Tissue Inhibitor of Metalloproteinase-1

Figure

  • Fig. 1 Recombinant adenovirus was transfected in fibroblast from the scar tissue of patients with adhesions around zone II flexor tendon after tendon repair by immunocytochemistry analysis, indicating highly efficient transduction rate of adenoviral marker gene construct when activated by transforming growth factor-beta 1 (TGF-β1). ‘+’ means ‘treatment’ and ‘−’ means ‘no treatment’. Ad-FST, adenovirus follistatin gene construct; DAPI, 4′,6-diamidino-2-phenylindole.

  • Fig. 2 Gene expressions of collagen I (A), collagen III (B), collagen IV (C), collagen V (D), and collagen XI (E) in fibroblast from adhesion tissue in patients with zone II flexor tendon repair and transfection with/without adenovirus follistatin gene construct (Ad-FST). Expression of collagen type I mRNA in fibroblast with Ad-FST showed a 25% decrease at 24 hours compared to control culture (TGF-β1+). ‘+’ means ‘treatment’ and ‘−’ means ‘no treatment’. *p<0.05. TGF-β1, transforming growth factor-beta 1; Ad-LacZ, adenovirus LacZ gene construct; SB505124, selective inhibitor of transforming growth factor-β type I receptor.

  • Fig. 3 Gene expressions of MMP-1 (A), MMP-2 (B), MMP-3 (C), MMP-9 (D), and MMP-13 (E) in fibroblast from adhesion tissue in patients with zone II flexor tendon repair and transfection with/without adenovirus follistatin gene construct (Ad-FST). Expressions of MMP-1 and -2 mRNA in fibroblast with Ad-FST showed a 31% and 59% decrease, respectively, at 24 hours compared to control culture (TGF-β1+). ‘+’ means ‘treatment’ and ‘−’ means ‘no treatment’. *p<0.05. TGF-β1, transforming growth factor-beta 1; MMP, matrix metalloproteinase; Ad-LacZ, adenovirus LacZ gene construct; SB505124, selective inhibitor of transforming growth factor-β type I receptor.

  • Fig. 4 Gene expressions of PAI-1 (A) and α-SMA (B) in fibroblast from adhesion tissue in patients with zone II flexor tendon repair and transfection with/without adenovirus follistatin gene construct (Ad-FST). Expression of α-SMA mRNA in the fibroblast with Ad-FST showed a 23% decrease at 24 hours compared to control culture (TGF-β1+). ‘+’ means ‘treatment’ and ‘−’ means ‘no treatment’. *p<0.05. TGF-β1, transforming growth factor-beta 1; Ad-LacZ, adenovirus LacZ gene construct; SB505124, selective inhibitor of transforming growth factor-β type I receptor.

  • Fig. 5 Protein expressions of MMPs (A), MMP-1 (B), MMP-2 (C), and MMP-13 (D) in fibroblast from adhesion tissue in patients with zone II flexor tendon repair and transfection with/without adenovirus follistatin gene construct (Ad-FST). Expression of MMP-1 protein in fibroblast with Ad-FST showed a 24% decrease at 24 hours compared to control culture (TGF-β1+). ‘+’ means ‘treatment’ and ‘−’ means ‘no treatment’. *p<0.05. TGF-β1, transforming growth factor-beta 1; MMP, matrix metalloproteinase; Ad-LacZ, adenovirus LacZ gene construct; SB505124, selective inhibitor of transforming growth factor-β type I receptor.

  • Fig. 6 Protein expressions of TIMP-1, -2, -4 (A–D) and fibronectin (E) in fibroblast from adhesion tissue in patients with zone II flexor tendon repair and transfection with/without adenovirus follistatin gene construct (Ad-FST). Expressions of TIMP-1 and fibronectin protein in fibroblast with Ad-FST showed a 23% and 24% decrease, respectively, at 24 hours compared to control culture (TGF-β1+). ‘+’ means ‘treatment’ and ‘−’ means ‘no treatment’. *p<0.05. TGF-β1, transforming growth factor-beta 1; Ad-LacZ, adenovirus LacZ gene construct; SB505124, selective inhibitor of transforming growth factor-β type I receptor.

  • Fig. 7 Protein expressions of TRPV4, PAI-1, and α-SMA (A) in fibroblast from adhesion tissue in patients with zone II flexor tendon repair and transfection with/without adenovirus follistatin gene construct (Ad-FST). Expressions of TRPV4 (B), PAI-1 (C), and α-SMA (D) protein in fibroblast with Ad-FST showed a 23%, 23%, and 28% decrease, respectively, at 24 hours compared to control culture (TGF-β1+). ‘+’ means ‘treatment’ and ‘−’ means ‘no treatment’. *p<0.05. TGF-β1, transforming growth factor-beta 1; Ad-LacZ, adenovirus LacZ gene construct; SB505124, selective inhibitor of transforming growth factor-β type I receptor.

  • Fig. 8 Fibroblasts with adenovirus follistatin gene construct (Ad-FST) showed decrease in α-SMA, desmin, and PAX7 expressions compared to control culture (TGF-β1+). TGF-β1, transforming growth factor-beta 1; DAPI, 4′,6-diamidino-2-phenylindole.


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