Yonsei Med J.  2019 Sep;60(9):854-863. 10.3349/ymj.2019.60.9.854.

Effect of Relaxin Expression from an Alginate Gel-Encapsulated Adenovirus on Scar Remodeling in a Pig Model

• Yun IS
• Kang E
• Ahn HM
• Kim YO
• Rah DK
• Roh TS
• Lee WJ
• Yun CO

• KMID: 2457481
• DOI: http://doi.org/10.3349/ymj.2019.60.9.854

Abstract

PURPOSE
Relaxin (RLX) is a transforming growth factor-β1 (TGF-β1) antagonist that is believed to function as a potent collagen re-arranger and a major suppressor of extracellular matrix components. Adenoviruses (Ads) are accepted vectors for cancer gene therapy. However, repeated treatments of Ad are limited by short-term biological activity in vivo. The efficacy of sustained RLX expression to scar remodeling was assessed using an injectable alginate gel-matrix system.
MATERIALS AND METHODS
Pig scar tissue was treated with relaxin-expressing Ad loaded in alginate gel (gel/Ad-RLX). Surface areas, color, and pliability of scars were compared, and various factors influencing scar formation and collagen arrangement were analyzed.
RESULTS
Gel/Ad-RLX decreased scar size, color index, and pliability. Immunohistochemistry showed decreased levels of major extracellular matrix proteins in the gel/Ad-RLX-treated group. Furthermore, treatment with gel/Ad-RLX reduced expression of tissue inhibitor of metalloproteinase-1 and alpha-smooth muscle actin and markedly increased expression of matrix metalloproteinase-1 in pig scar tissues. Gel/Ad-RLX also significantly downregulated TGF-β1 and upregulated TGF-β3 mRNAs in pig scar tissues.
CONCLUSION
These results support a prominent role for RLX in scar remodeling and suggest that gel/Ad-RLX may have therapeutic effects on scar formation.

Keyword

Gene therapy; scar remodeling; relaxin; adenovirus; alginate gel; pig scar model

MeSH Terms

Actins
Adenoviridae*
Cicatrix*
Collagen
Extracellular Matrix
Extracellular Matrix Proteins
Genes, Neoplasm
Genetic Therapy
Immunohistochemistry
Matrix Metalloproteinase 1
Pliability
Relaxin*
RNA, Messenger
Therapeutic Uses
Tissue Inhibitor of Metalloproteinase-1
Actins
Collagen
Extracellular Matrix Proteins
Matrix Metalloproteinase 1
RNA, Messenger
Relaxin
Therapeutic Uses
Tissue Inhibitor of Metalloproteinase-1

Figure

• 

  Fig. 1 Relaxin (RLX) expression reduces the size, color index, and pliability of pig scars. (A) Scars were created on the backs of pigs. The area of scars decreased, and the color improved at 50 days after the injection of relaxin-expressing Ad loaded in alginate gel (gel/Ad-RLX). (B) Scar size, (C) erythema index values, (D) melanin index values, and (E) pliability were significantly reduced (†p<0.05) compared to those of control groups [gel or LacZ-expressing Ad loaded in alginate gel (gel/Ad-LacZ)]. Data are expressed as a mean±standard error of the mean.

• 

  Fig. 2 Immunofluorescence staining to evaluate the effects of relaxin on scar reduction in pig scar tissues. Increased expression of relaxin was detected after treatment with relaxin-expressing Ad loaded in alginate gel (gel/Ad-RLX). In contrast, control groups [gel or LacZ-expressing Ad loaded in alginate gel (gel/Ad-LacZ)] showed lower levels of relaxin. Nuclei were visualized by DAPI (4',6-diamidino-2-phenylindole) staining; original magnification, ×400; scale bar=20 µm.

• 

  Fig. 3 Picrosirius red staining and immunohistochemical staining for collagen type-I, collagen type-III, elastin, and fibronectin in pig scar tissues. (A) Dense and coarse collagen fibers were replaced with closely packed collagen fibers after pig scar tissues were treated with gel/Ad-RLX; original magnification, ×100 and ×200; scale bar=500 µm and 200 µm. (B) The expression levels of the major extracellular matrix components collagen type-I, collagen type-III, elastin, and fibronectin were lower in pig scar tissues treated with gel/Ad-RLX than those in pig tissues treated with gel or gel/Ad-LacZ; original magnification, ×400; scale bar=100 µm. Semi-quantitative image analyses revealed that (C) collagen type-I, (D) collagen type-III, (E) elastin, and (F) fibronectin were significantly decreased in pig tissues treated with gel/Ad-RLX, compared to those in pig tissues treated with control virus (*p<0.01). Data are expressed as a mean±standard error of the mean.

• 

  Fig. 4 Immunohistochemical staining for matrix metalloproteinase-1 (MMP-1), tissue inhibitor of metalloproteinase-1 (TIMP-1), and alpha-smooth muscle actin (α-SMA) in pig scar tissues. (A) Treatment with gel/Ad-RLX markedly increased MMP-1 levels and decreased TIMP-1 and α-SMA levels in pig scar tissues; original magnification, ×400; scale bar=100 µm. Semi-quantitative image analyses revealed that (B) MMP-1 was significantly increased in pig tissues treated with gel/Ad-RLX versus that of tissues treated with control virus (*p<0.01). (C) TIMP-1 and (D) α-SMA levels were significantly decreased in pig tissues treated with gel/Ad-RLX, compared to those with control virus (†p<0.05). Data are expressed as mean±standard error of the mean.

• 

  Fig. 5 Relaxin-expressing Ad decreases transforming growth factor-β1 (TGF-β1) and increases TGF-β3 mRNA levels in pig scar tissues. (A) Quantitative analyses by qRT-PCR indicated that TGF-β1 mRNA levels were significantly decreased (†p<0.05) in scar tissues treated with gel/Ad-RLX, compared with those of tissue treated with control virus. (B) In contrast, TGF-β3 mRNA was significantly increased (†p<0.05) by relaxin overexpression in pig scar tissues. Data are expressed as a mean±standard error of the mean.

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