Adenovirus-relaxin gene therapy on keloid : attenuated proliferative response and collagen degradation

Abstract

[한글]

[영문]

Keloid or hypertrophic scars are pathologic proliferations of the skin dermal layer that results from excessive collagen deposition. This pathologic scarring is mainly due to the abnormalities of keloid fibroblast. Relaxin (RLX), a pleiotropic hormone, is a member of the insulin and insulin-like growth factor (IGF) superfamily, and it inhibits collagen synthesis and expression of stimulated fibroblasts with a pro-fibrotic agent including TGF-β and IL-1, 6, when collagen is over expressed, but it does not affect the basal levels of collagen expression. However, the half-life of RLX is very short. In order to overcome this drawback, we made a replication-incompetent relaxin-expressing adenovirus (dl-lacZ-RLX-RGD). The purpose of this study is to investigate the effect of dl-lacZ-RLX-RGD on type I and III collagen mRNA expression, matrix metalloproteinase (MMP) -1, 3, and Smad-2, 3 mRNA expressions of human dermal fibroblasts cell line (HDFCs) and keloid fibroblasts (KFs). In an ex vivo study, we investigated the expression levels of various ECM such as collagen type III, fibronectin, and elastin using immunohistochemisty on the keloid spheroids ex vivo cultures that were transduced with a relaxin-expressing adenovirus. Keloid fibroblasts (5×105 cells), which were obtained from tissues excised during surgical procedures, and HDFCs were transfected with dl-lacZ-RGD or dl-lacZ-RLX-RGD at multiplicity of infection (MOI) 100 for 72 hours. Three days after transfection and incubation, a β-galactosidase stain was performed. The expression level of RLX protein was assessed by ELISA. The mRNA expressions of collagen type 1 and 3, and matrix metalloproteinases 1 and 3 were examined by RT-PCR. In an ex vivo study, we used keloid dermal tissue that had become spherical (i.e., spheroids) which was approximately 2 mm in diameter (n=3). For transduction of tumor spheroids, 50 μL of adenovirus dl-lacZ-RGF or dl-lacZ-RLX-RGD in a culture medium (i.e., 1 × 10 9 viral particles) was added during the three days. We examined the expression levels of collagen type III, elastin, and fibronectin by immunohistochemistry and quantitatively analyzed the expression levels using the MetaMorph® image analyzer.In the human dermal fibroblast cell line (HDFCs), under basal conditions, we observed stimulation of type I collagen and MMP-1, 3 mRNA expressions by relaxin-expressing adenovirus, whereas it decreased the type I and III collagen mRNA expression and MMP-1, 3 mRNA expressions by the addition of TGF-ß1. However, relaxin-expressing adenovirus decreased collagen types I and III and MMP-1, 3 on mRNA levels regardless of the addition of TGF-ß1 in the keloid fibroblasts and could significantly (more than 69%) reduce Smad-3 mRNA expression. However, Smad-2 mRNA expression was not changed. We think that dl-lacZ-RLX-RGD was shown to inhibit the phosphorylation of Smad-3, which was a key event in TGF-β signaling for collagen synthesis. In an ex vivo study, we investigated the expression levels of collagen type III, fibronectin, and elastin by immunohistochemisty on the keloid spheroids and showed that the expression of collagen type III, elastin, and fibronectin were decreased in keloid spheroids transduced with dl-lacZ-RLX-RGD. Gene therapy using relaxin-expressing adenovirus could attenuate type I collagen mRNA expression and MMP-1, 3 mRNA expressions on keloid fibroblasts. We expect that relaxin gene transfer using adenovirus could be an effective treatment and may prevent keloid recurrence after surgical excision.