Anti-fibrotic Gene Therapy Effect of AP-1 Decoy ODN in Bleomycin-induced Sclerotic Animal Model / 대한피부과학회지

BACKGROUND: Scleroderma is a connective tissue disorder characterized by excessive collagen production by activated fibroblasts. TGF-beta plays key roles in fibrosis of dermsis. Although numerous studies have elucidated the pathogenesis of scleroderma, effective therapeutic strategies for improving the sclerosis of the skin have been underinvestigated. Recently several studies indicated that an animal model of sclerotic skin induced by bleomycin is useful for providing clues and therapeutic interventions for scleroderma. We previously reported that AP (Activator protein)-1 decoy ODN (oligodeoxynucleotides) suppresses the TGF-beta1-induced type I collagen gene expression in cultured scleroderma fibroblast in vitro studies. Therefore, it is necessary to confirm the anti-fibrotic effect of AP-1 decoy ODN in sclerotic animal model. OBJECTIVE: The purpose of this study is the establishment of a mouse model for scleroderma and confirmation of the anti-fibrotic effect of AP-l decoy ODN in vivo study. METHODS: Dermal sclerosis was induced by intradermal injection of bleomycin at a dose of 0.3, 1.5, 3 (mg/ml) into the back skin of BALB/C mice twice a week for 4 weeks. To confirm anti-fibrotic effect of AP1-decoy ODN, the AP-1 decoy ODN was transfected into subcutaneous tissue of mice with or without bleomycin. Dermal sclerosis was examined by hematoxylin and eosin (H&E) staining and Masson's trichrome staining. TGF-beta1 expression was detected by immunohistochemistry and type I collagen gene expression was also analyzed by dot blotting and western blot method. RESULTS: Histopathological examination revealed that the dermal sclerosis with the deposition of thickened and homogenous collagen bundles increased according to the concentration of bleomycin. The expressions of type I collagen and TGF-beta1 were markedly increased in bleomyin-injected mice. Furthermore transfection of AP-1 decoy ODN with bleomycin suppressed the dermal sclerosis and type I collagen gene expression as well as TGF-beta1 in mice. CONCLUSION: AP-1 decoy ODN inhibits the bleomycin-induced dermal sclerosis through down-regulation of type I collagen and TGF-beta1 expression in BALB/C mice. Thus the anti-fibrotic effect of AP-1 decoy ODN in bleomycin-induced sclerotic mouse model suggests the fundamental data for gene therapy of scleroderma.

The Effect of an AP-1 decoy on Collagen Synthesis in Wound Healing on Rat Skin: The use of Gene Therapy to Minimise Scarring / 대한피부과학회지

BACKGROUND: The wound healing process is composed of inflammation, cellular growth, migration, angiogenesis and an extracellular matrix composition. In this process, fibroblasts proliferate but leave scarring due to their excessive growth. The process is controlled by platelet derived growth factor (PDGF) and basic fibroblast growth factor (bFGF), while PDGF and transforming growth factor beta(TGF-beta) play a major role in controlling extracellular matrix composition. Numerous modalities have been tried to treat this abnormal response, but the results were unsatisfactory. TGF-beta is activated by AP-1 (Activator protein). Theregone, AP-1 decoy oligodeoxynucleotides(ODN) had recently been used for regulation of TGF-beta transcription as a gene treatment. OBJECTIVE: The purpose of this study was to examine the effect of blocking TGF-beta transcription by an AP-1 decoy ODN on collagen synthesis in wound healing on rat skin. METHODS: In this study, the effect of AP-1 decoy ODN on collagen synthesis was examined by Hematoxylin and eosin (H&E) staining, Masson's trichrome staining, and immunohistochemical staining for TGF-beta on damaged rat skin. RESULTS: In the H&E stain and Masson's trichrome stain of the damaged rat skin, the number of collagen fibers of AP-1 decoy ODN treated group had decreased in compared to the control group, especially on the 15th day after incision. With immunohistochemical stain, the expression of TGF-beta in fibroblasts, inflammatory cells and the endothelium of vessel walls in the dermis had also decreased, compared to the control group. TGF-beta was expressed in the dermis from the 3rd day, and predominantly in the fibroblasts on the 15th day after incision. CONCLUSION: These results indicate that AP-1 decoy ODN is a powerful down-regulator of collagen synthesis in wound healing through significant suppression of TGF-beta expression in damaged skin. Therefore, AP-1 decoy ODN can be used effectively to treat or minimize scarring on damaged skin.