Gene Therapy For Hypertrohic Scar By Hepatocyte Growth Factor And Studies On Its Related Mechanisms

Pathological scar formation is characterized by excessive dermal fibrosis, as a sequela of unnormal wound healing. Increased collagen synthesis and /or reduced matrix degradation is a major cause of this disorder which results in excessive scarring with a deformed appearance and dysfunction. Therefore, the basic principles of curing pathological scar are to accelerate wound repair, suppress fibroblasts hyperplasia and promote the degradation of extracellular matrix. Hepatocyte growth factor (HGF) displays mitogenic activities in a wide variety of cells including keratinocytes and endothelial cells, and recently it has been shown to strongly suppress the expression of TGF-β1, and promote the secretion of some matrix metalloproteinase. In view of the above-mentioned advances, the aim of this study was to characterize the role of HGF in wound healing and scar degradation by administering the HGF gene locally, and to unravel the related mechanisms.We treated the rabbit ear scar by gene therapy using plasmids containing HGF cDNAs. On day 15, the treated wounds became thin and flat, while the control wounds were still kept red, swelling and higher than the normal skin, i.e. the characters of hypertrophic scar. In addition, fibrotic change of scars was evaluated by hypertrophic index, histopathology, Sirius red stain and hydroxyproline detection .The hypertrophic index of pUDKH was 1.61, while the PBS and pUDK groups were 2.29 and 2.30 respectively. Histopathological observation revealed that HGF could promote the epidermis cell differentiation and reduce the fibrous tissue in the dermis. By means of polarizing microscopy, there were more abundant and gross type I collagen fibers in the PBS and pUDK groups. The content of hydroxyproline per unit area in pUDKH groups (1.08±0.11μg/ml skin wet weight) was lower than the PBS and pUDK groups (1.50±0.27 and 1.47±0.17μg/ml skin wet weight). Immunohistochemistry analysis showed that HGF could reduce the expression ofα-smooth muscle actin (α-SMA). Apoptotic cells in rabbit ear scar were identified by TUNEL. It was found that the number of apoptotic cells was increased about 50% in the HGF-treated animals, compared to the control groups. The results of in vivo study manifested the obvious effects of HGF on hypertrophic scar.Then, the cellular or molecular mechanisms of HGF for improving the hypertrophic scar have been investigated. Firstly, the primary fibroblasts isolated from normal skin (NFB) and hypertrophic scar (HFB) were infected with adenoviruses encoding human HGF gene (Ad-HGF) to determine the effect of HGF on these cells. The results of flow cytometry showed that infection efficiency was increasing along with the increase of multiplicity of infection (MOI). The expression level of HGF in supernatant was about 120ng in 1×10⁶ cells at 48h in NFB and HFB as measured by ELISA. There were no obvious differences in infection efficiency and HGF expression, as well as the expression of HGF and HGF receptor c-Met between NFB and HFB. Results of MTT assay revealed that Ad-HGF infection could inhibit the growth of HFB, but not NFB. By means of transmission electron microscopy, there appeared karyopyknosis, chromatic agglutination, short and small pustute, kytoplasm reductus, rich rough endoplasmic reticulum gradually dilated and gathered, and the degranulation occurred on membrane surface. Some vacuoles appeared in mitochondrium in HFB after infection with Ad-HGF, however, the NFB had not obvious changes. These observations demonstrated that HGF could induce the apoptosis of HFB. The concentration of free calcium ions in HFB cytoplasm after infecting with Ad-HGF was increased and more than that of NFB observed by laser scan confocal microscopy. As 1, 4, 5-inositol triphosphate (IP3), a second messenger, is a main inducer to intracellular Ca²⁺ release, and mRNA expression of IP3 receptor was up-regulated in NFB and HFB after infection with Ad-HGF as assayed by RT-PCR, our results thereby provide a clue that HGF may induce the apoptosis of HFB through the calcium ion signal pathway.Secondly, to explore the relation between the expression of oncogene c-Myb andα-SMA, the plasmid carrying the c-Myb cDNA were constructed and transfected into NIH3T3 cell line. Under G418 pressure, the single colony NIH3T3 cell line expressing c-Myb was obtained, then theα-SMA expression in NIH3T3 cell line was determined by immunofluorescence and Western blot. After infection with adenoviral HGF, the expression ofα-SMA reduced, as well as TGF-β1 and c-Myb. The same phenomena were observed in HFB. TGF-β1 plays a dominant role in scar formation, which can increases the expression of c-Myb, regulates the type I collagen andα-SMA gene expression. It is well known that HGF is an antagonist to this TGF-β1 activity. Thus, the results implied that HGF inhibited the TGF-β1 activity, subsequently down-regulated the oncogene c-Myb expression, finally affected the expression ofα-SMA and type I collagen in HFB. At the same time, HGF-induced HFB apoptosis might ascribe to the inhibition of c-Myb expression by HGF.Finally, in vitro study revealed that HGF induced the expression of urokinase-type plasminogen activator and facilitated the proliferation and migration of keratinocytes and neovascularization. This may be a possible explanation for rapid re-epithelialization and promotion of granulation tissue as well as blood vessel growth observed in vivo study.Conclusions:â‘ HGF has obvious curative effects on hypertrophic scar , through inhibiting the collagen andα-SMA expression, and promoting the apoptosis of myofibroblasts;â‘¡HGF induced the apoptosis of HFB through the calcium ion signal pathway;â‘¢HGF inhibited the oncogene c-Myb expression, subsequently downregulate the TGF-β1 activity, finally affected the expression ofα-SMA and type I collagen in HFB;â‘£HGF accelerates re-epithelialization and hastens neovascularization in granulation tissue through promotingurokinase type plasminogen activator expression .