| A higher incidence of hypertrophic scar has been demonstrated among Chinese whichare mainly caused by abnormal wound healing after skin injury, various kinds of burn,trauma and surgery. Although a great efforts have been made, the pathphysiologicalmechanism underlying the formation of hypertrophic scar is still not clear that hinderseffective prevention and treatment of this disease.Recent studies show that vascularization is closely related to the formation ofhypertrophic scars. There are multiple cytokines participating in the process ofvascularization including vascular endothelial growth factor (VEGF), which is known asone of the strongest pro-angiogenic factor. The high expression of VEGF is positivelyrelated to hypertrophic scar formation. Based on this theory, our research team has beendesigned, synthesized, and screened out the small interfering RNA fragment with excellentinhibitory effects against VEGF. Besides, we have constructed the lentiviral vector withthis interference fragment—Lentiviral-mediated silencing of vascular endothelial growth factor (LV-SiVEGF).On the basis of previous studies, we applied the LV-SiVEGF in therabbit models of hypertrophic scars in the experimentⅠ. The feasibility of vasculartargeting gene therapy was estimated by detecting the hypertrophic scar index, fibroblastproliferation, collagen synthesis, vascular endothelial cells and ultrastructural changes inrabbit ear tissues.Meanwhile, we also discovered in our previous study that recombinant angiogenesisinhibitor METH-1would obviously inhibit hypertrophic scars. Furthermore, weconstructed a lentiviral vector for METH-1---a lentivirus-mediated recombinantangiogenesis inhibitor METH-1(LV-METH-1).Therefore, we intended to explore whetherthere is a difference between the effects of stable interfering VEGF expression and theeffects of combined application of interfering VEGF expression and recombinantangiogenesis inhibitor METH-1in the rabbit ear hypertrophic scar animal models. In orderto investigate the feasibility of vascular targeting gene therapy in inhibiting the formationof hypertrophic scars and further explore the effective prevention and treatment methodsfor hypertrophic scars, we detected the hypertrophic scars index, fibroblast proliferation,collagen synthesis, and endothelial cells.This paper consists of two parts:Part I: The effects of targeted silencing VEGF on hypertrophic scars of rabbitearsObjective To investigate the effects and possible mechanisms of targeting silencingvascular endothelial growth factor (VEGF) mediated by lentivirus on hypertrophic scarformation. To explore the effects of vascular targeting gene therapy in preventing againstrabbit ears hypertrophic scar.Methods Ten healthy adult Japanese big ear rabbits were used to establish the rabbitears hypertrophic scar models. The left and the right ear of each individual were randomlydivided either experimental group or control. Rabbits in experimental group were injectedwith recombinant lentivirus carrying the targeting sequences LV-SiVEGF once the woundshealed, while Rabbits in control group were injected with loadless lentivirus carrying unrelated LV-NC. The resurfacing skin was resected for sectioning and staining30daysafter the wound healed. The resurfacing skin was resected for sectioning and staining, scarappearance was compared between the two groups, and resected scar tissue was handledwith HE staining, Masson staining, CD34staining, VEGF staining and the ultrastructurewas studied with transmission electron microscopy (TEM) so as to investigate the effect ofslencing target VEGF gene expression on the angiogenesis and fibroblast proliferation inthe rabbit ear scar tissue.Results In contrast with control group, the angiogenesis in the hypertrophic scar ofrabbit ear from experimental group was significantly reduced and the collagen synthesisand fibroblast proliferation were inhibited after30days’ vessels targeted gene therapy.Conclusion Application of LV-SiVEGF gene therapy in the early stage of scarformation could significantly inhibit the formation of hypertrophic scars of rabbit earsPart II: Effect of single or combination therapy of targeted silencing VEGF andrecombinant angiogenesis inhibitors METH-1on hypertrophic scars of rabbit earsObjective To investigate the effects and possible mechanism of single orcombination therapy of targeted silencing VEGF and recombinant angiogenesis inhibitorsMETH-1on rabbit ear hypertrophic scar formation and to explore the effects of vasculartargeting gene therapy on preventing against rabbit ear hypertrophic scar.Methods Twenty healthy adult Japanese big ear rabbits were used to establish therabbit ears hypertrophic scar models. The wounds were randomly divided into four groups:control group, single LV-SiVEGF group, single LV-METH-1group and combinationgroup. Once the wound healed, the control group was injected with loadless lentiviruscarrying unrelated LV-NC; the single LV-SiVEGF group was injected with LV-SiVEGF;the single LV-METH-1group was injected with LV-METH-1; the combination group wasinjected with LV-SiVEGF and LV-METH-1. The resurfacing skin was resected forsectioning and staining30days after the wound healed. We compared the changes of scarappearance between groups and then the resected scar tissue was handled with HE staining,Masson staining, CD34staining, VEGF staining and also the ultrastructure was studied with transmission electron microscopy (TEM) to investigate the effects of slencing targetVEGF gene expression on the angiogenesis and fibroblast proliferation in the rabbit earscar tissues.Results In contrast with control, the angiogenesis in the hypertrophic scar of rabbitear was significantly reduced and the collagen synthesis and fibroblast proliferation wereinhibited in single LV-SiVEGF group, single LV-METH-1group and combination groupafter30days’ vessels targeted gene therapy (count of vascular number: controlgroup>single LV-SiVEGF group>single LV-METH-1group> combination group). Besides,collagen synthesis and fibroblast proliferation are also inhibited in different degree (countof fibroblasts: control group>single LV-SiVEGF group>single LV-METH-1group>combination group).Conclusion Application of LV-SiVEGF and/or LV-METH-1gene therapy in theearly stage of scar formation may significantly inhibit the formation of hypertrophic scarsin rabbit ears. The potency of suppression from strong to weak were: the combinationgroup, single LV-SiVEGF group and single LV-METH-1group.The main purpose of the present study was to observe and verify the effects ofLV-SiVEGF and LV-METH-1in vivo on the development of hypertrophic scars. Theanimal models of this study were rabbit ear hypertrophic scar animal model which is wellcharacterized in our laboratory. We detected the hypertrophic scar index in rabbit ears’histopathology, fibroblast proliferation, collagen synthesis, vascular endothelial cells,ultrastructural changes after the application of LV-SiVEGF and LV-METH-1to investigatethe feasibility for vascular targeting gene therapy to inhibit the hypertrophic scars. |
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