An Experimental Study About The Effect Of PEDF On Collagen Synthesis Through Polarizing Macrophages In Skin Expansion

In plastic surgery,skin and soft tissue expansion is often applied to repair the defects in skin and reconstruct external organs.In repair and reconstruction,the application of skin expansion is safer and more effective compared with other methods,but it also has some disadvantages such as complications,long treatment cycles and great psychological burden of patients.Histologically,continuous tension caused by expansion can induce collagen breakage in the skin and thinning of the dermis,which can cause complications such as expander exposures.Therefore,the key to solve the problem of thinning of the expanded dermis is to regulate the collagen correctly.Collagen synthesis and decomposition in the skin are often regulated by a series of cytokines,which are not yet clear under the condition of expansion.Thereby,an efficient cytokine should be sought to regulate collagen in expansion.Pigment epithelium derived factor exists widely in normal adult tissues(brain,liver,bone,eye,ovary,etc.),and previous studies have shown that PEDF expression can be elevated to participate in the regulation of collagen synthesis in injured skin and other organs.Therefore,we hypothesize that PEDF may express at some level in the skin during expansion and function as a collagen regulator.The first part of this paper used the human skin samples,mouse head expansion model and rat back skin expansion model for skin expansion respectively,and we used immunofluorescence staining,PCR,Western Blot and ELISA methods to compare the differences of the expression of PEDF in the skin and exudation in experimental groups and control groups in order to explore whether expansion could stimulate the PEDF expression in skin and the microenvironment in skin.In the second part,based on the conclusion of the first part,we discussed whether PEDF can inhibit the synthesis of collagen in vitro under hypoxia induced by expansion.Meanwhile,we also explored that PEDF’s function on macrophages polarization into different directions and studied PEDF regulated collagen synthesis indirectly through polarizing macrophages.Part 1 The expression features of PEDF in human expanded skin and in mouse head expansion skin model and rat back skin expansion model Objecive: To characterize the pigment epithelium-derived factor expression in the skin during expansion.Methods: We collected 10 expanded skin and 6 normal skin of human.Besides,16 C57BL/6 mice and 20 SD rats were randomly divided into an experimental group and a control group,respectively,and the control group was a sham expansion group.The mice were implanted with a 1 mL skin expander between the subcutaneous and periosteum at the top of the head.One week after the operation,the saline was injected into the expander capsule twice a week with 0.5 mL a time.The intracapsular pressure was recorded before and after injection maintained within 6Kpa to ensure constant pressure expansion.There was no injection in control group.After 3 weeks of expansion,the mRNA content and protein level of PEDF in the mice skin were measured and we detected the expression level,distribution,and main positive expression cell types of PEDF in the skin after expansion in human samples and mice skin we got by immunofluorescence staining and photographed under a Nikon C2 confocal microscope.Image Pro Plus software was used for analysis;to mimic the expansion environment,the epidermal cell line HaCaT cells were treated in vitro in a hypoxic incubator to identify the major tissue microenvironment factor affecting the transcription level of PEDF gene in epidermal cells.We used a SD rat back expansion model since getting the exudate produced after expansion in the rat models was accessible and more numerous.A 10 mL skin expansion was implanted subcutaneously in the back of the rat and the injection into the expansion sac was started 1 week after the operation.We injected saline 2 times a week until the intracapsular pressure was maintained within 8Kpa each time.The protein content of PEDF in the exudate was measured by using the ELISA kit after the expansion.The data were statistically analyzed using SPSS 13.0 software.Results:After injection,the expression of PEDF in the skin of human and mice were both up-regulated and mainly expressed in the epidermal layer.The mRNA and protein level indicated that the expression of PEDF was statistically different between the two groups at the third week after expansion.In the exudate obtained after the skin expansion of the rats,compared with the control group,the expansion can cause the expression level of PEDF to be up-regulated,and the level of up-regulation increases with time.The in vivo results were confirmed in vitro through the HaCaT epidermal cell line.After hypoxia treatment,the cells at 0、1、3、6、9h were detected,and the expression level of PEDF mRNA was significantly up-regulated and maintained at a high level.Conclusion: Skin expansion can cause up-regulation of PEDF in the skin,and it is mainly distributed in the epidermal layer of the skin.In addition to the expression in the skin,PEDF can be secreted into the tissue microenvironment;in vitro,PEDF can be found under hypoxic conditions maintaining a high level,which indicates that under the simulated hypoxic microenvironment of skin expansion,PEDF expression could be maintained at the high level,and the characteristic expression of PEDF also lays a foundation for us to explore the regulation function of PEDF in the next step.Part 2 The regulation of collagen synthesis by PEDF under expansion Objecive: To investigate the effect of PEDF on the synthesis of collagen in the dermal layer of skin.Methods: The mouse macrophage cell line RAW264.7 and fetal rat fibroblast ICR were cultured in vitro.Under the condition of hypoxia,the cells were intervened by PEDF.We used PCR,flow cytometry and immunocytochemistry staining to ensure the polarization effects of PEDF on macrophages;under co-culture of macrophages and fibroblasts by Transwell assay,the combined effects of PEDF and macrophage on fibroblasts were explored.Furthermore,in order to further demonstrate the role of PEDF,we used the receptor inhibitors of PEDF to let them bind to the PEDF receptors on the surface of macrophages to inhibit the polarization of PEDF on macrophages in the same Transwell condition and it is confirmed the comprehensive effects of PEDF on collagen synthesis of fibroblasts combining macrophages.Results: Under hypoxic conditions,M1-marker(iNOS,TNF-α,MCP-1)was significantly increased in PEDF-treated macrophages,while M2-marker(Arg-1,Ym-1,IL-10)was not significantly increased.The addition of PEDF to fibroblasts revealed no significant changes in the synthesis of type I and type III collagen,whereas in the Transwell experiments,the final amounts of collagen synthesis were reduced and statistically significant.Furthermore,type I and type III collagen synthesis were increased following inhibition of PEDF receptors on macrophage membranes.Conclusion: PEDF can promote the polarization of macrophages to M1 by binding to the receptors on macrophage membranes under conditions of hypoxia,and target macrophages to reduce the synthesis of collagen indirectly.