| Background:Infantile hemangioma(IH)is the most common benign vascular tumor in infancy.It is characterized by an initial rapid proliferation of hemangioma-derived endothelial cells(Hem ECs)and formed hemangioma masses during infancy(proliferation phase),followed by gradual spontaneous regression over 1-5 years(involution phase),with continual improvement until 6-12 years of age(involuted phase).However,almost 10% to 15% of IH can cause serious complications and sequelae,such as ulceration,obstruction,disfigurement and life-threatening,which need to be treated in time and effectively.Because of these complications and sequelae will make a serious impact on the growth and health of physical and mental for children.The proliferation phase of IH(from birth to 1 year)is characterized by the active proliferation of Hem ECs and the formation of capillary-like structures.Later,vascular architecture are organised with endothelial cells and pericytes in the involution stage.After involuted,vascular tissue are replaced by fibro-fatty tissue.Therefore,Hem ECs play an important role in the occurrence and development of IH.The pathogenesis of IH is not completely clear.At present,relevant studies mainly support a possible molecular mechanism that is the activation of VEGFA/VEGFR-2signal pathway,which could promote the proliferation,migration and survival of Hem ECs.It also can promote angiogenesis and tumor formation in IH.Recent studies have found that CLEC14A(a single channel transmembrane glycoprotein)is highly expressed in the development vasculature of tumor.It can regulate endothelial cell proliferation,filopodia formation,cell adhesion,cell migration,lumen formation,angiogenesis and lymphangiogenesis through the VEGFA/VEGFR signal pathway.Then,we have analysis the GEO database of IH and found that CLEC14 A is highly expressed in IH tissues.Therefore,our study is try to explore and understand the interact molecular mechanism between CLEC14 A and Hem ECs and provide a new target for the therapy of IH.Objectives:1.To study the effects of CLEC14 A on the proliferation,migration,invasion and angiogenesis of Hem ECs.2.To study the effects of CLEC14 A on the VEGFA/VEGFR-2 signaling pathway in Hem ECs.3.To demonstrate the functions of VEGFA/VEGFR-2 signaling pathway and CLEC14 A in the angiogenesis of Hem ECs in IH.Methods:Part I: Primary cells are obtained from adherent culture of IH tissue,then endothelial cells are isolated and purified by magnetic-activated cell sorting(MACS)after CD31 dynabeads adsorbed endothelial cells.Cell purity is detected by flow cytometry and then the surface specific markers of proliferating Hem ECs are detected by cellular immunofluorescence to identify the Hem ECs phonetype.Finally,the ability of angiogenesis of our obtained Hem ECs is verified by endothelial cell tube formation assay.Part II: Firstly,the relative expression level of CLEC14 A in IH tissue from the GEO database is analyzed and verified by q PCR experiment.The expression localization of CLEC14 A in Hem ECs is determined by cellular immunofluorescence assay and the protein expression level is verified by western blotting experiment.The silencing and overexpression sequences of CLEC14 A are designed and add to plasmids.Then the plasmids are packed into lentivirus.The cells line with stable expression(silencing and overexpression)of CLEC14 A are constructed through the infection experiment of Hem ECs with recombinant lentivirus.The infection effects of recombinant lentivirus is verified by q PCR experiment.Part III: Ed U experiment was used to detect the effects of the proliferation in Hem ECs after silencing CLEC14 A.Wound healing experiment was used to detect the effects of the migration in Hem ECs after silencing CLEC14 A.Transwell experiment was used to detect the effects of the invasion in Hem ECs after silencing CLEC14 A.Tube formation assay was used to detect the ability of tubule formation in Hem ECs after silencing CLEC14 A.Then the relationship between silencing CLEC14 A and VEGFA/VEGFR-2 was detected by q PCR experiment.Finally,the effects of the protein expression level of PCNA,BAX and the related proteins(VEGFR-2,p-VEGFR-2,ERK,p-ERK)of VEGFA/VEGFR-2 signal pathway are detected by western blotting experiment after silencing CLEC14 A.Part IV: Ed U experiment was used to detect the effects of the proliferation in Hem ECs after overexpressing CLEC14 A.Wound healing experiment was used to detect the effects of the migration in Hem ECs after overexpressing CLEC14 A.Transwell experiment was used to detect the effects of the invasion in Hem ECs after overexpressing CLEC14 A.Tube formation assay was used to detect the ability of tubule formation in Hem ECs after overexpressing CLEC14 A.Then the relationship between overexpression of CLEC14 A and VEGFA/VEGFR-2 is detected by q PCR experiment.Finally,the effect of the protein expression level of PCNA,BAX and the related proteins(VEGFR-2,p-VEGFR-2,ERK,p-ERK)of VEGFA/VEGFR-2 signal pathway are detected by western blotting experiment after overexpressing CLEC14 A.Part V: The effects of the hemangioma formation of Hem ECs is observed by subcutaneous tumor formation model in nude mice after silencing and overexpressing CLEC14 A.The effects of the angiogenesis of Hem ECs is verified by Matrigel plug assay in nude mice after silencing and overexpressing CLEC14 A.Results:Part I: We have successfully cultured primary cells through adherent culture of IH tissue.And we have isolated and purified endothelial cells through magneticactivated cell sorting(MACS)after CD31 dynabeads adsorbed endothelial cells.The purity of Hem ECs was 94.04% through flow cytometry.Then,the results showed that CD31,GLUT1,VEGFA and VEGFR-2 are strongly positive expression on the surface of proliferating Hem ECs through cellular immunofluorescence experiment.Finally,it was found that our obtained Hem ECs have strong ability of angiogenesis through endothelial cell tube formation assay.Part II: We have found that CLEC14 A was highly expressed in IH tissue through the analysis of GEO database,which was verified by q PCR experiment.And we have found that CLEC14 A was localized on the cell-membrane of Hem ECs through cellular immunofluorescence assay.The CLEC14 A protein was highly expressed in Hem ECs through western blotting experiment,comparing to HUVECs.We have successfully constructed the cells line with stable expression(silencing and overexpression)of CLEC14 A after the infection experiment of Hem ECs with recombinant lentivirus.We have successfully transfected the silencing and overexpression sequences of CLEC14 A into Hem ECs and verified by q PCR experiment(P < 0.001).Part III: We have found that silencing CLEC14 A can inhibit the proliferation,migration,invasion and decrease the tubule formation of Hem ECs.The q PCR results showed that the relative expression of VEGFA and VEGFR-2 were downregulated after silencing CLEC14 A.Our western blotting results showed that the protein expression level of PCNA was decreased,the protein expression level of Bax was increased,and the related proteins(VEGFR-2,p-VEGFR-2,ERK,p-ERK)expression level of VEGFA/VEGFR-2 signal pathway were downregulated in Hem ECs after silencing CLEC14 A.We have found that silencing CLEC14 A can inhibit the secretion of VEGFA in Hem ECs and inhibit VEGFA/VEGFR-2 signaling pathway,and finally inhibit the angiogenesis of Hem ECs.Part IV: We have found that overexpressing CLEC14 A can inhibit the proliferation,migration,invasion and increase the tubule formation of Hem ECs.The q PCR results showed that the relative expression of VEGFA and VEGFR-2 were upregulated after overexpressing CLEC14 A.Our western blotting results showed that the protein expression level of PCNA was increased,the protein expression level of Bax was decreased,and the related proteins(VEGFR-2,p-VEGFR-2,ERK,p-ERK)expression level of VEGFA/VEGFR-2 signal pathway were upregulated in Hem ECs after overexpressing CLEC14 A.We have found that overexpressing CLEC14 A can promote the secretion of VEGFA in Hem ECs and activate VEGFA/VEGFR-2signaling pathway,and finally enhance the angiogenesis of Hem ECs.Part V: We have found that the VEGFA/VEGFR-2 signaling pathway of Hem ECs was inhibited after silencing CLEC14 A,resulting that the decreased of cell proliferation,the reduced viability,the decreased capacity of angiogenesis,in the subcutaneous tumor formation model and Matrigel plug model.On the contrary,we have found that Overexpressing CLEC14 A can activate VEGFA/VEGFR-2 signaling pathway of Hem ECs and enhance cell proliferation,survival and angiogenesis.We have found that Overexpressing CLEC14 A can promote the angiogenesis of Hem ECs by promoting the secretion of VEGFA and activating autocrine VEGFA/VEGFR-2loop.And CLEC14 A is an important molecule for differentiation,proliferation and angiogenesis of Hem ECs in vivo.Conclusion:Collectively,our study demonstrates that CLEC14 A could regulate the biological functions of Hem ECs in vivo and in vitro for the first time and CLEC14 A is an important molecule for differentiation,proliferation and angiogenesis of Hem ECs in vivo.And we have found that CLEC14 A regulates the proliferation and angiogenesis of Hem ECs through VEGFA/VEGFR-2 signaling pathway and provided a new therapeutic target for the anti-angiogenesis therapy of IH in the future. |
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