An In Vitro Study About The Effect Of Sildenafil On The Proliferation Of Human Lymphatic Malformations Endothelial Cells

BackgroundLymphatic malformations(LM)is a congenital malformation of the lymphatic system,often leads to the appearance of deformity and dysfunction,and even life-threatening.Although LM is benign lesions,it often invades the surrounding tissue or organs,with the border being unclear,so the treatment is difficult.In 2012,the New England Journal reported that oral administration of sildenafil was effective in the treatment of a patient with lymphatic malformations,and then sildenafil became a hotspot in the treatment of lymphatic malformations but the relevant mechanism remains unknown.In the pathological structure,lymphatic malformed endothelial cells(LMECs)are the main components of LM lesions.Studies have shown that abnormal proliferation of LMECs presented in lymphatic malformations,often accompanied by the increased expression of vascular endothelial growth factor C(VEGF-C)and vascular endothelial growth factor receptor 3(VEGFR-3),which played an important role in the development of LM.ObjectiveTo observe the effect of sildenafil on the proliferation of human lymphatic malformed endothelial cells(HLMECs)in vitro,to detect the changes of VEGF-C and VEGFR-3 expression.and to explore the potential and possible potential of sildenafil in the treatment of lymphatic malformations at the cellular level and to provide experimental evidence for the clinical treatment of LM.MethodsThe issues were selected from Oral and Maxillofacial Surgery Tongue lymphatic malformation resections in Shandong University Qilu Hospital and get pathologically confirmed.The endothelial cells of human lymphatic malformations were cultured in vitro;Immunocytochemical staining of CD-31 and lymphatic endothelium hyaluronan receptor-1(LYVE-1)was performed in cell identification;The morphological changes of HLMECs were observed by inverted microscope with different concentrations of sildenafil(1,2,5,10 ?mol/L)on HLMECs.The effect of cell proliferation was observed by MTT colorimetry and EdU.Western blotting was used to detect the expression of vascular endothelial growth factor C(VEGF-C)and its receptor VEGFR-3.Independent-samples T test and ANOVA(LSD method)were performed by SPSS 21.0 software to make statistic analysis.ResultsTissue block began to crawl cells after 72 h,cells were polygonal,monolayer adherent growth and look like cobblestones,the growth rate was slow.After passage of cells,the cells become long spindle shape,irregular shape,and accelerated proliferation rate.Immunocytochemical staining showed that LYVE-1 and CD31 were positive in the lymphatic endothelium hyaluronic acid receptor,and it was confirmed that the obtained cells were the lymphatic malformed endothelial cells.After the action of sildenafil,the cell density gradually decreased and the cell proliferation rate decreased with the increase of drug concentration,and the observed density decreased at 2 ?mol/L cells.The cytoplasm appeared granular,cell membrane became incomplete,and cells were of poor cell adhesion,cell expansion and irregular shape when at 10 ?mol/L.EdU staining showed that the cell proliferation rate decreased gradually with the increase of sildenafil concentration,and the cell proliferation rate decreased(P<0.05)at the concentration of 2 pmol/L group.The MTT results were consistent with one above and showed the inhibition rate of cell proliferation was increased over time with the same concentration of drug treatment(P<0.05),indicating that sildenafil inhibits the proliferation of lymphatic malformed endothelial cells in a concentration-dependent and time-dependent manner.Western blot showed that VEGF-C was down-regulated in the 5 ?mol/L dose group(P<0.05),while VEGFR-3 was down-regulated at 1 ?mol/L(P<0.05).ConclusionSildenafil inhibites the proliferation of HLMECs in vitro and reduces the expression of VEGF-C and VEGFR-3 in HLMECs,which may be one of the mechanisms of its action on LM.