Effects And Mechanistic Study Of Direct Current Electric Fields On Lymphatic Endothelial Cells And Lymphangiogenesis

Background:Lymphangiogenesis is process by which new lymphatic vessels grow from preexisting ones.It occurs during a number of physiological and pathological processes.During embryo development lymphatic progenitor cells form new lymphatic vessels through cell differentiation,proliferation and migration.Lymphangiogenesis are rare in the adult,but do occur during physiological and pathological processes,such as wound healing,lymphedema,inflammation and tumor.Stimulating or inhibiting lymphangiogenesis has very important clinical significance.Increasing lymphangiogenesis to improve lymphedema caused by local lymphatic reflux obstruction relating to lymphatic vasculature abnomality can lead to restoration of the lymph-transporting capacity and therefore have therapeutic effect.Inhibiting tumor lymphangiogenesis and thus blocking tumor growth and metastasis have clinical benefit of tumor therapy.Up to now,there is no effective ways of promoting lymphangiogenesis in clinical treatment of lympatic vasulature abnormality.In vitro and in vivo studies have showed that DC electric fields may play important roles in various processes such as wound healing,tissue regeneration,angiogenesis and tumor invasion.Our recent studies have demonstrated that the EF can induce angiogenic responses of vascular endothelial cells such as directional migration,alignment,elongation and tube-like structure formation.Since lymphatic endothelial cells have similar biological features of vascular endothelial cells,we hypothesized that the electric fields could also have critical effects on lymphangiogenic activity of lymphatic endothelial cells.Therefore,in this study physiological DC electric field is applied to cultured lymphatic endothelial cells to investigate the EF-induced lymphangiogenic responses of lymphatic endothelial cells,to quantify electric field induced cell migration and morphological changes,to determine the effects of electric field on cell proliferation and cell cycle progression and evalute tube-like structure formation of the cells.In addition,we also investigate the distribution and expression of membrane receptors and intracellur signaling molecules relating to lymphangiogenesis,and study profile changes of differentially expressed genes(DEGs)of the cells linking to lymphangiogenic activity by RNA-seq,therefore understanding the underlying mechanisms of electric field induced effects.Besides,we also explore EF-induced lymphangiogenic effect using rat thoracic duct ring model.Up to now,there are no other reports concerning the effects and mechanistic study of DC EF induced lymphangiogenic responses.This study has not only the theoretical significance in exploring EF stimulating lymphangiogenesis and underlying machanisms of it,but also therapeutic potential to apply EFs treating complications relating to lymphangiogenic abnormality.Method:Firstly,in order to investigate the effect of electric field on the behavior and morphology of lymphatic endothelial cells,the migratory behavior and morphological changes of lymphatic endothelial cells cultured in electric field were detected using primary culture of lymphatic endothelial cells and lymphatic endothelial cell line.1.The culture of lymphatic endothelial cells isolated from rat thoracic duct and human lymphatic endothelial cell line(HLEC).LYVE-1 and VEGFR-3,two specific markers of lymphatic endothelial cells,were stained by immunofluorescence.2.The primary lymphatic endothelial cells and HLECs were stimulated by EF at field strengths of 100mV/mm,200mV/mm and 300mV/mm,respectively.The stimulation durations were 4h,8h,12h and 24h.Cell images were analyzed by microscopy,and cell migration direction,migration speed and morphology were measured.3.Lymphatic endothelial tube formation model on metrigel was established.The cells cultured on the matrix were stimulated by EF at the field strength of 150mV/mm for 0～8h,and the formation of tube length,the number of branching points and the number of tubes were analyzed by microscopy.At the same time,in order to explore the underlying mechanism of electric fieldinduced lymphangiogenic responses in lymphatic endothelial cells,we used whole transcriptome analysis(RNA-seq)to analyze the profiles of differential expression of genes related to lymphangiogenic activity in the endothelial cells,and determined proteins and active molecules involved in electric field-induced lymphangiogenesis.4.Human lymphatic endothelial cells(HLECs)were stimulated with 200mV/mm DC EF for 24 h.The differentially expressed genes(DEGs)in HLECs were screened by RNA-seq method,and the DEGs were subjected to GO term enrichment analysis and KEGG pathway analysis.5.Some of the DEGs were randomly selected and validated by RT-qPCR.6.Cells were stimulated by 200mV/mm EF for 5 min,l0min,15 min,30 min and 1 h,and expression and activity of VEGFR-2 and VEGFR-3 were determined using confocal immunofluorescence analysis,7.Cells were treated with VEGFR-2 tyrosine kinase inhibitor(SU1498,1 μM)and VEGFR-3 tyrosine kinase inhibitor(SAR131675,1 μM),respectively.The cells were stimulated by 200mV/mm EF for 4h,and cell migration was quantified.8.PI3K/AKT and MAPK signaling pathways associated with lymphangiogenesis were selected,and the phosphorylation levels of AKT,p38,ERK1/2(p44/p42)and eNOS signaling molecules in lymphatic endothelial cells before and after EF exposure(200mV/mm)at 5,10,15,30min,and 1h time-points were detected by Western Blot analysis.9.Lymphatic endothelial cells were treated with AKT inhibitor(MK-2206 2HC1,10μM),ERK1/2 inhibitor(U0126,20μM),p38 inhibitor(SB203580,50μM)and eNOS inhibitor(L-Name HCl,100μM),respectively.The cells were stimulated by a 200mV/mm EF for 4h.The EF-induced cell migration in the present of the respective inhibitor was determined.10.Protein expression levels of eNOS in endothelial cells with and without EF exposure(200mV/mm)at 2,4,6,8,12 and 24h time-points were detected by Western Blot analysis.11.NO production in cell culture supernatant obtained from EF(200 mV/mm)stimulation at different time points from 5 min to 24h and with different EF strengths at 0,50,100,150,200 and 250 mV/mm were detected by chemiluminescence assay.12.The F-actin component of the cytoskeleton in lymphatic endothelial cells before and after EF exposure was stained with phalloidin.13.Effect of EF stimulation on cell cycle progression and proliferation of lymphatic endothelial cell was detected by Propidium iodide(PI)assay.We also investigated the effect of electric field on lymphangiogenesis using a model of rat thoracic duct ring culture.14.Immunofluorescence staining for lymphatic endothelial-specific markers in tissue sections of rat thoracic duct.15.Establishment of lymphangiogenic explant cultures using rat thoracic ducts.16.Stimulating the thoracic duct ring explant with 150mV/mm EF,and evaluating tubular structure formation and growth direction from the explant culture.Result:1.Specific lymphatic markers LYVE-1 and VEGFR-3 were expressed in primary culture of rat lymphatic endothelial cells and HLECs.2.The EFs induced directed migration of lymphatic endothelial cells.The rat lymphatic endothelial cells and HLECs apparently moved toward the anode in EF culture.The absence of EF exposure resulted in random migration direction with slow movement.At the field strengths of of 100mV/mm,200mV/mm and 300mV/mm,the two types of cells showed obvious migration toward the anode(p<0.001)with time and voltage dependency.3.The electric field can enhance migration speed of lymphatic endothelial cells.The migration velocity of rat primary lymphatic endothelial cells and HLECs stimulated by 200mV/mm and 300mV/mm were significantly faster than that of unstimulated control cells(p<0.05～p<0.001).The migration velocity of EF(200mV/mm)stimulated cells at 4h,8h,12h and 24h was significantly faster than that of the control cells(p<0.05 or p<0.01).4.The electric field can stimulate orientation and elongation of the two types of lymphatic endothelial cells.The cell asymmetry index of rat primary lymphatic endothelial cells and HLECs showed gradual increase in the culture of electric fields of 100mV/mm,200mV/mm and 300mV/mm,and the long axis of cells aligned gradually perpendicular to the EF vector.The increase of long/short axis of the cells in EF culture showed time and voltage dependency.5.The electric field stimulation can promote tube-like structure formation of two types of lymphatic endothelial cells.Compared with corresponding no EF stimulated cells,the lengths of tube-like structure in EF(150mV/mm)stimulated cells were significantly increased.The characteristics of EF enhanced tube-like structure formation in the two types of lymphatic endothelial cells were different.Duration of tube-like structure formation in primary rat lymphatic endothelial cells in EF was shorter than that of HLECs.6.Compared with control cells,the cells treat with 200 mV/mm EF for 24 h showed differential expression profiles and pathways of genes related to cell migration,cell proliferation,cell cycle,angiogenesis,cell adhesion and actin cy to skeleton organization.These differentially expressed genes were screened by RNA-seq as well as pathways such as PI3K/AKT,MAPK,and regulation of actin cytoskeleton.7.The up-regulated genes CORO1C,RTN4,PDCD10,CNBP,TMSB4X,RPS6,RAP1A,NACA,HSPA8,HSPA5,HMGB1,CNN3 and ANXA2,as well as downregulated genes BAB AM1,PHB2,CARM1,DGK2,FSCN1,HSF1,FLNA and DBN1 were validated by RT-qPCR,and the results were in accordance with the RNA-seq results.8.Activation of VEGFR-2 and VEGFR-3 involved in EF-induced cellular response.The level of VEGFR-2 phosphorylation was significantly increased at 10min and gradually increased from 15 to 60min after stimulation of the cells with 200 mV/mm EF(p<0.001),The phosphorylation level of VEGFR-3 was significantly increased at 10min(p<0.001),decreased at 15min and further decreased after 30min and later on stimulation of the cells.Inhibiting phosphorylation activity of VEGFR-2 and VEGFR3 could significantly inhibit migration of the cells.9.Activition of PI3K-AKT and MAPK involved in EF-induced cellular response.The level of AKT phosphorylation was significant increased at 5 min(p<0.05),and further increased to a higher level at 15min(p<0.001),and remained significantly increased level at 1h time point under EF(200 mV/mm)stimulation.The phosphorylation level of MAPK/ERK1/2,MAPK/p38 and eNOS showed rapid increase at 5 min time point in the EF stimulated cells(p all<0.05).Inhibiting phosphorylation activities of the AKT,ERK1/2,p38 and eNOS could significantly inhibit migration of the cells.10.EF stimulation of eNOS protein expression in the cells.Compared with No EF treated cells,EF(200 mV/mm)treated cells showed increased eNOS protein expression at 2,4,6,8,12 and 24h time points(p<0.05,p<0.01,p<0.001).11.EF stimulation of NO production in the cells.Compared with the corresponding controls,NO production in EF-stimulated cell culture supernatants at 5,15,30,60min and 2,4,6,8,12,and 24 h time points showed gradually increased fold changes from 13 to 811(all p<0.001).In addition,compared with No EF-treated control culture supernatant,NO production in culture supernatant at field strengths of 50,100,150,200 and 250 mV/mm(4h)also showed significantly increased(all p<0.001).12.EF(200mV/mm)stimulation of cells for 24 hours showed increased synthesis of cell stress fibers with even distribution,rearranged actin filaments,and elongated cells with perpendicular aligned microfilaments.13.EF stimulating cell proliferation activity.Compared with the No EF treated cells,EF(200mV/mm)stimulated cells showed increased values of cell proliferation index(PI)and increased S cell phase numbers based on cell cycle analysis.14.Lymphatic specific markers LYVE-1 and VEGFR-3 showed uniformly expressed in thoracic duct explant tissue.15.Culturing thoracic duct explant for two days,lymphotic endothelial cells showed migration from the explant tissue,and formation of tube-like structure.16.Stimulating the thoracic duct explant with 150mV/mm for 24h induced the growth of tube-like structure towards the anode.Compared with no EF stimulated culture tissue,EF enhanced the length of the tubular structures growed from the explant tissue.Conclusion:1.Physiological small DC EF can stimulate cell migration,morphological changes and alignment,as well as tube-like structure formation in cultured lymphatic endothelial cells.These cellular phenotypes are related to lymphangiogenesis of the cells.2.The differential expressed genes of HLECs stimulated by EF were enriched in celluar functions including cell migration,cell proliferation,cell cycle and angiogenesis,cell adhesion and actin cy to skeleton organization,and pathways including PI3K-AKT,MAPK and regulation of actin cytoskeleton.EF stimulation induced activation of VEGFR-2 and VEGFR-3 receptors,as well as intracellular AKT,ERK1/2,p38 and eNOS signaling molecules.EF enhanced expression of eNOS protein and stimulated NO production in cell culture supernatant.EF induced rearrangement of actin cytoskeleton.EF stimulated cell proliferation and induced increase of S phase cell numbers in cell cycle.3.Small physiological DC EF tended to induce tube-like structure aligned perpendicular to electric field and stimulate tubular growth in the explant model.