Mechanism Study On Inducing Human Umbilical Vein Endothelial Cell Transition By High Glucose

PurposeTo observe the biological characteristic changes of human umbilical vein endothelialcell line EA.hy 926 in vitro induced by high glucose, and explore whether the vascularendothelial-mesenchymal transition was involved in the pathogenesis of proliferativediabetic retinopathy.Methods1. Cell culture and experimental groupsHuman umbilical vein endothelial cell line EA.hy926 cells were cultured in highglucose Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetalbovine serum (FBS) in a humidified incubator(37℃, 5% CO₂). After 90% fusion, the cellswere passaged into culture plates, and randomly divided into two groups, control groupand high-glucose group.Control group: high glucose DMEM supplemented with 2% FBS, the concentration ofglucose in culture medium was 25mmol/L.High-glucose group: high glucose DMEM supplemented with 2% FBS and addition-nal glucose, the concentration of glucose in culture medium was 50mmol/L.2. Observe the change of cell morphology with contrast phase microscopeCell morphology was observed with contrast phase microscope every day.3. Evaluate cell proliferation ability with MTTThe cells (5×10³/well) were seeded in 96-well plate containing 100μl of medium, andserum starved overnight. The cells were divided into two groups(control and high-glucos-e), and the optical density were determined after treated 24 hours, 48 hours, 72 hours,and 96 hours.4. Evaluate cell migration ability with transwell and the scratch wound assaymethods(1) Transwell migration assay method: The cells were resuspended in medium supple-mented with 2% FBS, and the treated cells (10⁵/well) were added to the upper chambercontaining 100μl of medium and 600ul medium Containing 10%FBS was added into thebottom chamber. After 8h incubation at 37℃, the migrated cells on the back of themembrane were fixed, stained with crystal violet and extracted with 10% acetic acid. Thecells on the back of the membrane were counted and optical density was measured. (2) Scratch wound assay method: A scratch wound was made on cell monolayer witha pipet tip. After 8 hours, the migrated cells were counted within the scratch wound.5. Immunofluorescence stainingThe cells (1.0×10⁵) were cultured on sterile glass slides in 24-well plates. After90% confluence, The cells were randomly divided into two groups and cultured foradditional 3d, 7d and 15d to measure the expression of endothelial proteins (vascularendothelial cadherin (VE-cadherin) and a-catenin), mesenchymal proteins (neuralcadherin (N-cadherin), vimentin, fibronectin and a-smooth muscle actin (a-SMA)),vascular endothelial growth factor (VEGF) and cell transition controlling factor Twistprotein with immunofluorescence staining in the EA.hy926 cells.6. RT-PCRThe cells (1.0×10⁵) were seeded in 24-well plates. After 90% confluence, the cellswere randomly divided into two groups and cultured for additional 3d, 7d and 15d. Theexpression of VE-cadherin, N-cadherin, vimentin, VEGF and cell transition controllingfactor Twist protein in mRNA levels were determined with RT-PCR in the EA. hy926cells.Results1. Influence on cell morphology of EA. hy926 cells by high glucoseWith the extended incubation time, most of the EA.hy 926 cells became spindle-shaped, the lost 9f cell polarity in high-glucose group.2. Influence on cell proliferation of EA.hy926 cells by high glucoseGlucose can inhibit cell proliferation. Before cell confluence, inhibition rate increasewith the extended incubation time, and difference in two groups had statistical significance indifferent time (P＜0.05).3. Influence on cell migration ability of EA.hy926 cells by high glucoseIn high-glucose group, migrated cells on the lower surface of the filter membrane wasmore than these in the control group, so was opticaldensity and migrated cells in thescratch wound. Difference in two groups had statistical significance (P＜0.05).4. Immunofluorescence stainingVE-cadherin: VE-cadherin normally localizes to endothelial cell-cell junctions. Inhigh-glucose group, the expression of VE-cadherin had little change on day 3, decreasedon day 7 and 15 at endothelial cell-cell junctions, while increased in the cytoplasm onday 15.N-cadherin: N-cadherin is also found at intercellular junctions colocalized withVE-cadherin in addition to diffusive membrane expression. The expression of N-cadherinin high-glucose group was lower than that in control group on day 3, so was contrary on day 7, and was the same in two groups on day 15.a-catenin: a-catenin normally localizes to endothelial cell-cell junctions. In high-glucose group, its expression decreased with the extended incubation time at intercellularjunctions.Vimentin: vimentin is skeleton protein of stromal cell and localizes to cytoplasm. Inhigh-glucose group, its expression increased with the extended incubation time and wasmore than that in control group at each time point.Cell transition controlling factor Twist: The expression of Twist was the same in twogroups on day 3. Its expression of high-glucose group increased and was more than that incontrol group with the extended incubation time.Fibronectin: Its expression of high-glucose group increased with the extendedincubation time and was more than that in control group at each time point.VEGF: Its expression of high-glucose group was lower than that in control group onday 3, so was contrary on day 7, 15.a-SMA: Part of the cells only in high-glucose group expressd a-SMA on day 15.5. RT-PCRVE-cadherin: In high-glucose group, there was down-regulation of VE-cadherin gene onday 3 and so was contrary on day 7 compared to that of control group. Expression ofVE-cadherin gene was the same in two groups on day 15.N-cadherin: The change of its expression was the same to that of VE-cadherin at eachtime point.Vimentin: In high-glucose group, there was up-regulation of vimentin gene with theextended incubation time and was more than that in control group at each time point.Cell transition controlling factor Twist: The expression of Twist gene in mRNAlevels was the same in two groups on day 3, 15. Its expression of high-glucose groupup-regulated with the extended incubation time and was more than that in control groupon day 7.VEGF: Its expression of high-glucose group was lower than that in control group onday 3, so was contrary on day 7, 15.ConclusionHigh glucose can destroy adherent junctions of vascular endothelial cells, promote cellmigration, increase the expression of mesenchymal proteins, VEGF and Twist, induceendothelial-mesenchymal transition and might directly contribute to retinal neovasculariz-ation and fibrous membrane in diabetic retinopathy.