The Effect Of Fosinopril On High Glucose-induced Apoptosis And Expression Of ICAM-1and VCAM-1in Human Umbilical Vein Endothelial Cells

Objective: In this study, high-glucose induced cultured human umbilicalvein endothelial cells (HUVECs) to build damage model. To investigatewhether an angiotension-converting enzyme inhibitor (ACEI), Fosinopril hasprotective effect on high-glucose induced HUVECs injure, we assessed theeffect of Fosinopril on cell apoptosis and the expression level of intercellularadhesion molecule-1(ICAM-1) and vascular cell adhesion molecule-1(VCAM-1) on HUVECs surface induced by high-glucose.Methods: HUVECs were isolated by0.1%type I collagenase digestionfor15-20min at37℃, and cultured in low serum endothelial cell medium.After passage cultivation, only cells at passage1to2were used in the study toavoid age-dependent injury in human umbilical vein endothelial cells.1Building damage modelHUVECs were seeded at equal density in6-well plates and each onereceiving following fresh media every24h, respectively:①continuousnormal glucose medium(5mmol/L D-glucose),②continuous high-glucosemedium(20mmol/L D-glucose),③Intermittent high-glucose medium（alterna-ting5mmol/L and20mmol/L D-glucose medium every24h）.We took48hours to complete experiment.2Medicine interventionHUVECs seeded in6-well plates received following fresh media every24.④⑤⑥groups added0.1umol/L、1umol/L、10umol/L Fosinopril tocontinuous high-glucose medium. We took48hours to complete experiment.3Analysis of HUVECs apoptosis rate.HUVECs were exposed to the experimental conditions for48hours,0.25%trypsase digestion and collected cells at the end of the experiment and transferred into inflow-pipe, washed cells by forecooling4℃PBS for onetime, and added300μL1×Binding every pipe to suspend cells, then added5μLAnnexin V-FITC, incubated at room temperture and dark15minites, lastadded5μL PI and200μL1×Binding Buffer. Flow cytometry detectedHUVECs apoptosis rate in each tube, each counting30000cells.4Learning about the HUVECs Apoptosis morphologyHUVECs were cultured in different experimental condition, fixed cells10-20minites by0.5ml4%Paraformaldehyde, washed cells by PBS two timesabout three minutes, then added100ul Hoechst33258to each well, incubatedat room temperture and dark10minites, at last PBS washed cell two timesagain about three minutes. Fluorescence staining examined morphologicalchanges of HUVECs apoptosis.5ICAM-1and VCAM-1expression on the HUVECs surface.HUVECs were stimulated in different concentration，0.25%trypsasedigestion and collected cells and cell suspension transferred into inflow-pipe,washed cells one time by PBS, remained100ul supernatant to get cellsuspension, and added20ul FITC labeled ICAM-1antibody and20ul PElabeled VCAM-1antibody to each pipe, at last PBS washed cells three times.The expression of ICAM-1and VCAM-1were detected by using flowcytometry, and each counting30000cells, the results expressed in meanfluorescence intensity (MFI).Results:1Flow cytometry to detect HUVECs apoptosis rate was significantdifference between the①②③groups (F=10.697, p=0.004). Compared with①c ontinuous normal glucose,②continuous high-glucose increased apoptosisrate (2.22±1.38VS5.23±0.98，p=0.007),③I ntermittent high-glucose alsoincreased significantly（2.22±1.38VS5.96±1.25，p=0.002）.2Flow cytometry to detect HUVECs apoptosis rate was significantdifference between the②④⑤⑥groups (F=3,656, p=0.044). Compared with②c ontinuoushigh-glucose,⑤high-glucose+1umol/L Fosinopril (5.23±0.98VS3.22±1.12, p=0.033) and⑥h igh-glucose+10umol/L Fosinopril apoptosis rate decreased significantly (5.23±0.98VS2.71±1.50, p=0.011). However,there was no significant difference of apoptosis rate between different dosagesof Fosinopril groups (p>0.05).3Fluorescence staining examined morphological changes of HUVECsapoptosis and took photos. Compared with①c ontinuous normal glucose,②continuous high-glucose and③I ntermittent high-glucose enhanced fluoresce-nce intensity of cell nucleus, and was visible to the nuclear lobes and fragment.(Fig.6)4Using flow cytometry analysis the expression of ICAM-1was signifi-cant difference between the①②③groups (ⅹ2=6.86, p=0.032). Comparedwith①continuous normal glucose,②continuous high-glucose(p=0.048) and③intermittent high-glucose （p=0.013）induced the expression of ICAM-1significantly.(Fig.7)5Using flow cytometry analysis the expression of ICAM-1was signific-ant inhibited only after the⑥high-glucose+10umol/L Fosinopril Intervention（49.44±21.87VS111.93±56.07, p=0.04）. However, there was no significantdifference of ICAM-1expression between different dosages of Fosinoprilgroups (p>0.05).6Using flow cytometry analysis the expression of VCAM-1was nosignificant difference between the①②③groups (F=0.522, p=0.61), and alsono difference after the Fosinopril Intervention（F=1.69, p=0.209）.Conclution:1Continuous normal glucose induced HUVECs apoptosis, and intermit-tent high-glucose induced cell apoptosis significantly.2Fosinopril inhibited high-glucose induced HUVECs apoptosis.3Continuous normal glucose and intermittent high-glucose induced theexpression of ICAM-1significantly, but did not increase the expression ofVCAM-1due to the short observation time.4Fosinopril intervention could significantly inhibit the expression ofICAM-1on the HUVECs surface induced by high-glucose.