The role of vitronectin in human umbilical vein endothelial cells conditioned by high glucose

Abstract

Background Vitronectin is a glycoprotein that has a variety of functions.Its expression was markedly higher in the retina of oxygen induced mice,which was confirmed in our animal model,and also increased in human umbilical vein endothelial cells (人 UVECs) that were cultured in high glucose.However,there was no evidence that showed vitronectin was involved in retinal neovascularization.Objective This study was to observe the influence of vitronectin on cytoskeleton remodeling,cell migration and blood vessel formation in 人 UVECs conditioned by high glucose.Methods 人 UVECs were cultured in high glucose and the expression of vitronectin was knocked down using RNA interference technology.The experiments were divided into the high glucose group (人 UVECs were conditioned with DMEM medium that contained 50 mmol/L glucose),negative interference group (人 UVECs were transfected with control siRNA in advance,and then were conditioned with DMEM medium that contained 50 mmol/L glucose) and positive interference group (HUVEC were transfected with vitronectin siRNA in advance,and then were conditioned with DMEM medium that contained 50 mmol/L glucose).The protein expression of vitronectin was measured by Western blot,and the microfilament cytoskeleton of 人 UVECs was examined by immunofluorescence cytochemical staining followed by fluorescence microscopy.Cell migration ability in a scratch wound assay and blood vessel formation ability in a matrigel assay of 人 UVECs were evaluated.The general differences were analysed by One-Way ANOVA ;further contrasts of the two groups were analysed by the LSD-t test.Results The differences in vitronectin expression of the three groups were not obvious at 0 hour (F=1.064,P＞0.05).After 24 hours,vitronectin expression was highest in the high glucose group,lower in the negative interference group,and the lowest in the positive interference group,and the differences were significant (F =15.519,P＜0.05).After 48 hours,vitronectin expression of the three groups displayed the same pattern,and the differences were also significant (F=37.521,P＜0.05).Immunofluorescence showed that the cytoskeleton structure was most obvious in the high glucose group,moderate in the negative interference group,and was the least obvious in the positive interference group,after both 24 hours and 48 hours.In the scratch wound assay,the cell migration ability of the high glucose group was the highest,lower in the negative interference group,and the lowest in the positive interference group after 24 hours,and the differences were significant (F=90.685,P＜0.05).After 48 hours,the cell migration abilities of the three groups displayed the same pattern,and the differences were also significant (F=67.880,P＜0.05).In the matrigel assay,after 6 hours,the number of blood vessels formed in the high glucose group was more than that in the negative interference group,and the least amount was found in the positive interference group.The differences among of them were significant (F =86.653,P＜0.05).The number of blood vessel formed in the positive interference group was also the lowest after 12 hours,and the differences were also significant (F=18.992,P＜0.05).Conclusions Vitronectin can bring about cytoskeleton remodeling,increase in cell migration,and enhancement of blood vessel formation in 人 UVECs conditioned in high glucose.It may be one of the important influence factors of diabetic retinopathy. Key words: Vitronectin; High glucose; Cytoskeleton; Cell migration; Human umbilical vein endothelial cells