The effect of hyperglycaemia on expression and re‐distribution of VE‐cadherin in human endothelial cells

In vitro studies using animal vascular endothelial cells have shown that hyperglycaemia increases monolayer permeability. The mechanism underlying this increase is thought to involve disruption of adherens junctions by phosphorylation of the transmembrane molecule vascular‐endothelium cadherin (VE‐cadherin) and loss of its perijunctional actin anchorage. Recently an alternate mechanism of disruption, i.e. inflammation induced loss of actin independent VE‐cadherin, has been proposed (Lim et al. Microvas. Res. 2001 on IDEAL). The purpose of this study was to expose human endothelial cells to high glucose and follow the fate of VE‐cadherin. Human vein umbilical endothelial cells (HUVEC) were grown to confluence before being exposed to media containing different concentrations of glucose: 5 mm d‐glucose as euglycaemic control, 25 mm d‐glucose as hyperglycaemic medium, and 25 mm l‐glucose as osmotic control. VE‐cadherin localisation was studied using immunocytochemistry and systemic randomised count of junctional profiles which allowed classification of the VE‐cadherin immunoreactivity at cell‐cell borders as continuous or discontinuous. Immunoblot studies and selective protein extraction into Triton‐soluble and Triton‐insoluble fractions were performed in order to measure total protein levels of expression and actin association of VE‐cadherin under the different experimental conditions. Immunoprecipitation studies were used to investigate tyrosine phosphorylation status of VE‐cadherin. Hyperglycaemic exposure of HUVEC for 2 h resulted in a statistically significant (one‐way Anova, P < 0.01) increase in the percentage of discontinuous VE‐cadherin at cell‐cell contacts of HUVEC monolayers, this significance was lost after longer durations (4 d). After 2 h of glucose insult, the total VE‐cadherin expression was not altered, whilst the selective extraction studies showed VE‐cadherin had a greater association with the actin‐cytoskeleton. The tyrosine phosphorylation level of VE‐cadherin was not altered. Our studies reveal that hyperglycaemia does cause redistribution of VE‐cadherin. However the pattern of VE‐cadherin redistribution favours the hypothesis of a loss of VE‐cadherin from actin‐independent membrane microdomains. Furthermore we find this loss is not mediated by the tyrosine kinase signalling pathway. Funded by the European Social Fund.