A role for cadherin-5 in regulation of vascular endothelial growth factor receptor 2 activity in endothelial cells

FLK-1/vascular endothelial growth factor receptor 2 (VEGFR-2) is one of the receptors for VEGF. In this study we examined the effect of cell density o n activation of VEGFR-2. VEGF induces only very slight tyrosine phosphoryla tion of VEGFR-2 in confluent (95-100% confluent) pig aortic endothelial (PA E) cells. In contrast, robust VEGF-dependent tyrosine phosphorylation of VE GFR-2 was observed in cells plated in sparse culture conditions (60-65% con fluent). A similar cell density-dependent phenomenon was observed in differ ent endothelial cells but not in NIH-3T3 fibroblast cells expressing VEGFR- 2. Stimulating cells with high concentrations of VEGF or replacing the extr acellular domain of VEGFR-2 with that of the colony-stimulating factor 1 re ceptor did not alleviate the sensitivity of VEGFR-2: to cell density, indic ating that the confluent cells were probably not secreting an antagonist to VEGF. Furthermore, in PAE cells, ectopically introduced platelet-derived g rowth factor alpha receptor could be activated at both high and low cell de nsity conditions, indicating that the density effect was not universal for all receptor tyrosine kinases expressed in endothelial cells. In addition t o lowering the density of cells, removing divalent cations from the medium of confluent cells potentiated VEGFR-2 phosphorylation in response to VEGF. These findings suggested that cell-cell contact may be playing a role in r egulating the activation of VEGFR-2. To this end, pretreatment of confluent PAE cells with a neutralizing anti-cadherin-5 antibody potentiated the res ponse of VEGFR-2 to VEGF. Our data demonstrate that endothelial cell densit y plays a critical role in regulating VEGFR-2 activity, and that the underl ying mechanism appears to involve cadherin-5.