VEGF receptor 2/-3 heterodimers detected in situ by proximity ligation on angiogenic sprouts

The vascular endothelial growth factors VEGFA and VEGFC are crucial regulators of vascular development. They exert their effects by dimerization and activation of the cognate receptors VEGFR2/KDR/Flk-1 and VEGFR3/Flt-4. Here, we have used in situ proximity ligation to detect receptor complexes in intact endothelial cells. We show that both VEGFA and VEGFC potently induce formation of VEGFR2/KDR/Flk-1/-3 heterodimers. Receptor heterodimers were found in both developing blood vessels and immature lymphatic structures in embryoid bodies. We present evidence that heterodimers frequently localize to tip cell filopodia. Interestingly, in the presence of VEGFC, heterodimers were enriched in the leading tip cells as compared with trailing stalk cells of growing sprouts. Neutralization of VEGFR3/Flt-4 to prevent heterodimer formation in response to VEGFA decreased the extent of angiogenic sprouting. We conclude that VEGFR2/KDR/Flk-1/-3 heterodimers on angiogenic sprouts induced by VEGFA or VEGFC may serve to positively regulate angiogenic sprouting.