Selective Inhibition of Vascular Endothelial Growth Factor Receptor‐2 (VEGFR‐2) Identifies a Central Role for VEGFR‐2 in Human Aortic Endothelial Cell Responses to VEGF

Abstract

Vascular endothelial growth factor receptors (VEGFR) are considered essential for angiogenesis. The VEGFR‐family proteins consist of VEGFR‐1/Flt‐1, VEGFR‐2/KDR/Flk‐1, and VEGFR‐3/Flt‐4. Among these, VEGFR‐2 is thought to be principally responsible for angiogenesis. However, the precise role of VEGFRs1–3 in endothelial cell biology and angiogenesis remains unclear due in part to the lack of VEGFR‐specific inhibitors. We used the newly described, highly selective anilinoquinazoline inhibitor of VEGFR‐2 tyrosine kinase, ZM323881 (5‐[[7‐(benzyloxy) quinazolin‐4‐yl]amino]‐4‐fluoro‐2‐methylphenol), to explore the role of VEGFR‐2 in endothelial cell function. Consistent with its reported effects on VEGFR‐2 [IC(50) < 2 nM], ZM323881 inhibited activation of VEGFR‐2, but not of VEGFR‐1, epidermal growth factor receptor (EGFR), platelet‐derived growth factor receptor (PDGFR), or hepatocyte growth factor (HGF) receptor. We studied the effects of VEGF on human aortic endothelial cells (HAECs), which express VEGFR‐1 and VEGFR‐2, but not VEGFR‐3, in the absence or presence of ZM323881. Inhibition of VEGFR‐2 blocked activation of extracellular regulated‐kinase, p38, Akt, and endothelial nitric oxide synthetase (eNOS) by VEGF, but did not inhibit p38 activation by the VEGFR‐1‐specific ligand, placental growth factor (PlGF). Inhibition of VEGFR‐2 also perturbed VEGF‐induced membrane extension, cell migration, and tube formation by HAECs. Vascular endothelial growth factor receptor‐2 inhibition also reversed VEGF‐stimulated phosphorylation of CrkII and its Src homology 2 (SH2)‐binding protein p130Cas, which are known to play a pivotal role in regulating endothelial cell migration. Inhibition of VEGFR‐2 thus blocked all VEGF‐induced endothelial cellular responses tested, supporting that the catalytic activity of VEGFR‐2 is critical for VEGF signaling and/or that VEGFR‐2 may function in a heterodimer with VEGFR‐1 in human vascular endothelial cells.