A novel function of VEGF receptor-2 (KDR): Rapid release of nitric oxide in response to VEGF-A stimulation in endothelial cells

VEGF-A induces angiogenesis and regulates endothelial function via producti on and release of nitric oxide (NO), which is produced by endothelial nitri c oxide synthase (eNOS), While the upregulation of eNOS expression has been shown to be mediated via VEGF receptor KDR, there is controversy about whi ch of the VEGF receptors triggers the release of nitric oxide in endothelia l cells. In order to determine the levels of NO produced in response to VEG F-A stimulation in different endothelial cells, a reporter assay measuring the formation of cGMP as the direct product of NO-induced activation of gua nylate cyclase was performed. Using two independent experimental strategies , we were able to prove that VEGF receptor KDR, but not VEGF receptor Flt-1 , can induce NO release in endothelial cells. First, we made use of porcine aortic endothelial cells (PAE) expressing either KDR or Flt-1. While KDR-e xpressing PAE/KDR cells responded to VEGF-A stimulation with a significant elevation of intracellular cGMP already after 2 min, Flt-1-expressing PAE/F lt-1 cells did not show any signal in this RIA-based cGMP assay. In a secon d experimental strategy freshly isolated human umbilical vein endothelial c ells (HUVEC) were stimulated either with the KDR-specific ligand VEGF-E or with the Flt-1-specific ligand PIGF-2, VEGF-E induces cGMP elevation in thi s setting, while PIGF-2 was unable to do so, clearly demonstrating that KDR is responsible for NO release in endothelial cells. In our assays cGMP for mation is fully dependent on NO generation since the NOS inhibitor L-NAME c an block this VEGF-A-induced action. These data show that the VEGF receptor KDR is responsible for NO release in endothelial cells, highlighting a new function of KDR and further supporting the importance of KDR in the regula tion of the vasculature. (C) 1999 Academic Press.