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 production and release of nitric oxide (NO), which is produced by endothelial nitric oxide synthase (eNOS). While the upregulation of eNOS expression has been shown to be mediated via VEGF receptor VEGFR2/KDR/Flk-1, there is controversy about which of the VEGF receptors triggers the release of nitric oxide in endothelial cells. In order to determine the levels of NO produced in response to VEGF-A stimulation in different endothelial cells, a reporter assay measuring the formation of cGMP as the direct product of NO-induced activation of Guanylate Cyclase was performed. Using two independent experimental strategies, we were able to prove that VEGF receptor VEGFR2/KDR/Flk-1, 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 VEGFR2/KDR/Flk-1 or Flt-1. While KDR-expressing PAE/VEGFR2/KDR/Flk-1 cells responded to VEGF-A stimulation with a significant elevation of intracellular cGMP already after 2 min, Flt-1-expressing PAE/Flt-1 cells did not show any signal in this RIA-based cGMP assay. In a second experimental strategy freshly isolated human umbilical vein endothelial cells (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 this setting, while PIGF-2 was unable to do so, clearly demonstrating that VEGFR2/KDR/Flk-1 is responsible for NO release in endothelial cells. In our assays cGMP formation is fully dependent on NO generation since the NOS inhibitor L-NAME can block this VEGF-A-induced action. These data show that the VEGF receptor VEGFR2/KDR/Flk-1 is responsible for NO release in endothelial cells, highlighting a new function of VEGFR2/KDR/Flk-1 and further supporting the importance of VEGFR2/KDR/Flk-1 in the regulation of the vasculature.