Submitted December 24, 2008
Accepted June 25, 2009
Phosphorylation of endothelial nitric oxide synthase by atypical PKC
contributes to angiopoietin-1-dependent inhibition of VEGF-induced endothelial permeability in vitro
Malika Oubaha and Jean-Philippe Gratton*
Laboratory of Endothelial Cell Biology, Institut de recherches cliniques de Montreal (IRCM), Universite de Montreal, Montreal, QC, Canada
* Corresponding author; email: jean-philippe.gratton{at}ircm.qc.ca.
Vascular endothelial growth factor (VEGF) is a potent angiogenic cytokine that also increases vascular permeability. Nitric oxide (NO) released from endothelial cells, following activation of endothelial NO synthase (eNOS), contributes to proangiogenic and permeability effects of VEGF. Angiopoietin-1 (Ang-1), via Tie2 receptors, shares many of the proangiogenic properties of VEGF on endothelial cells. However, in contrast to VEGF, Ang-1 protects blood vessels from increased plasma leakage which contributes to their stabilization. Since eNOS-derived NO is central to increased permeability in response to VEGF, we investigated if Ang-1 interferes with VEGF signaling to eNOS. We demonstrate that Ang-1 stimulation of endothelial cells inhibits VEGF-induced NO release and transendothelial permeability. In contrast to VEGF stimulation, Ang-1 causes a marked PKC-dependent increase in phosphorylation of eNOS on the inhibitory threonine 497. Furthermore, using pharmacological inhibitors, overexpression studies and siRNA-mediated gene silencing, we demonstrate that atypical PKC
is responsible for phosphorylation of eNOS on Thr497 in response to Ang-1. In addition, PKC knockdown abrogates the capacity of Ang-1 to inhibit VEGF-induced NO release and endothelial permeability. Thus, inhibition of NO production by Ang-1, via phosphorylation of eNOS on Thr497 by PKC is responsible, at least in part, for inhibition of VEGF-stimulated endothelial permeability by Ang-1.
