Oxidized LDL Inhibits Vascular Endothelial Growth Factor–Induced Endothelial Cell Migration by an Inhibitory Effect on the Akt/Endothelial Nitric Oxide Synthase Pathway

Abstract

Background —Oxidized LDL (oxLDL) inhibits endothelial cell (EC) migration. Stimulating ECs with vascular endothelial growth factor (VEGF) leads to the activation of Akt/protein kinase B, which in turn activates endothelial nitric oxide synthase (eNOS) by phosphorylation on serine 1177. VEGF-induced cell migration is dependent on the generation of nitric oxide (NO). Therefore, we investigated whether oxLDL affects EC migration by an inhibitory effect on the Akt/eNOS pathway. Methods and Results —During an in vitro “scratched wound assay,” oxLDL dose-dependently inhibited the VEGF-induced migration of human umbilical vein endothelial cells. Western blot analysis revealed that oxLDL dose- and time-dependently led to dephosphorylation and thus deactivation of Akt. Moreover, oxLDL inhibited the VEGF-induced generation of NO, as detected and quantified using a fluorescent NO indicator, 4,5-diaminofluorescein diacetate. Overexpression of a constitutively active Akt construct (Akt T308D/S473D) or a phosphomimetic eNOS construct (eNOS S1177D) almost completely reversed the inhibitory effect of oxLDL on VEGF-induced EC migration and NO generation. Conclusions —Our data indicate that oxLDL-induced dephosphorylation of Akt, followed by impaired eNOS activation, reduces the intracellular level of NO and thereby inhibits VEGF-induced EC migration.