The effect of oxidant stress on VEGF stimulated phosphorylation of Akt in human umbilical vein endothelial cells

Baijun Kou1, Manu Vatish2, Donald R. J. Singer1. 1Clinical Pharmacology and 2Obstetrics & Gynaecology, Clinical Sciences Research Institute, Warwick Medical School, University of Warwick, Coventry CV2 2DX.

Reduced capillary density (rarefaction) is an early event associated with cardiovascular risk factors as well as established cardiovascular disease. The PI-3K-PKB/Akt signalling pathway is a key player in endothelial cell survival or anti-apoptosis. VEGF is a key growth factor for angiogenesis by ECs. However, the effect of oxidant stress on VEGF-stimulated human endothelial cells Akt signalling are unclear. In this study, we investigated effects of oxidant stress on ECs survival signalling Akt pathway.

We grew primary cultures of endothelial cells obtained from human umbilical cords (HUVECs) obtained following elective Caesarean Section with written informed consent and local ethics committee approval. Phosphorylated-Akt (p-Akt) signalling was studied by semi-quantitative Western blot analysis. HUVECs were incubated with xanthine250uM/XOD(20-80U/L) for 5min-1hour with or without VEGF stimulation. To evaluate the role of oxidant stress, ECs were pre-treated for 30 min with the superoxide scavenger Tiron or the xanthine oxidase inhibitor oxypurinol before adding other agents to the HUVECs. Data are means ±SEM, analyses by ANOVA.

Xanthine/XOD(X/XOD) caused a dose and time-dependent decrease in p-Akt signalling with XOD 80U/L, with at 1h only 6.0±0.5% p-Akt vs. basal(p<0.0001).VEGF 50ng stimulated p-Akt signalling to a peak at 4.5±0.2 fold after 15min incubation(p<0.0001). This was inhibited by X/XOD dose and time-dependently, with complete inhibition of p-Akt after 1h of incubation(p<0.0001). The inhibition of p-Akt signalling by 5-15min oxidant stress in HUVECs was reversed on pre-treatment with Tiron 1.0mM. Tiron 5-10mM improved p-Akt signalling at 15min by 1.8±0.1 fold(p<0.001). Oxypurinol, dose-dependently blocked inhibition by X/XOD of p-Akt signalling. At 10-6M oxypurinol, the level of p-Akt recovered to basal values. At 10-3M oxypurinol, the p-Akt level increased 1.8±0.1 fold compared with baseline. Tiron 5.0mM, but not oxypurinol, protected VEGF-stimulated p-Akt signalling from external X/XOD oxidant stress (p<0.05). Interestingly, incubation with oxypurinol (10-4M)alone for 30min attenuated p-Akt signalling ca. 57% and attenuated VEGF-stimulated p-Akt signalling(p<0.01). Incubation with Tiron (5.0mM) for 30 min enhanced p-Akt signalling 1.6 fold; it also enhanced VEGF-stimulated p-Akt signalling compared with VEGF alone (p<0.01).

External oxidant stress inhibits both basal and VEGF stimulated p-Akt signalling in HUVECs. Inhibitors of oxidant stress selectively attenuate inhibition of Akt signalling by exogenous oxidant stress. However, that inhibition of endogenous oxidant stress by oxypurinol but not by Tiron suppressed VEGF-stimulated p-Akt signalling, implicates a superoxide-independent but xanthine oxidase-sensitive mechanism in mediating VEGF-stimulated p-Akt signalling.