The effect of angiotensin II on VEGF stimulated Akt phosphorylation 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 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 endothelial cells. The effects of angiotensin II (Ang II) on VEGF-stimulated human endothelial cell Akt survival pathway signalling are unclear.

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. Akt phosphorylation (p-Akt) was studied by semi-quantitative Western blot analysis. HUVECs were incubated with angiotensin II (10-8-10-4M) for 5min-2hours with or without VEGF stimulation. PD123319(10-7M), was used to assess the role of the angiotensin II type 2 receptor in Akt phosphorylation. ECs were pre-treated with PD123319 for 30 min before adding other agents. Data are means±SEM, analyses by ANOVA.

VEGF stimulated p-Akt signalling in cultured HUVECs, maximally 4.5±0.2 fold after 15 min stimulation by VEGF 50ng/ml (p<0.0001). Ang II had a biphasic effect on p-Akt level in cultured HUVECs. At 10-8-10-6M, Ang II caused a dose-dependent increase in p-Akt level, maximal 2.3±0.1 fold after 10 min Ang II stimulation (p<0.0001). Thereafter, p-Akt signalling decreased rapidly. However, within the range of 10-5-10-4M, Ang II caused a dose-dependent decrease in p-Akt level to 66±7% below baseline after 30 min of incubation. When PD123319 was added to medium, Ang II stimulated Akt phosphorylation increased up to 4.3±0.3 fold after 10 min exposure to Ang II ((P<0.001). At low concentration, Ang II 10-8-10-6M enhanced VEGF-stimulated Akt phosphorylation (p<0.05). Higher concentrations of Ang II (10-5-10-4M) inhibited VEGF-stimulated Akt phosphorylation to 58% below baseline (P<0.001). Both in the presence of low and high concentration of Ang II, PD123319 increased VEGF-stimulated Akt phosphorylation compared with Ang II alone (p<0.05).

Our study indicates a bimodal effect of Ang II both on basal and VEGF-stimulated Akt phosphorylation, with stimulation at low and inhibition at high Ang II concentration. The Ang II type-2 receptor mediates inhibitory regulation of both basal and VEGF-stimulated Akt phosphorylation.