VEGF protects from apoptosis induced by oxidant stress and angiotensin ii in huvecs by PI-3K dependent and P-38 mapk independent pathways

Baijun Kou1, Jinsong Ni1, Manu Vatish2, Donald RJ Singer1. Clinical Pharmacology1 & Obstetrics & Gynaecology2, Warwick University Medical School, CV2 2DX. 

Reduced capillary density (rarefaction) occurs with cardiovascular risk Antonios et al, 2001. Vascular endothelial growth factor (VEGF) is a key player in angiogenesis, with PI-3K/PKB/Akt a critical signalling pathway. Angiotensin II and oxidant stress are implicated in endothelial cell apoptosis, a major candidate mechanism for vascular rarefaction, with PI-3K and p-38MAPK involved in anti- and pro-apoptotic signalling respectively. We investigated VEGF-mediated promotion of endothelial cell survival.

We grew primary cultures of endothelial cells from human umbilical cords obtained after elective Caesarean Section (HUVEC), with written informed consent and local ethics approval. HUVECs were seeded on 6-well plates 5×104 cells/well. The pro-apoptotic protein, caspase-3 and its 17KD & 19KD cleaved forms were studied by Western blots. Apoptotic changes in nuclear chromatin were determined by bis-benzimide (Hoechst 33342) and propidium iodide staining. A minimum of 500 cells were scored for chromatin changes % apoptotic cells/total cells cell viability ratio. Cell-survival signalling was probed by 2h pre-treatment with the PI-3/Akt inhibitor LY294002 (25uM) or the p38 MAPK inhibitor SB202190 (10uM). Cells were incubated for 24hr with VEGF 50ng/ml and oxidant stress induced by 24hr incubation with xanthine 250mM/xanthine oxidase 40U/L [X/XOD). To evaluate specificity of oxidant reponses, ECs were pre-treated 30 min with the superoxide scavenger Tiron ( 5.0mM) or the XOD inhibitor oxypurinol 10-4M before adding other agents. Results are mean±SEM for data in triplicate, with analysis by ANOVA.

24hr oxidant stress decreased caspase-3 by 27±6% [p<0.001 vs. basal; cleaved. Tiron or oxypurinol prevented cleaved caspase-3 expression by X/XOD. VEGF inhibited oxidant stress induced cleaved caspase-3 expression cleaved. Addition of LY 294002 further decreased CVR to 18 +5% p<0.001 vs. oxidant stress alone. In contrast, pre-treatment with SB202190 reversed the X/XOD induced decrease in CVR to 63 +4% P<0.001 vs. oxidant stress alone. Ang II 10-8M increased CVR to 82 +11% P<0.05 vs. basal whereas Ang II 10-4M decreased CVR to 52 +7% P<0.001 vs. basal. This was further decreased by pre-treatment with LY294002 p<0.01 vs. Ang 10-4M alone but unaffected by SB 202190. VEGF 50ng/ml increased CVR to 89 +9% P<0.05 vs. basal. LY294002 completely blocked the VEGF-induced increase in CVR, with no effect of SB 202190.

Oxidant stress and high concentration Ang II activated the caspase-3 pro-apoptotic pathway and reduced human endothelial cell viability. VEGF protected ECs from these effects. Protective effects of VEGF on CVR were mediated by the PI-3 kinase pathway and were p38-MAPK independent.Oxidant decreased CVR was p38-MAPK mediated and inhibitable by basal and VEGF stimulated PI-3 kinase.

Antonios TF et al.Eur. Heart J. 2001; 22:1144-8.