BS20 The opposing effects of chronic interleukin-1β on TIE2:TIE1 ratio and angiopoietin1 induced PI3 Kinase/AKT signaling in endothelial cells

Introduction Angiopoietin-1 (Ang-1) is a growth factor that plays a crucial role in maintaining normal vascular function. The main role of Ang-1 is to maintain endothelial survival. Ang-1 exerts its protective effect by activating Tie-2 receptors and subsequently the Phosphatidylinositol 3-kinase (PI3Kinase)/AKT pathway. Tie-1 regulates Ang-1 signalling with high levels of the receptor reducing Ang-1-induced Tie-2 activation. Proinflammatory cytokines including Tumor Necrotic factor (TNF-α) Interleukin 1β (IL-1β) have been implicated in a range of vascular pathologies including vascular inflammation and atherosclerosis. While TNF-α has shown to regulate Tie receptors, the chronic impact IL-1β has on Ang-1/Tie receptor signalling pathway and vascular function has not been investigated. Aim To examine the impact IL-1β has on Tie receptor expression and Angiopoietin-1 induced PI3Kinase/AKT activity in endothelial cells. Method Primary Human Umbilical Vein Endothelial Cells (HUVEC) were stimulated with 25ng/ml of IL-1β in the presence or absence of 100ng/ml of human recombinant Ang-1. The treatment times ranged from 0 to 48h. Cell lysates from the treated cells were then subjected to Western blotting to analyze Tie receptor levels and phospho-AKT (pAKT), a signaling molecule associated with Ang-1 cellular transduction. The levels of target proteins were compared between reactions by quantifying mean intensity of bands. In addition, cells were treated with Ang-1 in the absence or presence of IL-1β at various time points. The cell viability assay was performed on the treated cells by following the manufactures protocol. The mean percentage of live to dead cells was calculated from three random fields for each treatment. Data for the Tie receptor and AKT analysis is presented as means and SEM of three independent experiments. Statistical significance represented with p Results A significant reduction in the levels of Tie-1 receptor was observed at 3h (58.6±8.6%) in HUVECs treated with IL-1β. The cytokine was able to maintain significant low levels of Tie1 up until 48h of treatment, whereas the changes in levels of Tie-2 were insignificant. Interestingly, IL-1β significantly reduced Ang1-induced pAKT activity from 3h onwards with maximum reduction of 73.4±12.8% observed at 48h. The cell viability assays showed reduction in the percentage live to dead cells between Ang-1 and Ang1 + IL-1β for chronic time points tested. Conclusion Long term exposure of Interleukin-1β is capable of altering Tie-1 levels and increasing the Tie-2: Tie-1 ratio in endothelial cells. In contracts, IL-1β reduces Ang-1-PI3Kinase/AKT signalling and endothelium cell viability. This opposing phenomenon observed where IL-1β impairs Ang-1 protective ability suggests and different mechanism of regulation, independent of the Tie-1 receptor. Conflict of Interest None