Diabetes-associated Proinflammatory Mediators: The Influence on Tie Receptor Signalling in Endothelial Cells.

Vascular integrity is vital to maintain normal vascular function. Diabetes is a global issue which affects hundreds of millions of individuals, and if left untreated can lead to long-term vascular complications. Inflammatory mediators associated with diabetes, impact on the vasculature and can contribute to the development of atherosclerosis and coronary artery disease. Vascular stability is controlled by several signalling pathways, however, the Angiopoietin-1/Tie receptor pathway functions to serve multiple purposes that relate to the overall function of endothelial cells, in turn the function of the vasculature. Angiopoietins are a group of glycoproteins that bind exclusively to a family of receptor tyrosine kinase receptors known as Tie receptors. The functions of activated signalling, through Ang-1 binding to Tie-2, include vascular stability, vessel remodelling and enlargement, migration, and the mediation of anti-inflammatory processes. Ang-1 mediates its protective effect by activation the PI3K/AKT pathway. The ratio between the Tie receptors and their associated ligand, Ang-1, and Ang-2, are crucial regulators of the Ang-1/Tie-2/AKT signalling pathway that can cause various vascular diseases. Inflammatory mechanisms have been noted to have an impact on vascular dysfunctions, including the dysfunction in diabetic patients. IL-1β and TNF-α have been reported to be significant pro-inflammatory cytokines that affect the vasculature through the mechanisms of the immune response. The aim of this study is to analyse the impacts of diabetes-associated inflammatory cytokines on the Ang-1/Tie signalling pathway. This was addressed by first profiling patient samples for cytokines and Ang-2, using RANDOX multiplex ELISA as well as traditional sandwich ELISA. The acute and chronic impact of IL-1β and TNF-α on Tie receptor expression, and on Ang-1-induced PI3K/AKT activation, was examined in HUVECs using Western blot techniques. The functional impact of cytokine exposure on HUVECs was further analysed by performing a cell viability assay using a fluorescent LIVE/DEAD assay. Profiling of TNF-α only showed an increase in type 2 diabetic patients. IL-6 analysis returned results of both type 1 and type 2 diabetic patients to have increase levels to that of the control. Ang-2 returned significant differences in between the volunteer groups. Type 1 diabetics were seen to have lower levels of Ang-2 and type 2 diabetics reported an increase in Ang-2 levels (p = 0.039). Acute exposures to IL-β and TNF-α up until 3hours in HUVECs reported an increase in Tie-2 levels, with the addition of increase in Ang-1-induced Tie-2 phosphorylation. IL-1β further displayed increases in Ang-1-induced AKT activity. For results of acute exposures, the following time points displayed significance for IL-1β and TNF-α (IL-1β Tie-2 1hour p = 0.045; IL-1β Tie-2 2hour p = 0.043; IL-1β pTie-2 0.5hour p = 0.001; IL-1β pTie-2 4hour p = 0.001; TNF-α pAKT 1hour p = 0.050; TNF-α pAKT 4hour p = 0.006). The chronic exposures of IL-1β up to 48hours displayed reduced Tie-2 levels, as well as decreases in AKT levels, however, significance was not observed. On the other hand, the activity of Ang-1-induced AKT was seen to show significant decreases in phosphorylation of AKT (3hour p = 0.025; 6hour p = 0.014; 24hour p = 0.002; 48hour p = 0.002). TNF-α displayed increase in Tie2 levels with decrease shown in AKT levels, though not significant. Similar to IL-1β, TNF-α display significant decreases in Ang-1-mediated AKT activity the longer the HUVECs were exposed to the cytokine (6hour p = 0.033; 12hour p = 0.016; 24hour p = 0.006; 48hour p < 0.001). Both IL-1β and TNF-α displayed significant decreases in mature Tie-1 levels as well as a decrease in the ratio of mature: immature Tie-1 (p ≤ 0.05). The functional cell survival assay performed on HUVECs showed significant increase in cell death over the length of 24hours when incubated with IL-1β (p = 0.007). Even though not significant, HUVECs exposed to IL-1β in the presence of Ang-1 displayed an increase in cell death compared to cells treated with Ang-1 alone. In conclusion, the increase in levels of TNF-α and IL-6 observed in certain diabetic patient samples, alongside the changes to the Tie receptors and Ang-1-induced AKT activity in HUVECs exposed to TNFα and IL-1β over a chronic period, suggests potential disruption to the Ang-1/Tie signalling pathway in disease conditions including diabetes and inflammatory diseases