P701The role of a phosphoinositide-3 kinase inhibitor in vascular inflammation

Abstract

Abstract Background/Introduction Cardiovascular inflammation is associated with endothelial damage, resulting in leukocyte trafficking and oedema formation, which results in a positive-feedback loop of inflammatory events. Purpose The phosphoinositide 3-kinase (PI3K) pathway has been shown to be upregulated in vascular disease conditions whereby its inhibitors potentially have beneficial effects. Hence this study was designed to examine the actions of PI-103, a PI3K non-selective inhibitor, on inflammatory responses. Methods Permeability assays were undertaken to quantify the effect of TNF-a in the presence and absence of PI-103 using FITC-dextran (40 kDa, 0.1 mg/ml) to measure levels of permeability in trans-wells plates using human microvascular endothelial cells (HMVEC). In vivo analysis was carried out per the UK Home Office Animals (Scientific Procedures) Act 1986. CD1 mice were anaesthetised, to test PI-103 in a zymosan-induced model of dorsal skin inflammation on neutrophil accumulation (measured by myeloperoxidase) and oedema formation (measured by Evans Blue accumulation). All data were analysed using 1-way or 2-way ANOVA with post-hoc test. Results PI-103 significantly reduced TNF-α induced microvascular endothelial cell permeability (Graph), thus impeding in vitro cytokine-induced inflammation. Whilst there was no effect on neutrophil accumulation in vivo, there was significant reduction in weight of treated areas and oedema formation, which was inhibited by PI-103 (Table 1). Effect of PI-103 in vivo Control 30 mg/kg PI-103 Tyrode solution Zymosan (μg/ml) Tyrode solution Zymosan (μg/ml) 10 30 100 10 30 100 MPO/Protein (U/mg) 0.66±0.26 1.42±0.07 1.40±0.14 1.68±0.12* 0.60±0.11 1.23±0.32 1.36±0.25 1.59±0.21* Oedema Volume (mm3) 0.00±0.00 3.26±1.76 11.70±2.21 21.50±4.67 2.20±1.53 1.05±1.05 3.27±0.74 5.52±1.58 Mice were pre-treated with PI-103 i.p. for 30 min, i.d injected with zymosan (50 μl/site) for 4 hr, followed by ex vivo MPO assay, weight and oedema volume measurements. Oedema was quantified by measuring: width (x), height (y) and depth (z) from each of the i.d. sites; volume of oedema = ((π/6)xyz). N=6 independent experiments in duplicates; *p<0.05 between Tyrode solution vs zymosan groups. Effect of PI103 in vitro on permeability Conclusions Our findings show that the PI3K non-selective inhibitor, PI-103 (as well as a PI3K α-selective inhibitor), reduces endothelial activation and inhibits inflammatory oedema formation. We conclude that there is a potential for PI3K inhibitors to act as anti-oedema agents in cardiovascular-related inflammatory conditions. Acknowledgement/Funding British Heart Foundation