The role of an alpha-selective phosphoinositide-3 kinase inhibitor in vascular inflammation

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Abstract Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): British Heart Foundation Introduction Cardiovascular inflammation is associated with endothelial cell (EC) damage, resulting in leukocyte trafficking and oedema formation. Inflammation disrupts EC junctions, increasing microvascular permeability, and resulting in a positive-feedback loop of inflammatory events. The phosphoinositide 3-kinase pathway stimulates this endothelial response and this study examined the actions of BYL-719, a PI3K-α selective inhibitor, on inflammatory responses. Method Confocal imaging using immunofluorescence and permeability assays were undertaken to quantify the effect of inflammatory cytokines, TNF-α and IL-1β, in the presence of the PI3K inhibitor using fluorescein isothiocyanate-dextran (40 kDa, 0.1 mg/ml) on human microvascular endothelial cells (HMVEC) (Lonza, derived from dermal tissue). Cells were treated with either cytokine for 16-18 h, followed by a 1 h treatment of drug and a final 1 h treatment with FITC-dextran. In vivo analysis was carried out per the UK Home Office Animals (Scientific Procedures) Act 1986. Male WT CD1 mice were anaesthetised initially using 5% isoflurane and maintained at 2% for procedures, to test BYL-719 in a model of dorsal skin inflammation, to determine neutrophil accumulation (measured by myeloperoxidase) and oedema formation (measured by Evans Blue accumulation). All statistical significance was determined using one-way or two-way ANOVA followed by Tukey’s post hoc test. Results BYL-719 significantly reduced cytokine-induced EC permeability and shape changes, including cell area and elongation (Table 1), impeding in vitro cytokine-induced inflammation. The inhibitor, abrogated effects of the inflammatory cytokines in vivo of both TNF-α and IL-1β, but interestingly had no effect on the neutrophil accumulation or oedema formation in the presence of C5a (Figure 1). Figure 1: The effect of BYL-719 in the dorsal skin inflammation model. Mice were pre-treated with 50 mg/kg BYL-719 intraperitoneally for 30 min, injected intravenously with Evans Blue dye, intradermally injected with TNF-α (100ng/50ul), IL-1β (10ng/50ul) and C5a (300ng/50ul) for 4 h, followed by ex vivo MPO assay (A) and oedema volume measurements (B). Data are mean ±SEM two-way ANOVA by Tukey test., n=6 independent experiments in duplicates; p* < 0.05 and p**** < 0.0001 between control (DMSO) and BYL-719 treated groups. Conclusions Our findings show that the PI3K-α inhibitor, BYL-719, reduces endothelial activation and inhibits inflammatory oedema formation in the presence of TNF-α and IL-1β. We conclude that there is a potential for PI3K inhibitors to act as anti-neutrophil and oedema agents in cardiovascular-related inflammatory conditions.

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