Background
Inflammation is a driver of thrombosis but the phenomenon of thromboinflammation has been defined only recently, bringing together the multiple pathways involved. In vitro models can support the development of new therapeutics targeting the endothelium also assess the existing immunomodulatory drugs such as hydroxychloroquine in modulating the inflammation driven endothelial prothrombotic phenotype.
Objectives
Developing a model for thrombin generation (TG) on the surface of human endothelial cells to assess pro/antithrombotic properties in response to inflammation. Furthermore, to elucidate the mechanisms of TG regulation and its modulation by immunomodulatory therapies.
Methods
Cytokine-induced (TNF-α, IL-1β, IFN-γ) effects on endothelial cells isolated from umbilical vein or human aortic tissue were assessed using calibrated automated thrombography in platelet-poor plasma. Expression of key coagulant and inflammatory regulators was measured at mRNA level. Tissue factor (TF) protein levels were further assessed by flow cytometry.
Results
Endothelial stimulation with TNF-α or IL-1β caused EC to trigger TG without addition of exogenous TF, with higher TG observed after 6hrs stimulation compared to 24 hrs. IL-1β induced higher peak thrombin (170±5.9nM vs 115±4.9nM), endogenous thrombin potential (1632±35nM*min vs 1370±23nM*min), and TF expression (∼2.8-fold higher), compared to TNF-α at 6hrs. IFN-γ stimulation failed to induce TG and TF expression. The immunomodulatory hydroxychloroquine reduced cytokine-induced TG and downregulated TF expression.
Conclusions
We provide a detailed optimisation of a robust in vitro model to assess induction of an inflammation-driven endothelial prothrombotic phenotype that is also sensitive to immunomodulatory therapies, providing a tool for investigating mechanisms of disease and new drugs.