Arachchillage Deepa J, Mobayen Golzar, Laffan Mike, Randi Anna M, Ahnström Josefin, Pericleous Charis
Centre for Haematology, Department of Immunology and Inflammation, Imperial College London, London, United Kingdom.
Department of Haematology, Imperial College Healthcare NHS Trust, London, United Kingdom.
Res Pract Thromb Haemost. 2024 Dec 25;9(1):102665. doi: 10.1016/j.rpth.2024.102665. eCollection 2025 Jan.
Inflammation is a driver of thrombosis, but the phenomenon of thromboinflammation has been defined only recently, bringing together the multiple pathways involved. models can support the development of new therapeutics targeting the endothelium and also assess the existing immunomodulatory drugs, such as hydroxychloroquine, in modulating the inflammation-driven endothelial prothrombotic phenotype.
To develop a model for thrombin generation (TG) on the surface of human endothelial cells (ECs) to assess pro/antithrombotic properties in response to inflammation. Furthermore, to elucidate the mechanisms of TG regulation and its modulation by immunomodulatory therapies.
Cytokine-induced (tumor necrosis factor [TNF]-α, interleukin [IL]-1β, and interferon-γ) effects on ECs isolated from umbilical veins or human aortic tissue were assessed using calibrated automated thrombography in platelet-poor plasma. The expression of key coagulant and inflammatory regulators was measured at the mRNA level. Tissue factor (TF) protein levels were further assessed by flow cytometry.
Endothelial stimulation with TNF-α or IL-1β caused ECs to trigger TG without the addition of exogenous TF, with higher TG observed after 6 hours of stimulation than 24 hours. IL-1β induced higher peak thrombin (170 ± 5.9 nM vs 115 ± 4.9 nM), endogenous thrombin potential (1632 ± 35 nM ∗ min vs 1370 ± 23 nM ∗ min) presented as mean ± SD, and TF expression (∼2.8-fold higher) compared with TNF-α at 6 hours. Interferon-γ stimulation failed to induce TG and TF expression. The immunomodulatory drug, hydroxychloroquine, reduced cytokine-induced TG and downregulated TF expression.
We provide detailed optimization of a robust model to assess the 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.
炎症是血栓形成的驱动因素，但血栓炎症现象直到最近才被定义，它整合了多个相关途径。模型可以支持针对内皮细胞的新疗法的开发，还能评估现有免疫调节药物（如羟氯喹）对炎症驱动的内皮促血栓形成表型的调节作用。
建立一种在人内皮细胞（ECs）表面生成凝血酶（TG）的模型，以评估炎症反应下的促/抗血栓形成特性。此外，阐明TG调节机制及其受免疫调节疗法的调控情况。
使用校准自动血栓成像技术，在少血小板血浆中评估细胞因子（肿瘤坏死因子 [TNF]-α、白细胞介素 [IL]-1β 和干扰素-γ）对从脐静脉或人主动脉组织分离的ECs的影响。在mRNA水平上测量关键凝血和炎症调节因子的表达。通过流式细胞术进一步评估组织因子（TF）蛋白水平。
用TNF-α或IL-1β刺激内皮细胞可使其在不添加外源性TF的情况下触发TG，刺激6小时后的TG高于24小时。与TNF-α相比，IL-1β在6小时时诱导的凝血酶峰值更高（170±5.9 nM对115±4.9 nM）、内源性凝血酶潜力更高（1632±35 nM∗min对1370±23 nM∗min），以平均值±标准差表示，且TF表达更高（约高2.8倍）。干扰素-γ刺激未能诱导TG和TF表达。免疫调节药物羟氯喹可降低细胞因子诱导的TG并下调TF表达。
我们提供了一个强大模型的详细优化方法，以评估炎症驱动的内皮促血栓形成表型的诱导情况，该表型对免疫调节疗法也敏感，为研究疾病机制和新药提供了一种工具。
