基于细胞的模型研究内皮依赖性凝血酶在炎症反应中的产生机制及其羟氯喹的调节作用。

IF 3.4 3区医学 Q2 HEMATOLOGY

Research and Practice in Thrombosis and Haemostasis Pub Date : 2025-01-01 DOI:10.1016/j.rpth.2024.102665

Deepa J. Arachchillage , Golzar Mobayen , Mike Laffan , Anna M. Randi , Josefin Ahnström , Charis Pericleous

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引用次数: 0

摘要

背景：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，将涉及的多种途径结合在一起。体外模型可以支持针对内皮的新疗法的开发，也可以评估现有的免疫调节药物，如羟氯喹，在调节炎症驱动的内皮血栓前表型方面的作用。目的：建立人内皮细胞（ECs）表面凝血酶生成（TG）模型，以评估炎症反应中的促/抗血栓特性。进一步阐明TG调控机制及免疫调节疗法对其的调节作用。方法：使用校准的血小板贫乏血浆自动血栓造影术评估细胞因子诱导（肿瘤坏死因子[TNF]-α，白细胞介素[IL]-1β和干扰素-γ）对脐静脉或人主动脉组织分离的ECs的影响。在mRNA水平上检测关键凝血因子和炎症调节因子的表达。流式细胞术进一步检测组织因子（TF）蛋白水平。结果：内皮细胞TNF-α或IL-1β刺激可导致内皮细胞在不添加外源性TF的情况下触发TG，刺激6小时后TG高于刺激24小时。IL-1β诱导的凝血酶峰值更高（170±5.9 nM vs 115±4.9 nM），内源性凝血酶电位（1632±35 nM∗min vs 1370±23 nM∗min）以平均±SD表示，与TNF-α相比，6小时时TF表达（高2.8倍）。干扰素γ刺激不能诱导TG和TF的表达。免疫调节药物羟氯喹可降低细胞因子诱导的TG，下调TF的表达。结论：我们提供了一个强大的体外模型的详细优化，以评估炎症驱动的内皮血栓形成前表型的诱导，该表型对免疫调节疗法也很敏感，为研究疾病机制和新药提供了工具。

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A cell-based model to study mechanisms of endothelial-dependent thrombin generation in response to inflammation and its modulation by hydroxychloroquine

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 and also assess the existing immunomodulatory drugs, such as hydroxychloroquine, in modulating the inflammation-driven endothelial prothrombotic phenotype.

Objectives

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.

Methods

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.

Results

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.

Conclusion

We provide detailed optimization of a robust in vitro 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.

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