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 immun...
Saved in:
| Main Authors: | , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Elsevier
2025-01-01
|
| Series: | Research and Practice in Thrombosis and Haemostasis |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2475037924003601 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1841544027440152576 |
|---|---|
| author | Deepa J. Arachchillage Golzar Mobayen Mike Laffan Anna M. Randi Josefin Ahnström Charis Pericleous |
| author_facet | Deepa J. Arachchillage Golzar Mobayen Mike Laffan Anna M. Randi Josefin Ahnström Charis Pericleous |
| author_sort | Deepa J. Arachchillage |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-f37e547c9b8b4f659ad0d75461964757 |
| institution | Kabale University |
| issn | 2475-0379 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Research and Practice in Thrombosis and Haemostasis |
| spelling | doaj-art-f37e547c9b8b4f659ad0d754619647572025-01-13T04:19:04ZengElsevierResearch and Practice in Thrombosis and Haemostasis2475-03792025-01-0191102665A cell-based model to study mechanisms of endothelial-dependent thrombin generation in response to inflammation and its modulation by hydroxychloroquineDeepa J. Arachchillage0Golzar Mobayen1Mike Laffan2Anna M. Randi3Josefin Ahnström4Charis Pericleous5Centre for Haematology, Department of Immunology and Inflammation, Imperial College London, London, United Kingdom; Department of Haematology, Imperial College Healthcare NHS Trust, London, United Kingdom; Correspondence Deepa J. Arachchillage, Centre for Haematology, Department of Immunology and Inflammation, Imperial College London, 5th Floor, Commonwealth Building, Du Cane Road, London W12 0NN, UK.Centre for Haematology, Department of Immunology and Inflammation, Imperial College London, London, United KingdomCentre for Haematology, Department of Immunology and Inflammation, Imperial College London, London, United KingdomNational Heart and Lung Institute, Imperial College London, London, United KingdomCentre for Haematology, Department of Immunology and Inflammation, Imperial College London, London, United KingdomNational Heart and Lung Institute, Imperial College London, London, United KingdomBackground: 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.http://www.sciencedirect.com/science/article/pii/S2475037924003601coagulationcytokinesendotheliuminflammationthrombin generation |
| spellingShingle | Deepa J. Arachchillage Golzar Mobayen Mike Laffan Anna M. Randi Josefin Ahnström Charis Pericleous A cell-based model to study mechanisms of endothelial-dependent thrombin generation in response to inflammation and its modulation by hydroxychloroquine Research and Practice in Thrombosis and Haemostasis coagulation cytokines endothelium inflammation thrombin generation |
| title | A cell-based model to study mechanisms of endothelial-dependent thrombin generation in response to inflammation and its modulation by hydroxychloroquine |
| title_full | A cell-based model to study mechanisms of endothelial-dependent thrombin generation in response to inflammation and its modulation by hydroxychloroquine |
| title_fullStr | A cell-based model to study mechanisms of endothelial-dependent thrombin generation in response to inflammation and its modulation by hydroxychloroquine |
| title_full_unstemmed | A cell-based model to study mechanisms of endothelial-dependent thrombin generation in response to inflammation and its modulation by hydroxychloroquine |
| title_short | A cell-based model to study mechanisms of endothelial-dependent thrombin generation in response to inflammation and its modulation by hydroxychloroquine |
| title_sort | cell based model to study mechanisms of endothelial dependent thrombin generation in response to inflammation and its modulation by hydroxychloroquine |
| topic | coagulation cytokines endothelium inflammation thrombin generation |
| url | http://www.sciencedirect.com/science/article/pii/S2475037924003601 |
| work_keys_str_mv | AT deepajarachchillage acellbasedmodeltostudymechanismsofendothelialdependentthrombingenerationinresponsetoinflammationanditsmodulationbyhydroxychloroquine AT golzarmobayen acellbasedmodeltostudymechanismsofendothelialdependentthrombingenerationinresponsetoinflammationanditsmodulationbyhydroxychloroquine AT mikelaffan acellbasedmodeltostudymechanismsofendothelialdependentthrombingenerationinresponsetoinflammationanditsmodulationbyhydroxychloroquine AT annamrandi acellbasedmodeltostudymechanismsofendothelialdependentthrombingenerationinresponsetoinflammationanditsmodulationbyhydroxychloroquine AT josefinahnstrom acellbasedmodeltostudymechanismsofendothelialdependentthrombingenerationinresponsetoinflammationanditsmodulationbyhydroxychloroquine AT charispericleous acellbasedmodeltostudymechanismsofendothelialdependentthrombingenerationinresponsetoinflammationanditsmodulationbyhydroxychloroquine AT deepajarachchillage cellbasedmodeltostudymechanismsofendothelialdependentthrombingenerationinresponsetoinflammationanditsmodulationbyhydroxychloroquine AT golzarmobayen cellbasedmodeltostudymechanismsofendothelialdependentthrombingenerationinresponsetoinflammationanditsmodulationbyhydroxychloroquine AT mikelaffan cellbasedmodeltostudymechanismsofendothelialdependentthrombingenerationinresponsetoinflammationanditsmodulationbyhydroxychloroquine AT annamrandi cellbasedmodeltostudymechanismsofendothelialdependentthrombingenerationinresponsetoinflammationanditsmodulationbyhydroxychloroquine AT josefinahnstrom cellbasedmodeltostudymechanismsofendothelialdependentthrombingenerationinresponsetoinflammationanditsmodulationbyhydroxychloroquine AT charispericleous cellbasedmodeltostudymechanismsofendothelialdependentthrombingenerationinresponsetoinflammationanditsmodulationbyhydroxychloroquine |