Thrombosis Research Group (TREC), Department of Clinical Medicine, UiT - The Arctic University of Norway, Tromsø, Norway.
Science for Life Laboratory, Department of Gene Technology, KTH Royal Institute of Technology, Stockholm, Sweden.
Front Immunol. 2023 Sep 11;14:1240597. doi: 10.3389/fimmu.2023.1240597. eCollection 2023.
Venous thromboembolism (VTE) is a leading cause of preventable deaths in hospitals, and its incidence is not decreasing despite extensive efforts in clinical and laboratory research. Venous thrombi are primarily formed in the valve pockets of deep veins, where activated monocytes play a crucial role in bridging innate immune activation and hemostatic pathways through the production of inflammatory cytokines, chemokines, and tissue factor (TF) - a principal initiator of coagulation. In the valve pocket inflammation and hypoxia (sustained/intermittent) coexist, however their combined effects on immunothrombotic processes are poorly understood. Inflammation is strongly associated with VTE, while the additional contribution of hypoxia remains largely unexplored. To investigate this, we modelled the intricate conditions of the venous valve pocket using a state-of-the-art hypoxia chamber with software-controlled oxygen cycling. We comprehensively studied the effects of sustained and intermittent hypoxia alone, and in combination with VTE-associated inflammatory stimuli on primary monocytes. TF expression and activity was measured in monocytes subjected to sustained and intermittent hypoxia alone, or in combination with IL-1β. Monocyte responses were further analyzed in detailed by RNA sequencing and validated by ELISA. Stimulation with IL-1β alone promoted both transcription and activity of TF. Interestingly, the stimulatory effect of IL-1β on TF was attenuated by sustained hypoxia, but not by intermittent hypoxia. Our transcriptome analysis further confirmed that sustained hypoxia limited the pro-inflammatory response induced by IL-1β, and triggered a metabolic shift in monocytes. Intermittent hypoxia alone had a modest effect on monocyte transcript. However, in combination with IL-1β intermittent hypoxia significantly altered the expression of 2207 genes and enhanced the IL-1β-stimulatory effects on several chemokine and interleukin genes (e.g., IL-19, IL-24, IL-32, MIF), as well as genes involved in coagulation (thrombomodulin) and fibrinolysis (VEGFA, MMP9, MMP14 and PAI-1). Increased production of CCL2, IL-6 and TNF following stimulation with intermittent hypoxia and IL-1β was confirmed by ELISA. Our findings provide valuable insights into how the different hypoxic profiles shape the immunothrombotic response of monocytes and shed new light on the early events in the pathogenesis of venous thrombosis.
静脉血栓栓塞症（VTE）是医院中可预防死亡的主要原因，尽管在临床和实验室研究方面进行了广泛的努力，但其发病率并未降低。静脉血栓主要在深静脉的瓣膜口袋中形成，其中激活的单核细胞通过产生炎症细胞因子、趋化因子和组织因子（TF）——凝血的主要启动子，在桥接先天免疫激活和止血途径方面发挥关键作用。然而，在瓣膜口袋炎症和缺氧（持续/间歇）共存的情况下，它们对免疫血栓形成过程的综合影响仍知之甚少。炎症与 VTE 密切相关，而缺氧的额外贡献在很大程度上仍未得到探索。为了研究这一点，我们使用具有软件控制氧气循环的最先进的缺氧室来模拟静脉瓣膜口袋的复杂情况。我们全面研究了持续和间歇缺氧单独以及与 VTE 相关的炎症刺激联合作用对原代单核细胞的影响。在单独持续和间歇缺氧或与白细胞介素-1β（IL-1β）联合作用下，测量单核细胞中 TF 的表达和活性。通过 RNA 测序进一步分析单核细胞反应，并通过 ELISA 进行验证。单独用 IL-1β刺激可促进 TF 的转录和活性。有趣的是，IL-1β 对 TF 的刺激作用被持续缺氧减弱，但不受间歇缺氧影响。我们的转录组分析进一步证实，持续缺氧限制了 IL-1β 诱导的促炎反应，并触发了单核细胞的代谢转变。单独间歇缺氧对单核细胞转录的影响较小。然而，与 IL-1β 联合使用时，间歇缺氧会显著改变 2207 个基因的表达，并增强 IL-1β 对几种趋化因子和白细胞介素基因（如 IL-19、IL-24、IL-32、MIF）以及参与凝血（血栓调节蛋白）和纤维蛋白溶解（VEGFA、MMP9、MMP14 和 PAI-1）的基因的刺激作用。ELISA 证实，间歇性缺氧和 IL-1β 刺激后 CCL2、IL-6 和 TNF 的产生增加。我们的研究结果提供了有价值的见解，了解不同的缺氧特征如何塑造单核细胞的免疫血栓形成反应，并为静脉血栓形成发病机制的早期事件提供了新的认识。
