Development and Characterisation of a Novel, Endothelialised in vitro Model of Human Atherothrombosis

动脉血栓形成是世界范围内死亡的主要原因，是动脉粥样硬化病变破裂或侵蚀的结果（Cate和Hemker， 2016）。斑块成分暴露于循环血小板，血小板粘附、激活和聚集，以响应血栓形成蛋白，如血管性血友病因子（vWF）和胶原蛋白（Olie et al., 2018）。目前动脉粥样硬化血栓形成的治疗方法包括抗血小板治疗，阿司匹林和氯吡格雷的联合治疗已被证明可将主要血管事件的风险降低10%以上（Patrono等人，2017），但有效性受到高度个体间差异的限制，12个月后复发事件/死亡的风险仍在20%左右（Olie等人，2018）。从历史上看，对动脉粥样硬化血栓形成的研究主要集中在小鼠体内模型的使用上，其中内皮被人为破坏，显示出健康的细胞外基质（ECM）。通过使用Apoe-/-小鼠，已经开发了更多与疾病相关的模型，但是有充分的证据表明，小鼠的动脉粥样硬化病变在组织学上与人类斑块在大小、脂质核心和纤维帽方面不同。如果不进行干预，它们也不太可能破裂。用于破坏血管壁的方法因研究而异，因此斑块破裂和血栓形成因使用的方法而异（Mastenbroek et al., 2015）。此外，细胞之间的物种差异意味着该模型不具有人类动脉粥样硬化血栓的代表性。体外研究目前被用于更深入地了解血栓形成的机制。涂有I型胶原蛋白的平行板流室用于评估各种出血性疾病患者的血小板粘附、活化和聚集（brows等人，2018），然而，这些模型没有纳入斑块破裂和侵蚀中观察到的功能失调基质，并且缺乏内皮细胞。最近对动脉粥样硬化相关基质的研究表明，斑块破裂和侵蚀中的ECM成分不同，每种斑块相关的血栓组成也不同。与斑块侵蚀相关的血栓似乎富含血小板，而破裂血栓则颜色更深，含有更多的红细胞和纤维蛋白（Otsuka et al., 2016）。斑块破裂和侵蚀中基质成分的组成和血栓形成性，以及它如何影响内皮和平滑肌细胞功能以及血小板反应是一个新的研究领域。需要进一步的研究来更深入地了解不同斑块表型的动脉血栓形成。病变特异性模型的发展将允许评估现有和新型抗血栓药物在不同临床情况下的疗效，为更个性化的抗血栓治疗方法提供信息。目的本项目旨在建立动脉粥样硬化血栓形成的体外模型，包括内皮细胞、平滑肌细胞和与斑块破裂和侵蚀相关的功能失调基质。开发具有斑块破裂和侵蚀代表性的ECM复合材料，并确定其血栓形成性。检查内皮细胞和平滑肌细胞在侵蚀性和破裂性ECM复合材料上的血栓形成性。开发并验证一种在微流控室中局部消融不同ECM复合材料上培养的内皮细胞的方法。使用最终侵蚀和破裂模型评估和比较现有和新型抗血栓药物的疗效方法功能失调细胞外基质的发展MMU心血管研究小组现有文献和目前正在进行的研究将用于表征斑块破裂和侵蚀中的基质成分。初步实验将在市售的单个co上进行

BackgroundAtherothrombosis is a leading cause of mortality worldwide, and occurs as a consequence of the rupture or erosion of atherosclerotic lesions (Cate and Hemker, 2016). Plaque components are exposed to circulating platelets, which adhere, activate and aggregate in response to thrombogenic proteins such as von Willebrand Factor (vWF) and collagen (Olie et al., 2018). Current treatments for atherothrombosis include antiplatelet therapy, where a combination of aspirin and clopidogrel has been shown to reduce the risk of major vascular events by over 10% (Patrono et al., 2017), however effectiveness is limited by high inter-individual variability, and the risk of recurrent events/death after 12 months remains at around 20% (Olie et al., 2018).Historically, research into atherothrombosis has primarily focused on the use of murine in vivo models, where the endothelium is artificially damaged, revealing a healthy extracellular matrix (ECM). More disease relevant models have been developed through the use of Apoe-/- mice, however it is well documented that atherosclerotic lesions in mice are histologically different from human plaques in terms of size, lipid core and fibrous cap. They are also unlikely to rupture without intervention. The methods used to damage the vessel wall varies between studies, and consequently plaque disruption and thrombus formation differs depending on the methodology used (Mastenbroek et al., 2015). Additionally, the species variation between cells means that this model is unrepresentative of human atherothrombosis. In vitro studies are currently utilised to gain a deeper insight into the mechanisms of thrombus formation. Parallel-plate flow chambers coated with Type I collagen are used to assess platelet adhesion, activation and aggregation for individuals with various bleeding disorders (Brouns et al., 2018), however these models do not incorporate the dysfunctional matrices observed in plaque rupture and erosion and are devoid of endothelial cells.Recent studies investigating the matrices associated with atherosclerosis have shown that the ECM components in plaque rupture and erosion differ, as does the composition of thrombi associated with each plaque. Thrombi associated with plaque erosion appear platelet-rich, whereas rupture thrombi appear darker, containing more erythrocytes and fibrin (Otsuka et al., 2016). The composition and thrombogenicity of the matrix components in plaque rupture and erosion, and how this affects endothelial and smooth muscle cell function, as well as platelet responses is a novel area of research. Further investigations are needed to gain a deeper insight into arterial thrombosis in different plaque phenotypes. The development of lesion specific models would allow assessment of the efficacy of both existing and novel antithrombotic drugs in the different clinical scenarios, informing a more personalised approach to antithrombotic treatment.AimThe aim of this project is to create in vitro models of atherothrombosis, incorporating endothelial cells, smooth muscle cells, and the dysfunctional matrices associated with plaque rupture and erosion.Objectives1. Develop ECM composites representative of plaque rupture and erosion and determine their thrombogenicity.2. Examine the thrombogenicity of endothelial cells and smooth muscle cells cultured on the erosion and rupture ECM composites3. Develop and validate a method to focally ablate endothelial cells cultured on the different ECM composites in microfluidic chambers4. Evaluate and compare the efficacy of existing and novel antithrombotics using the final erosion and rupture modelsMethodsDevelopment of dysfunctional extracellular matricesExisting literature and current ongoing research in the cardiovascular research group at MMU will be used to characterise the matrix components in plaque rupture and erosion. Initial experiments will be performed on commercially available individual co