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

人类动脉粥样硬化的新型内皮化体外模型的开发和表征

• 批准号: 2817469
• 金额: --
• 依托单位: Manchester Metropolitan University
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2019
• 资助国家: 英国
• 起止时间: 2019 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2817469
• 关键词: Development; Characterisation; Novel; Endothelialised; vitro

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

背景动脉粥样硬化血栓形成是全球死亡的主要原因，是动脉粥样硬化病变破裂或糜烂的结果（Cate和Hemker，2016）。斑块组分暴露于循环血小板，其响应于血栓形成蛋白如血管性血友病因子（vWF）和胶原蛋白而粘附、活化和聚集（Olie et al.，2018年）。目前动脉粥样硬化血栓形成的治疗包括抗血小板治疗，其中阿司匹林和氯吡格雷的组合已显示将主要血管事件的风险降低超过10%（Patrono等人，2017），然而，有效性受到高个体间变异性的限制，并且12个月后复发事件/死亡的风险仍保持在20%左右（Olie et al.，从历史上看，对动脉粥样硬化血栓形成的研究主要集中在使用鼠体内模型，其中内皮被人为破坏，揭示了健康的细胞外基质（ECM）。已经通过使用Apoe-/-小鼠开发了更多的疾病相关模型，然而，有充分证据表明，小鼠中的动脉粥样硬化病变在大小、脂质核心和纤维帽方面在组织学上不同于人类斑块。如果不进行干预，它们也不太可能破裂。用于损伤血管壁的方法在研究之间不同，因此斑块破裂和血栓形成根据所使用的方法而不同（Mastenbroek等人，2015年）的报告。此外，细胞之间的物种差异意味着该模型不能代表人类动脉粥样硬化血栓形成。体外研究目前用于更深入地了解血栓形成的机制。使用涂有I型胶原的微孔板流动室来评估患有各种出血性疾病的个体的血小板粘附、活化和聚集（Brouns等人，2018），然而，这些模型不包括斑块破裂和糜烂中观察到的功能失调基质，并且缺乏内皮细胞。最近研究动脉粥样硬化相关基质的研究表明，斑块破裂和糜烂中的ECM成分不同，与每个斑块相关的血栓成分也不同。与斑块侵蚀相关的血栓看起来富含血小板，而破裂血栓看起来更暗，含有更多的红细胞和纤维蛋白（Otsuka等人，2016年）。斑块破裂和糜烂中基质成分的组成和致血栓性，以及这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现有的和新的抗血栓药物在不同的临床情况下的疗效评估，通知一个更个性化的方法，以抗血栓treatment.AimThe的目的，这个项目的目的是建立动脉粥样硬化血栓形成的体外模型，纳入内皮细胞，平滑肌细胞，和功能失调的基质与斑块破裂和糜烂。开发代表斑块破裂和侵蚀的ECM复合物，并确定其血栓形成性。检查在侵蚀和破裂ECM复合物上培养的内皮细胞和平滑肌细胞的血栓形成性3。开发并验证一种方法，以局部消融微流体室中不同ECM复合材料上培养的内皮细胞4。评价和比较现有的和新的抗血栓药物的疗效，使用最终的侵蚀和破裂modelsMethodsDevelopment的功能障碍的细胞外基质现有的文献和目前正在进行的研究，在MMU心血管研究组将被用来验证斑块破裂和侵蚀的基质成分。最初的实验将在商业上可获得的单个CO上进行。