Development and characterisation of a novel, endothelialised in vitro model of human atherothrombosis

Drysdale, Amelia ORCID: https://orcid.org/0009-0002-4978-550X (2024) Development and characterisation of a novel, endothelialised in vitro model of human atherothrombosis. Doctoral thesis (PhD), Manchester Metropolitan University.

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Abstract

Atherothrombosis is a leading cause of mortality worldwide and occurs following rupture or erosion of an atherosclerotic plaque. Current treatments focus on the use of antiplatelet therapy, which reduces but does not eliminate the risk of major vascular events. Historically, research to identify and develop novel antithrombotics has depended heavily on the use of murine in vivo models, where the endothelium is artificially damaged, exposing a healthy extracellular matrix. The lack of disease relevance and species variation however, render these models unrepresentative of human atherothrombosis. In vitro studies which usually comprise of flow chambers coated with Type I collagen overcome some of these issues by using human blood and relevant haemodynamic conditions, however they do not incorporate the dysfunctional matrices observed in plaque rupture and erosion and are often devoid of endothelial cells. Recent studies investigating the matrices associated with atherosclerosis have shown that matrix components in plaque rupture and erosion differ, as does the composition of thrombi associated with each plaque. Thrombi associated with plaque rupture are often occlusive and appear more fibrin- and erythrocyte-rich. In contrast, erosion thrombi, are platelet-rich and less occlusive. The composition and thrombogenicity of the matrix components in plaque rupture and erosion, and how this affects endothelial cell function and platelet responses, is a novel area of research. The aim of this project was to develop lesion specific, in vitro flow models of atherothrombosis, incorporating endothelial cells and the dysfunctional matrices observed in plaque rupture and erosion. Firstly, recombinant disease-relevant matrices were established and incorporated into a flow model to investigate platelet responses to extracellular matrices associated with plaque rupture and erosion. It was observed that platelet thrombus formation and activation marker expression were significantly enhanced on Types I and III collagen compared with individual proteoglycans associated with plaque rupture and erosion. However, when added to collagen, vascular proteoglycans had a role in regulating thrombus formation on Type I collagen. To further explore the role of a multicomponent extracellular matrix in arterial thrombosis, native extracellular matrices, derived from endothelial and smooth muscle cells, were isolated and characterised. Proteomic analysis identified key differences between the composition of endothelial and smooth muscle cell matrices. Additionally, stimulating cells with common stimuli associated with cardiovascular disease, including cigarette smoke extract and TNF-α, significantly altered the composition and thrombotic properties of extracellular matrices generated from endothelial and smooth muscle cells. Prior to the incorporation of endothelial cells into the model, comparisons were performed between human umbilical vein endothelial cells (HUVECs) and human coronary artery endothelial cells (HCAECs) to determine the most appropriate cell type to use. Transcript analysis demonstrated similarities in up- and down-regulation of key markers in the regulation of thrombosis in response to inflammatory stimuli, but the resting gene levels of both cell types differed significantly, demonstrating heterogeneity in their capacity to regulate thrombosis. HCAECs were therefore taken forward as the most disease relevant cell type, and successfully incorporated into the flow model under arterial shear stress. Different types of focal damage were evaluated including ferric chloride, needle stick injury and cell dissociation buffer, to enable translation of current in vivo protocols and to create a model of plaque erosion. The final thrombosis model developed throughout the project incorporates vascular endothelial cells, a relevant shear stress and a disease-relevant extracellular matrix to enable investigations into arterial thrombosis as a result of plaque rupture or erosion. The findings from the project demonstrate that a multi-component extracellular matrix can regulate thrombus formation on Type I collagen, and that these components alter in response to different stimuli, demonstrating the importance of using a diseaserelevant matrix when performing thrombus formation assays and evaluating antiplatelet efficacy. Uptake of this novel human model of thrombosis as an alternative to current in vivo models has the potential to significantly reduce the number of animals used in research, while providing a more relevant model to human atherothrombosis.