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

Background
Atherothrombosis 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.



Aim
The 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.

Objectives
1. 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 composites
3. Develop and validate a method to focally ablate endothelial cells cultured on the different ECM composites in microfluidic chambers
4. Evaluate and compare the efficacy of existing and novel antithrombotics using the final erosion and rupture models

Methods
Development of dysfunctional extracellular matrices
Existing 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