Development of an in vitro thrombosis model to assess endothelial regulation of thrombus formation

Riley, Ryan (2022) Development of an in vitro thrombosis model to assess endothelial regulation of thrombus formation. Doctoral thesis (PhD), Manchester Metropolitan University.

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Abstract

Arterial thrombosis culminating in myocardial infarction and stroke remains one of the leading causes of death and disability in the western world. Both in vitro and in vivo thrombosis models have been beneficial tools in furthering our understanding of arterial thrombosis. However, these models are not without limitations, as a result there is not currently a gold standard thrombosis model. Endothelial cells play a key role in regulating platelet activation and thrombus formation, through the activity of endothelial derived platelet inhibitors such as nitric oxide, prostacyclin and CD39. Despite the clear role of endothelial cells in thrombus formation many in vitro thrombosis models lack endothelial cells. These models therefore do not consider the role of the endothelium and endothelial dysfunction in thrombus formation which in turn, inhibits their ability to accurately model atherothrombosis. The aim of this project was to develop a model which incorporates endothelial cells and allows for the assessment of endothelial cell regulation of thrombus formation. Utilising a basic microfluidic model set up, it was successfully demonstrated that incubation of endothelial derived platelet mediators in human whole blood had significant effect on thrombus formation on exposed collagen. Next, endothelial cells were incorporated into the model, the endothelialised model consisted of two separate compartments one lined with endothelial cells, upstream of a second compartment coated with collagen. This novel model setup allowed for the assessment of the effects of endothelial derived mediators on thrombus formation. Incorporation of endothelial cells into the model significantly reduced thrombus formation, thus supporting the hypothesis that endothelial cells should be incorporated into thrombosis models. Accurate replication of the proinflammatory atherothrombotic vessel environment observed in cardiovascular disease was also a key component that needed to be incorporated into the model. TNF-alpha stimulation resulted in an increase in cell adhesion molecules, in addition to a decrease in endothelial nitric oxide synthase phosphorylation. Furthermore, TNF-alpha stimulation of endothelial cells incorporated into the model lead to the abolishment of the previous anti-thrombotic effects observed with endothelial cell incorporation. This confirmed the sensitivity of the model to changes in the endothelial cell compartment, in addition to showing TNF-alpha stimulation could achieve a prothrombotic environment. Optimisation of the model was important to enable a higher throughput and to allow multiple experimental conditions to be modelled simultaneously. The utilisation of a six-syringe pump enabled this optimisation step and enabled the assessment of anti-platelet drug efficacy in the presence of healthy and activated endothelial cells. Further optimisation compared differences between endothelial cells from different vessel beds and demonstrated a significant difference in the levels of platelet mediators in venous and arterial endothelial cells. Finally, the model was used to investigate the ACE2/ANG1-7/MAS signalling axis as a potentially novel anti-thrombotic pathway. The unique dual channel setup, with an endothelial cell channel upstream of a collagen channel, enabled the analysis of the effects of ACE2/ANG1-7/MAS stimulation on endothelial cell regulation of thrombus formation. No significant effects on thrombus formation were observed following ACE2/ANG1-7/MAS stimulation; further studies are required to evaluate the anti-thrombotic effects of this pathway. In conclusion, an in vitro thrombosis model was successfully developed during this project, which included the incorporation of endothelial cells and allowed for the assessment of endothelial cell regulation of platelet activity. It was successfully demonstrated that the model was sensitive to changes in the endothelial compartment both in terms of response to cytokine stimulation and the incorporation of endothelial cells from different vessel beds. In addition, the model’s ability to assess anti-platelet drug efficacy, in the presence of a healthy or activated endothelium was also successfully demonstrated.