Cardiovascular disease causes more than a quarter of all deaths in the UK, costing the health service £11 billion each year. Atherosclerosis, the underlying mechanism potentiating Coronary Heart Disease, develops focally at regions of disturbed (athero-prone) flow within coronary arteries and not within regions that experience laminar (athero-resistant) flow, clearly implicating the endothelium in the initiation of disease. Athero-prone flow activates the endothelium, priming them for inflammation, increasing permeability and leukocyte trafficking. The PREDICTION study, which used intravascular imaging to study disease development in patients with coronary heart disease, demonstrated that atherosclerotic plaques progressed in areas of low endothelial wall shear stress. Furthermore, 30% of heart attacks are caused by endothelial erosion of plaques, where the endothelium detaches, precipitating thrombus formation and arterial occlusion. Therefore, the endothelium and its response to flow and cardiovascular risk factors, plays a significant contribution to initiation, progression and clinical presentation of coronary heart disease. This suggests that creation of an endothelial-based in vitro detection system (E-Sense) will provide an ideal platform for detecting agents that cause vascular harm and promote atherosclerosis and coronary heart disease.
To replace the use of animal models of cardiovascular disease for screening compounds that might contribute to coronary heart disease, we will develop a scalable modular endothelial-based system to monitor key aspects of cell function, known to be involved in atherosclerosis. These include inflammation, cellular stress and downregulation of protective pathways. This will be performed in immortalised human coronary artery endothelial cells, using our existing Transcription Factor Activated Reporter (TFAR) gene cassettes. The E-sense system makes use of key transcription factors that assimilate multiple cellular signals. This allows accurate quantification of the cellular response to agents potentially involved in atherosclerosis, while concomitantly allowing optimisation of targeted therapeutic strategies.
The E-sense system will make use of our existing custom-made parallel plate flow apparatus (capable of running 20 independent flow cells at a time), to culture the engineered E-Sense iHCAEC lines under disturbed (athero-prone) flow and normal (athero-protective) flow for 24 hours to adapt cells to their flow environment and measure baseline TFAR measurement of VLuc and NLuc in the circulating medium (CM). Addition of oxidised LDL, TNFα, or cigarette smoke extract to the CM (dose escalation, with 3 doses, 24 hours apart, with measurement of VLuc and NLuc every 12 hours) will validate the cell assay against published and our unpublished data sets. Parallel development of the flow system will deliver a semi-automated in vitro platform for medium to high throughput screening.
This system has several advantages of screening compounds in hypercholesterolemic mice. It uses cells to detect the toxic dose, allowing the generation of accurate dose response curves and valuable information to suggest maximal environmental exposure limits. The TFARs have a large dynamic range and are very sensitive to subtle changes in activation. The TFARs also identify the molecular basis of cellular toxicity, from activation of pro-inflammatory, or cellular stress pathways to inhibition of protective pathways. The modular nature of the system allows continued expansion if required. When fully developed and validated, E-Sense will replace the need for animal testing of compounds for vascular harm. This will be especially useful for rapid screening of novel agents, including potential new risk factors (e.g. components of energy drinks, dietary supplements, electronic cigarette additives, ‘legal highs’ etc.) to screen for indications of vascular damage and alert health authorities to emerging risk.
This Studentship was co-awarded with the British Heart Foundation (BHF).