R.X. Smith, Alex Langford-Smith, Tristan McKay, Stephen J White
Introduction Coronary heart disease (CHD) is the leading cause of mortality and morbidity globally. Atherosclerosis, a key underlying mechanism in the potentiation of CHD, is often defined as a chronic inflammatory disease provoked by the oxidation of lipids retained in arterial walls resulting in plaque formation. Plaque formations have been demonstrated to preferentially form at bifurcations and arcs exposed to disturbed blood flow patterns (athero-prone), whereas uniform sections exposed to laminar flow (athero-resistant) promote an athero-protective response, implicating the endothelium in the initiation of the disease. Various genes are known to regulate atherosclerosis development; NFκB and AP-1 (inflammatory cytokine response), IRF3 (toll-like receptor signalling), XBP1 (unfolded protein response/ER stress) and KLF2 and NRF2 (protective signalling pathways).
Methods Human coronary artery endothelial cells (HCAECs) were immortalised via lentiviral overexpression of anti-senescence polycomb protein, BMI1, which allowed for the extended proliferative lifespan of the primary cells, without affecting their morphology. Immortalised human coronary artery endothelial cells (iHCAECs) were then integrated within our parallel-plate flow apparatus, in which the response to varying flow conditions was observed.
Results From extensive published and unpublished data, the overexpression of BMI1 enables prolonged cell proliferation whilst inhibiting cell senescence. Utilising lentiviral overexpression, we were able to immortalise HCAEC with BMI1. Isolated iHCAEC clones were placed under varying hemodynamic conditions in order to screen their response. These iHCAEC clones lined up in conjunction with HCAEC counterparts demonstrating a continued mechanosensitivity. Current work investigating gene expression related to the differing hemodynamic environments is underway with full benchmarking against HCAEC controls in progress. TFAR gene constructs have been developed known to regulate atherosclerosis. The TFARs; NFκB, AP-1, IRF3, XBP1, KLF2 and NRF2 have been developed along with lentiviral vectors with secretable luciferase reporters (VLuc and NLuc) allowing for incorporation into validated iHCAECs. Validating these reporter constructs in response to ox-LDL, TNFα and cigarette smoke extract via luciferase activity measurement is currently underway, with selected constructs already cloned.
Conclusion Our initial results indicate the overexpression of BMI1 in HCAEC results in an extended lifespan and inhibition in cell senescence, with morphology unaffected. The resulting iHCAECs exhibit mechanosensitivity to a changing hemodynamic environment comparable to primary HCAECs. This highlights the advantages of this cell line for future CHD modelling, with its integration with our E-Sense system allowing for the development of a novel research tool and the potential to replace pre-existing methods.
Conflict of interest no
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