Investigating endothelial cell pim kinase as a novel anti-thrombotic target

Karim, Eima (2023) Investigating endothelial cell pim kinase as a novel anti-thrombotic target. Masters by Research thesis (MSc), Manchester Metropolitan University.

Preview

Available under License Creative Commons Attribution Non-commercial No Derivatives. Download (1MB) | Preview

Abstract

Cardiovascular disease is the most common cause of mortality worldwide, presenting a significant burden on healthcare costs globally. Atherothrombosis, the development of an occlusive clot in an artery, is triggered by atherosclerotic plaque rupture/erosion, and involves the interaction of multiple cell types in the blood and vasculature, with endothelial cells and platelets playing significant roles. Blood clots formed during thrombotic events are rich in platelets, making them a suitable target for anti-thrombotic therapies. However, the most widely prescribed anti-platelet drugs for arterial thrombosis prevention, aspirin and clopidogrel, only provide ~20% protection against cardiovascular disease related events. Simultaneously targeting platelets and the endothelium could provide an effective novel anti-thrombotic therapeutic approach. Pim kinase, a family of serine/threonine kinases, have shown to modulate platelet function, and whilst their expression is confirmed in endothelial cells, their role in the endothelium remains unknown. This project aimed to investigate the role of Pim kinase in regulating the inflammatory pathways involved in endothelial cell control of thrombus formation. The role for Pim kinase in endothelial cells in response to cigarette smoke extract and Tumour Necrosis Factor alpha, initiators of endothelial cell damage, was determined using human umbilical vein endothelial cells as a model platform of endothelial cell function. Human umbilical vein endothelial cells were treated for 24 hours with cigarette smoke extract and/or Tumour Necrosis Factor alpha +/- pan Pim kinase inhibitor AZD1208, and techniques including enzyme-linked immunosorbent assay, fluorescence microscopy, qPCR, and Western Blotting used to monitor gene and protein expression of Pim kinases and mediators of thrombo-inflammation. mRNA expression of all three Pim kinase isoforms, and protein expression of Pim-1 was confirmed. Human umbilical vein endothelial cells treated with cigarette smoke extract and Tumour Necrosis Factor alpha combined demonstrated a decrease in endothelial nitric oxide synthase levels, a protective mediator of cardiovascular homeostasis. Human umbilical vein endothelial cells treated with AZD1208 demonstrated a decrease in the expression of von Willebrand factor, a pro-coagulant mediator, and release of inflammatory markers, Interleukin-6, and Page | 7 Interleukin-8 were observed. Collectively, these findings identify a potential role for Pim kinase in atherothrombosis and indicate that Pim kinase inhibitors could be repurposed for use alongside other anti-thrombotic agents for the prevention of cardiovascular-related events.