COLLEGE OF MEDICINE, BIOLOGICAL SCIENCES AND PSYCHOLOGY SCHOOL OF MEDICINE DEPARTMENT OF INFECTION, IMMUNITY & INFLAMMATION Microparticles as biomarkers of early changes leading to Cardiovascular disease in chronic kidney disease © Nima Abbasian This thesis submitted for the degree of Doctor of Philosophy at the University of Leicester- 2015 Abstract Microparticles as biomarkers of early changes leading to cardiovascular disease in chronic kidney disease Nima Abbasian Hyperphosphataemia in patients with advanced chronic kidney disease (CKD) is thought to be an important contributor to cardiovascular risk, in part because of endothelial cell (EC) dysfunction induced by inorganic phosphate (Pi). Such patients also have an elevated circulating concentration of pro-coagulant endothelial microparticles (MPs), leading to a pro-thrombotic state, which may contribute to acute occlusive events. It is hypothesised that hyperphosphataemia leads to MP formation from ECs via an elevation of intracellular Pi, which directly inhibits phosphoprotein phosphatases, triggering a global increase in phosphorylation and cytoskeletal changes. Using cultured human endothelial cells (EAhy926), incubation with elevated extracellular Pi (2.5mM) led to a rise in intracellular Pi concentration within 90min. This was mediated by PiT-1/slc20a1 Pi transporters; and led to global accumulation of Tyr- and Ser-Thr phosphorylated proteins, a marked increase in cellular Tropomyosin-3, plasma membrane blebbing and release of 0.1 – 1 micron diameter MPs. The effect of Pi was independent of oxidative stress or apoptosis. Similarly, global inhibition of phosphoprotein phosphatases with orthovandate or fluoride also yielded a global protein phosphorylation response and rapid release of MPs. The Pi-induced MPs expressed VE-cadherin and superficial phosphatidylserine, and in a thrombin generation assay were significantly more pro-coagulant than particles derived from cells incubated in medium with a physiological level of Pi (1mM). These data demonstrate a mechanism of Pi-induced cellular stress and signalling which may be widely applicable in mammalian cells; and in ECs provides a novel pathological link between hyperphosphataemia, generation of MPs and thrombotic risk. Keywords: hyperphosphataemia, chronic kidney disease, cardiovascular disease, endothelial cells, Cell Signaling, Microparticle i | P a g e Important note on the layout of this thesis The main scientific findings and the methods that were used to obtain them are presented in Chapters 2 to 5 inclusive of this thesis. Most of this material was submitted as a paper to Journal of the American Society of Nephrology and has been published (please see the Publications List below). Supplementary data, and additional detail on the methods are presented in the Appendix to the thesis. Publications, Presentations, Posters, & Academic Awards Publications Abbasian N, Burton JO, Herbert KE, Tregunna BE, Brown JR, Ghaderi-Najafabadi M, Brunskill NJ, Goodall AH, Bevington A. Hyperphosphataemia, phosphoprotein phosphatases and microparticle release in vascular endothelial cells. (Paper from this PhD project; J Am Soc Nephrol, 2015. doi: 10.1681/ASN.2014070642). Batool S, Abbasian N, Burton JO and Stover CM. Microparticles and their Roles in Inflammation: A Review. The Open Immunology Journal, 2013, 6, 1-14 Burton JO, Hamali HA, Singh R, Abbasian N, Parsons R, Patel AK, Goodall AH and Brunskill NJ. (2013) Elevated Levels of Procoagulant Plasma Microvesicles in Dialysis Patients. PLoS ONE 8(8): e72663.doi:10.1371/journal.pone.0072663 Abbasian N, Herbert KE, Bevington A, Brunskill NJ, and Burton JO. Hyperphosphataemia and microvesicle formation: a novel mechanism for cardiovascular risk in chronic kidney disease. Nephrology Dialysis Transplantation, vol. 27, suppl no. 2, pp. ii241, 2012 (Abstract) Singh R, Abbasian N, Stover CM, Brunskill NJ and Burton JO. Pro-Inflammatory and Pro-Coagulant Characteristics of Platelet Derived Microvesicles in Haemodialysis Patients. Nephrology Dialysis Transplantation, vol. 27, suppl no.2, pp. ii241, 2012 (Abstract) Oral presentations Abbasian N, Burton J, Herbert K, Goodall AH, Brunskill NJ, Bevington A. Endothelial inorganic phosphate signalling to Tropomyosin: A molecular basis for generation of acutely pro-coagulant endothelial microparticles in uraemic cardiovascular disease. (Renal research seminar- Department of Infection, Immunity & Inflammation, University of Leicester, UK, March 2015) ii | P a g e Abbasian N, Burton J, Herbert K, Brunskill NJ, Goodall AH, Bevington A. Hyperphosphataemia signalling and pro-coagulant microparticle release in human vascular endothelial cells. (M.V. Lomonosov Moscow State University, Moscow, Russia, March 2015) Abbasian N, Burton J, Herbert K, Brunskill NJ, Goodall AH, Bevington A. Hyperphosphataemia signalling and pro-coagulant microparticle release in human vascular endothelial cells. (University of Leicester Microvesicles Group Meeting, Department of Cardiovascular Sciences, Glenfield Hospital, University of Leicester, UK, September 2014) Abbasian N, Burton J, Herbert K, Goodall AH, Brunskill NJ, Bevington A. Endothelial inorganic phosphate signalling to Tropomyosin: A molecular bases for generation of acutely pro-coagulant endothelial microparticles in uraemic cardiovascular disease. (University of Leicester Microvesicles Group Meeting, Department of Cardiovascular Sciences, Glenfield Hospital, University of Leicester, UK, September 2014) Abbasian N, Burton JO, Herbert KE, Alison Goodall, Bevington A. Inorganic phosphate toxicity and cardiovascular death in chronic kidney disease: More than just rubble in the arteries. (Sixth Annual Postgraduate Student Conference, University of Leicester, UK, April 2013) Abbasian N, Burton JO, Herbert KE, Brunskill NJ, Bevington A. Hyperphosphataemia and microvesicle formation: a novel mechanism for cardiovascular risk in chronic kidney disease. (Department of Cardiovascular Sciences, University of Leicester, UK, February 2014) Abbasian N, Burton JO, Herbert KE, Brunskill NJ, Bevington A. Hyperphosphataemia and microvesicle formation: a novel mechanism for cardiovascular risk in chronic kidney disease. (Renal research seminar- Department of Infection, Immunity & Inflammation, University of Leicester, UK, January 2014) Abbasian N, Burton JO, Herbert KE, Brunskill NJ, Bevington A. Hyperphosphataemia and microvesicle formation: a novel mechanism for cardiovascular risk in chronic kidney disease. (Fifth Annual Postgraduate Student Conference, University of Leicester, UK, April 2013) Abbasian N, Burton JO, Herbert KE, Brunskill NJ, Bevington A. Hyperphosphataemia and microvesicle formation: a novel mechanism for cardiovascular risk in chronic kidney disease. (East Midlands Renal Research Showcase, Loughborough, UK, April 2012) Abbasian N, Burton J, Herbert K, Bevington A. Microparticles as biomarkers of early changes leading to cardiovascular disease in chronic kidney disease (CKD). (Department of Infection, Immunity, & Inflammation, University of Leicester, UK, February 2012) iii | P a g e Abbasian N, Burton J, Herbert K, Bevington A. Microparticles as biomarkers of early changes leading to cardiovascular disease in chronic kidney disease (CKD). (University of Leicester Microparticles Group Meeting, Department of Cardiovascular Sciences, Glenfield Hospital, University of Leicester, UK, February 2012) Abbasian N, Burton J, Herbert K, Bevington A. Microparticles as biomarkers of early changes leading to cardiovascular disease in chronic kidney disease (CKD). (Renal research seminar- Department of Infection, Immunity & Inflammation, University of Leicester, UK, April 2011) Abbasian N, Burton J, Herbert K, Bevington A. Microparticles as biomarkers of early changes leading to cardiovascular disease in chronic kidney disease (CKD). 15th April, 2011; (University of Leicester Microparticles Group Meeting, Department of Cardiovascular Sciences, Glenfield Hospital, University of Leicester, UK, April 2011) Posters Abbasian N, Burton JO, Herbert KE, Brunskill NJ, Bevington A. Microvesicle formation and hyperphosphataemia: a novel mechanism for cardiovascular risk in chronic kidney disease. (The Renal Association Conference, Newcastle, UK, June 2012) Singh R, Abbasian N, Stover CM, Brunskill NJ and Burton JO. Pro-Inflammatory and Pro-Coagulant Microvesicles: A Novel Mechanism for Cardiovascular Disease in Haemodialysis Patients. (Renal Association Conference, Newcastle, UK, June 2012) Abbasian N, Herbert KE, Bevington A, Brunskill NJ, and Burton JO. Hyperphosphataemia and microvesicle formation: a novel mechanism for cardiovascular risk in chronic kidney disease. (49th ERA-EDTA congress, Paris, France, May 2012) Singh R, Abbasian N, Stover CM, Brunskill NJ and Burton JO. Pro-Inflammatory and Pro-Coagulant Characteristics of Platelet Derived Microvesicles in Haemodialysis Patients. (49th ERA-EDTA Congress, Paris, France, May 2012) Singh R, Abbasian N, Stover CM, Brunskill NJ and Burton JO. Pro-inflammatory and Pro-coagulant Microvesicles: A Novel Mechanism for Cardiovascular Risk in Chronic Kidney Disease. (East Midlands Renal Research Showcase, Loughborough, UK, April 2012) Research highlight(s) Carney, Ellen F. "Chronic kidney disease: Procoagulant microparticles provide a novel pathogenic link between hyperphosphataemia and cardiovascular risk." Nature Reviews Nephrology. doi:10.1038/nrneph.2015.34, Published online 24 March 2015 (Research highlights) iv | P a g e Press Release: http://www2.le.ac.uk/offices/press/press-