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Identification of suPAR cleaved products as potential new biomarkers of heart disease

A 2020/2021 Summer Studentship research project

Student: Fergus Allan
Supervisors: Dr Janice Chew-Harris, Christopher Pemberton
Sponsor: Heart Foundation NZ

Introduction

Introduction Cardiovascular disease is highly complex and is the leading cause of death in New Zealand. Recently, a multi-domain protein called soluble urokinase plasminogen activator receptor (suPAR), has been discovered to be highly expressed in the human atherosclerotic wall and to have a key role in cellular mechanisms that lead to plaque vulnerability. We have measured circulating suPAR in patients with cardiovascular disease. We have found suPAR to be highly predictive of 1-year death in patients presenting to hospital with acute breathlessness, and separately it could also predict long-term mortality in patients with acute chest pain. Although these initial results show promise, greater understanding of suPAR as a biomarker is warranted prior to application in clinical practice.

To date, little is known about the profiles of circulating suPAR in relation to heart disease progression. suPAR can exist in the full-length form consisting all domains of DI-DII-DIII or in the cleaved forms of DI and DII-DIII. These cleaved forms may be present to carry out distinct functions for compensatory mechanisms related to cardiovascular disease progression. Our preliminary work has further confirmed that these cleaved forms of suPAR exist in the circulation of patients with acute breathlessness, but measurement systems currently do not provide information regarding the abundance or differences in suPAR forms that may be present in circulation. The objective of this project is to extend on this work, and to uncover suPAR cleaved-forms in the human plasma of individuals after onset of acute coronary syndrome. This may identify a new class of biomarkers that is specific to cardiovascular disease.

Aim

This study will provide the first description of suPAR cleaved forms in heart disease. This may lead to a new class of blood markers that are more specific for heart disease to better guide patient management.

Method

suPAR cleaved products are expected to be present as either full length; suPARI-II-III, or suPARI and suPARII-III, represented by the following molecular weights (MW); 37-60kDa, ~10kDa and ~25kDa, respectively. We will investigate the presence of the various circulating suPAR forms in the regional samples of 30 patients undergoing clinically invasive procedures as part of their acute coronary syndrome management. In these patients, blood samples have been collected from the femoral, renal and hepatic veins, high inferior vena cava, internal jugular vein and pulmonary artery, with matching arterial samples (9 samples per patient, stored in the Christchurch Heart Institute’s Biobank).

Firstly, we will measure suPAR concentrations using the CE-marked ELISA (ViroGates suPARnostic ®). We will compare suPAR concentrations between samples to determine potential trans-organ differences so that any uptake or production across tissue beds can give important directions towards potential bioactivity and functions underlying biomarker efficacy. To detect the presence of suPAR forms in these samples, we will further perform immunoblotting experiments. Firstly, plasma samples of patients will be immunopurified using a polyclonal suPAR antibody immobilised to a resin column (AminoLink, ThermoFisher). Eluates will be collected for further Western Blot analyses (15% SDS-PAGE gels under reducing conditions, BIORAD Clarity Max Western ECL Substrates, complete with Molecular Weight markers), and detection will be conducted using separate antibodies to suPAR-I, suPAR-II and suPAR-III (Hycult Biotech and ThermoFisher). We will document the presence suPAR cleaved forms in these regional samples, and any transorgan venous and arterial marker concentrations showing differences will be compared using paired students t-test.

Student researcher’s component of the study

To perform immunopurification of study samples and Western blotting procedures.

Student prerequisites

Students with laboratory knowledge will be advantageous, although training will be provided by Dr Chew-Harris.