Introduction: Ischaemic heart disease (IHD) is the leading cause of death in Australia. Its onset is due to abnormal pathology such as blockages or narrowing in the arteries resulting in poor perfusion. This is usually caused by atherosclerotic plaque development and can result in a myocardial infarction (MI). Through exploration of molecular pathways, we hope to identify novel biomarkers and/or therapies for cardiovascular disease (CVD). I have approached this by utilising patient-derived EPCs from the BioHEART study for molecular and functional characterisation.
1. To develop methodology to isolate EPCs from participants in the BioHEART study.
2. To functionally characterise EPCs and compare them to standard vascular endothelial cells.
3. To collate molecular and functional data from EPCs with participant coronary artery disease (CAD) and CVD risk profile.
Methods: Patients were recruited to the BioHEART study at North Shore Private Hospital, and data was collected on age, sex, medication and cardiovascular risk factors. Blood samples were collected in heparin-coated vacuettes and peripheral blood mononuclear cells were extracted via Ficoll gradient centrifugation and cultured in 0.1% gelatin-coasted flasks in endothelial cell growth medium. Following the emergence of EPCs, cells were passaged 3 times and cryopreserved. Cells were stained with fluorescent tagged antibodies and surface marker expression was detected through flow cytometry. Angiogenesis was assessed by migration and tubule formation assays. Redox signalling was assessed by superoxide generation in live cells and expression of reactive oxygen species (ROS) and other proteins by immunoblotting.
Results: The spontaneous development of EPCs was assessed in BioHEART participants. Body mass index (BMI) of participants affected the capacity for EPCs to develop in culture. Samples where EPCs spontaneously appeared came from patients with BMI of 25.9 ± 0.3, (n=215) whereas samples where EPCs failed to grow were from patients with BMI 30.6 ± 0.4, (n=728; p<0.0001). Having a BMI ≥ 30, diabetes and taking beta-blockers negatively affected growth, while taking statins positively contributed to growth. EPCs had an expression and functional profile similar to human umbilical vein endothelial cells (HuVECs). NADPH oxidase (Nox)-2 protein expression was increased two-fold in EPCs of participants that were classified to have CVD based on their coronary calcification measure by computer tomography (n=13-14, p<0.05). Interestingly, I observed disparity in between EPCs extracted from males and females. In females with CVD there was elevated eNOS expression in comparison to EPCs from healthy females, but this was not evident in male EPCs. This correlated with increased tubule formation in females with CVD but not males. However, I found that migration of EPCs were not affected by CVD in females, yet slightly accelerated in males. These functional changes in capacity for angiogenesis were seemingly not caused by the common angiogenesis signalling pathways, as expression and phosphorylation of AKT and ERK were not altered in EPCs from CVD patients.
Conclusion: Patient-derived EPCs have characteristics similar to vascular endothelial cells and are a robust source of molecular information relevant to CVD. They may be useful in predicting disease and/or developing personalised therapies in the future.