Identification and characterisation of human endothelial progenitor cells in health and cardiac disease

Abstract

Endothelial progenitor cells (EPCs) offer promise for autologous revascularisation and as biomarkers of vascular health, but their identity is controversial and few studies have evaluated the significance of ‘late’ EPCs in patients with chronic ischaemic heart disease (CIHD). This study aimed to characterise putative EPC populations, primarily in adult peripheral blood (PB), from patients with severe CIHD (n=29) and non-ischaemic controls (n=20). We established methods for the in vitro detection of early and late outgrowth EPCs. MACS isolated CD133+ cells derived from adult PB and G-CSF-mobilised PB were also examined. Endothelial differentiation was assessed following cell culture in angiogenic media by flow cytometry, qRT-PCR, immunohistochemical staining, a Matrigel tube forming assay and uptake of acetylated low-density lipoprotein. CD133+ cells did not differentiate into endothelial cells (ECs) under a range ‘angiogenic’ growth conditions (n=20), but readily differentiated into haematopoietic lineages in methylcellulose colony-forming assays. Early EPCs also lacked endothelial differentiation potential and displayed monocytic characteristics. In contrast, late EPCs demonstrated robust endothelial differentiation potential. Cell sorting revealed that late EPCs reside in the CD34+/CD45- fraction of adult PB mononuclear cells. Late EPC cultures were effectively established from patients with severe IHD but were not distinguishable from those in controls. Examination of late EPC colony numbers revealed no difference between IHD and control subjects, however the number of late EPC colonies was a weak positive predictor of patient age in IHD (r2=0.22, p=0.046), but not control subjects. These findings indicate that late EPCs might be useful for autologous revacularisation therapies in patients with severe IHD. We also examined the number of early ‘EPCs’, as assessed by colony forming units. No difference between IHD and control EPC counts was observed, and colony counts did not correlate with major cardiac risk factors. These findings are supported by recent suggestions that early ‘EPC’ colonies do not reflect early EPC numbers and serve to highlight the pitfalls associated with EPC research. In conclusion, of the three putative EPC populations examined, only late EPCs display characteristics of bona fide EPCs because they readily differentiate into ECs. These findings highlight the complexity associated with the current field.