Circulating Angiogenic Cell Phenotyping

Angiogenic cells were enumerated by flow cytometer (LSRFortessa™ X20; BD Biosciences, USA) within 4 hours of blood draw. Whole blood collected in EDTA vacutainer (Becton, Dickinson and Company, UK) was incubated with anti-CD34 FITC, anti-VEGFR2 APC, anti-CD45 BV421, anti-CXCR4 APC-Cy7, and anti-CXCR7 PE (BioLegend, USA) in a Trucount™ tube (BD Biosciences, USA) with red blood cell lysis buffer (BD PharmLyse^TM, BD Biosciences, USA). Samples were analysed for 45 minutes or until 500,000 CD45⁺ events had been enumerated.
Flow cytometry files were analysed using FCS Express 7 (De Novo, California, USA). Counts of HPCs (CD34⁺) and EPCs (CD34⁺VEGFR2⁺) and subsequent cell surface expression of chemokine receptor 4 (CXCR4) and 7 (CXCR7) were converted to cells/mL using BD Trucount™, and adjusted for changes in blood volume [further methodology details available elsewhere (31 (link))]. Briefly, these phenotypes were chosen as low circulating CD34⁺ HPCs and CD34⁺VEGFR2⁺ counts are proven as risk factors to future adverse cardiovascular outcomes and death (25 (link), 27 (link)). Surface expression of CXCR4 and CXCR7 gives insight into how these cells respond to a stimuli such as exercise (31 (link)), characterising their ability to home to hypoxic environments, and may be a better predictor of mortality than HPCs and EPCs alone (27 (link)).