Flow cytometric measurement of circulating endothelial cells: the effect of age and peripheral arterial disease on baseline levels of mature and progenitor populations.

BACKGROUND: Age and cardiovascular disease status appear to alter numbers and function of circulating endothelial progenitor cells (EPCs). Despite no universal phenotypic definition, numerous studies have implicated progenitors with apparent endothelial potential in local responses to vascular injury and with cardiovascular disease in general. To further define the role of this lineage in peripheral artery disease (PAD), we developed a multiparameter flow cytometry assay to analyze multiple phenotypic definitions of progenitor cells (PCs), EPCs, and mature endothelial cells (ECs) and evaluate effects of age and PAD on baseline levels of each subset. METHODS: Blood was collected from young healthy subjects (N = 9, mean age 33 +/- 8 years), older healthy subjects (N = 13, mean age 66 +/- 8 years), and older subjects with PAD (N = 15, mean age 69 +/- 8 years). After ammonium chloride lysis, cells were stained and analyzed on a Becton-Dickinson LSR II with a 5-color antibody panel: FITC-anti-CD31, PE-anti-CD146, PE-anti-CD133, PerCP-Cy5.5-anti-CD3,-CD19,-CD33 (lineage panel), PE-Cy7-anti-CD34, and APC-anti-VEGF-R2. Viability was assessed by propidium iodide exclusion, and only viable, low to medium side scatter lineage-negative singlets were analyzed. In some studies, cells were sorted for morphological studies. Subsets were defined as indicated later. RESULTS: Our results, using a comprehensive flow cytometric panel, indicate that CD133+, CD34+, and CD133+/CD34+ PCs are elevated in younger healthy individuals compared to older individuals, both healthy and with PAD. However, the number of EPCs and mature ECs did not significantly differ among the three groups. Assessment of endothelial colony forming units and dual acLDL-lectin staining supported the flow cytometric findings. CONCLUSIONS: We describe a comprehensive flow cytometric method to detect circulating mature and progenitor endothelial populations confirmed by conventional morphological and functional assays. Our findings suggest that aging may influence circulating levels of PCs, but not EPCs or ECs; PAD had no effect on baseline levels of any populations investigated. This study provides the basis for evaluating the potential effects of acute stress and therapeutic intervention on circulating progenitor and endothelial populations as a biomarker for cardiovascular status.

Effect of acute exercise on endothelial progenitor cells in patients with peripheral arterial disease.

To determine whether exercise increases endothelial progenitor cells (EPCs) in patients with peripheral vascular disease, we developed a multi-parameter flow cytometry assay to rigorously assess EPCs and mature endothelial cells (ECs) in control subjects and patients with peripheral artery disease (PAD) subjected to graded exercise. Blood was collected from young healthy subjects (n = 9, mean age 33 years), older healthy subjects (n = 13, mean age 66 years), and older subjects with PAD (n = 15, mean age 69 years) before and 10 minutes after exercise. White blood cells were isolated and stained with a five-color antibody panel: FITC-anti-CD31, PE-anti-CD146, PE-anti-CD133, PerCP-Cy5.5-anti-CD3,-CD19,-CD33, PE-Cy7-anti-CD34, and APC-anti-VEGF-R2. Viability was assessed by propidium iodide exclusion. Viable, low, side scatter singlets that were CD3-, 19-, and 33-negative were counted. While baseline levels of EPCs and ECs were similar among all subjects, young healthy subjects demonstrated significantly greater (p < 0.05) levels of progenitor cells (PCs) than older healthy and PAD subjects. Levels of EPCs and ECs tended to increase in all subjects after exercise; however, increases in PCs were only observed in young healthy and PAD subjects. Further, trends in the magnitude of change of subsets with exercise were most similar between young and PAD subjects. Our findings suggest that aging may reduce baseline circulating levels of PCs, but not EPCs or ECs, and that exercise-induced mobilization of subsets may differ depending on age and presence of PAD.