title: Flow cytometric measurement of circulating endothelial cells: the effect of age and peripheral arterial disease on baseline levels of mature and progenitor populations.
abstract: 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.
authors.affiliation: Department of Medicine, Cardiovascular Division, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA.
authors.forename: Rebecca Gusic
authors.initials: RG
authors.lastname: Shaffer
authors.forename: Sam
authors.initials: S
authors.lastname: Greene
authors.forename: Arash
authors.initials: A
authors.lastname: Arshi
authors.forename: Greg
authors.initials: G
authors.lastname: Supple
authors.forename: Andrew
authors.initials: A
authors.lastname: Bantly
authors.forename: Jonni S
authors.initials: JS
authors.lastname: Moore
authors.forename: Emile R
authors.initials: ER
authors.lastname: Mohler
mesh_terms: D000071916:AC133 Antigen; D000328:Adult; D000368:Aged; D000375:Aging / Q000097:blood*; D015703:Antigens, CD / Q000032:analysis; D018952:Antigens, CD34 / Q000032:analysis; D015415:Biomarkers / Q000097:blood; D002452:Cell Count; D003114:Colony-Forming Units Assay; D042783:Endothelial Cells / Q000166:cytology* / Q000502:physiology; D005434:Flow Cytometry / Q000379:methods*; D006023:Glycoproteins / Q000032:analysis; D006801:Humans; D008875:Middle Aged; D010455:Peptides / Q000032:analysis; D016491:Peripheral Vascular Diseases / Q000097:blood*; D010641:Phenotype; D013234:Stem Cells / Q000166:cytology* / Q000502:physiology
chemical_list: D000071916:AC133 Antigen; D015703:Antigens, CD; D018952:Antigens, CD34; D015415:Biomarkers; D006023:Glycoproteins; C000606023:PROM1 protein, human; D010455:Peptides