title: The constituents and mechanisms of generation of 'endothelial cell--colony forming units'.
abstract: OBJECTIVE
  The formation of endothelial cell-colony forming units (EC-CFUs) is increased by vascular injury, although their function in vivo is unclear. We, therefore, examined the constituents of EC-CFUs and the mechanisms of their generation.
  
  
  RESULTS
  We performed immunohistochemical characterization of EC-CFUs and their mononuclear precursors. Using fluorescent-activated cell sorting, we evaluated the capacity of mononuclear subpopulations to generate EC-CFUs, and monitored their migratory behaviour when co-incubated with EC-CFUs. Time-lapse microscopy was used to observe colony maturation. Cellular proliferation within EC-CFUs was assessed using bromodeoxyuridine (BrdU) and anti-proliferative agents. EC-CFUs exhibited typical endothelial characteristics; however, several endothelial markers were weakly expressed or absent. Macrophage and lymphocyte antigens were intensely expressed. EC-CFUs readily incorporated BrdU, and failed to develop in the presence of anti-proliferative agents (P < 0.01; n = 12). Time-lapse microscopy demonstrated that the characteristic EC-CFU 'spindle cells' are not EC-CFU progeny, but are mononuclear cells migrating towards, and incorporating into colonies. Only CD14(+) monocytes were necessary for EC-CFU formation. CD14 expression was progressively down-regulated during colony maturation (P < 0.001; n = 6). Although unable to generate EC-CFUs directly, CD34(+) cells could differentiate into CD14(+) cells and potentiate EC-CFU formation.
  
  
  CONCLUSIONS
  EC-CFUs exhibit endothelial characteristics, but are predominantly CD14(+) derived macrophages and are a potent stimulus for lymphocyte migration. Proliferation is necessary for EC-CFU generation; however, colony growth also occurs as a result of leucocyte migration. Although confirmatory in vivo studies are required, EC-CFU formation likely reflects leucocyte activation as a reparatory response to vascular denudation or tissue ischaemia.
authors.affiliation: British Heart Foundation Centre for Cardiovascular Science, University of Edinburgh, Chancellor's Building, Edinburgh EH16 4SU, UK.
authors.forename: Gareth J
authors.initials: GJ
authors.lastname: Padfield
authors.forename: Abigail
authors.initials: A
authors.lastname: Short
authors.forename: Nicholas L
authors.initials: NL
authors.lastname: Mills
authors.forename: Kay
authors.initials: K
authors.lastname: Samuel
authors.forename: Marc
authors.initials: M
authors.lastname: Turner
authors.forename: David E
authors.initials: DE
authors.lastname: Newby
authors.forename: G Robin
authors.initials: GR
authors.lastname: Barclay
authors.forename: Olga
authors.initials: O
authors.lastname: Tura-Ceide
mesh_terms: D015703:Antigens, CD / Q000032:analysis; D018952:Antigens, CD34 / Q000032:analysis; D015415:Biomarkers; D002465:Cell Movement; D049109:Cell Proliferation; D000071063:Endoglin; D042783:Endothelial Cells / Q000166:cytology*; D006801:Humans; D018950:Lipopolysaccharide Receptors / Q000032:analysis; D008214:Lymphocytes / Q000502:physiology; D009000:Monocytes / Q000502:physiology; D011956:Receptors, Cell Surface / Q000032:analysis; D013234:Stem Cells / Q000502:physiology*
chemical_list: D015703:Antigens, CD; D018952:Antigens, CD34; D015415:Biomarkers; C064206:ENG protein, human; D000071063:Endoglin; D018950:Lipopolysaccharide Receptors; D011956:Receptors, Cell Surface
keywords: Cell migration; Endothelial colony forming units; Endothelial progenitor cells; Leucocytes; Proliferation