The constituents and mechanisms of generation of 'endothelial cell - colony forming units'

Aims The formation of endothelial cell-colony forming units (EC-CFU) is increased by vascular injury, although their function in-vivo is unclear. We therefore examined the constituents of EC-CFU and the mechanisms of their generation.

Methods We performed immunohistochemical characterisation of EC-CFU and their mononuclear precursors. Using fluorescent activated cell sorting we evaluated the capacity of mononuclear subpopulations to generate EC-CFU, and monitored their migratory behavior when co-incubated with EC-CFU. Time-lapse microscopy was used to observe colony maturation. Cellular proliferation within EC-CFU was assessed using bromodeoxyuridine (BrdU) and anti-proliferative agents.

Results EC-CFU exhibited typical endothelial characteristics, however several endothelial markers were weakly expressed or absent. Macrophage and lymphocyte antigens were intensely expressed. EC-CFU 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 toward, 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-CFU directly, CD34+ cells could differentiate into CD14+ cells and potentiate EC-CFU formation.

Conclusions EC-CFU 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 leukocyte migration. Although confirmatory in-vivo studies are required, EC-CFU formation likely reflects leukocyte activation as a reparatory response to vascular denudation or tissue ischaemia.