Circulating Apoptotic Endothelial Cells

To the Editor:

In their article published in the May 1, 1999 issue of Blood,Mutin et al1 find no circulating endothelial cells in their control group of patients and are unable to demonstrate activated or apoptotic endothelial cells. These results disagree with previously published data on the characteristics of circulating nucleated endothelial cells in normal healthy persons.2 3 

There are several possible reasons for the failure to identify apoptotic endothelial cells in this study. (1) Neither the antibody nor the methods used by the authors have ever been validated for their ability to reliably identify and retrieve apoptotic endothelial cells from peripheral blood. (2) The morphologic criteria used by the investigators for identification of endothelial cells after staining with acridin orange1 exclude cells with characteristics of apoptotic endothelial cells after such staining.4-6 (3) They use a kit for detection of apoptosis1 that fails to identify cells in early stages of apoptosis.7 (4) All patients are on aspirin, heparin, and a β-blocker.1Aspirin prevents activation of endothelial cells in vitro8and reduces endothelial detachment9 and numbers of circulating endothelial cells in vivo.10 It also stimulates ferritin production in endothelial cells,11 and iron chelation was shown to prevent apoptosis of these cells.12Heparin reduces endothelial cell detachment9 and numbers of circulating endothelial cells in vivo.13 It also activates the constitutive nitric oxide synthase in endothelial cells,14 an effect that prevents apoptosis in these cells.15 β-Blockers may reduce the influence of endogenous adrenergic stimulants that are known to increase the number of circulating endothelial cells.16 They also prevent the formation of angiotensin II,17 and the latter has a known ability to induce apoptosis in endothelial cells.18 

The use of poorly validated methods for detection and isolation of apoptotic cells, the exclusion of cells in early and late stages of apoptosis, and administration of antiapoptotic factors may have significantly influenced the results as reported by Mutin et al.1