Cell-derived microparticles contain caspase 3 in vitro and in vivo.
BACKGROUND
Microparticles (MP) from endothelial cells (endothelial microparticles; EMP) circulate in disease states, but the processes such as apoptosis or cell activation underlying their release are unclear.


OBJECTIVE
We investigated whether adherent (viable) or detached (apoptotic) endothelial cells are the possible source of EMP in vitro, i.e. under control and interleukin (IL)-1alpha activation conditions, and in vivo.


METHODS
Adherent and detached endothelial cells, and EMP, were isolated from human umbilical vein endothelial cell cultures (n = 6), treated without or with IL-1alpha (5 ng mL(-1); 24 h). Cell fractions were analyzed by flow cytometry for annexin V binding, propidium iodide (PI) and caspase 3 staining (n = 3). Caspase 3 in EMP was studied using Western blot (n = 6) and flow cytometry (n = 6). Plasma from healthy subjects and systemic lupus erythematosus patients (both n = 3) were analyzed for caspase 3-containing (E)MP.


RESULTS
Detached but not adherent cells double-stained for annexin V and PI, confirming the apoptotic conditions of the detached cells and the viable nature of the adherent cells. Caspase 3 was solely present in the detached cells and procaspase 3 in the adherent cells. Caspase 3 was present in EMP from both control and IL-1alpha-treated cultures. Counts of EMP and detached cells, but not adherent cells, highly correlated (r = 0.959, P < 0.0001). In vivo circulating MP from nucleated (endothelial cells, monocytes) and anucleated cells (platelets, erythrocytes) contained caspase 3.


CONCLUSIONS
EMP contain caspase 3 and may be mainly derived from detached (apoptotic) endothelial cells in vitro. The presence of caspase 3 in MP from anucleated cell types, however, suggests that its presence may not necessarily be related to apoptosis in vivo but may be associated with caspase 3 activation unrelated to apoptosis.
Abid Hussein
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MN
Department of Clincial Chemistry, Academic Medical Center of the University of Amsterdam, Netherlands. m.n.abidhussein@amc.uva.nl
Nieuwland
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Hau
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Evers
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Meesters
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Sturk
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D017304:Annexin A5 / Q000494:pharmacology; D017209:Apoptosis; D001777:Blood Coagulation; D001792:Blood Platelets / Q000378:metabolism; D015153:Blotting, Western; D016022:Case-Control Studies; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism / Q000494:pharmacology / Q000502:physiology*; D002448:Cell Adhesion; D002470:Cell Survival; D002478:Cells, Cultured; D017077:Culture Media, Conditioned / Q000494:pharmacology; D042783:Endothelial Cells / Q000166:cytology*; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism; D004789:Enzyme Activation; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D066298:In Vitro Techniques; D007375:Interleukin-1 / Q000378:metabolism; D008180:Lupus Erythematosus, Systemic / Q000097:blood; D008833:Microcirculation; D008875:Middle Aged; D009389:Neovascularization, Pathologic; D015539:Platelet Activation; D011419:Propidium / Q000494:pharmacology; D014471:Umbilical Veins / Q000166:cytology
D017304:Annexin A5; D017077:Culture Media, Conditioned; D007375:Interleukin-1; D011419:Propidium; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases