Cell‐derived microparticles contain caspase 3 in vitro and in vivo

• Published in: Journal of thrombosis and haemostasis Vol. 3; no. 5; pp. 888 - 896
• Main Authors: ABID HUSSEIN, M. N., NIEUWLAND, R., HAU, C. M., EVERS, L. M., MEESTERS, E. W., STURK, A.
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Science Inc 01.05.2005
• Subjects: Annexin A5 - pharmacology; Apoptosis; Blood Coagulation; Blood Platelets - metabolism; Blotting, Western; Case-Control Studies; Caspase 3; Caspases - metabolism; Caspases - pharmacology; Caspases - physiology; Cell Adhesion; Cell Survival; Cells, Cultured; Culture Media, Conditioned - pharmacology; endothelial cells; Endothelial Cells - cytology; Endothelium, Vascular - cytology; Endothelium, Vascular - metabolism; Enzyme Activation; Female; Flow Cytometry; Humans; HUVEC; In Vitro Techniques; Interleukin-1 - metabolism; interleukin‐1α; Lupus Erythematosus, Systemic - blood; Microcirculation; microparticle; Middle Aged; Neovascularization, Pathologic; Platelet Activation; Propidium - pharmacology; Umbilical Veins - cytology
• ISSN: 1538-7933; 1538-7836; 1538-7836
• DOI: 10.1111/j.1538-7836.2005.01240.x

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. Objectives: 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)‐1α 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‐1α (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‐1α‐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.