Cell-derived Microparticles Contain Caspase 3 in Vitro and in Vivo

Abstract number: P0573

Abid Hussein¹ MN, Nieuwland¹ R, Hau¹ RCM, Evers¹ LM, Meesters² EW, Sturk¹ A

¹¹Academic Medical Center, Amsterdam, The Netherlands ¹¹Academic Medical Center, Amsterdam, The Netherlands ²²Slotervaart Hospital, Amsterdam, The Netherlands

Background:  Microparticles (MP) from endothelial cells (endothelial microparticles; EMP) circulate in disease states, but the processes underlying their release such as apoptosis or cell activation remain 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)-1a 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-1a (5 ng/mL; 24 h). Cell fractions were analyzed by flow cytometry for annexin V binding, propidium iodide (PI) and caspase 3 staining (n= 3), and by Western blot for the presence of (pro-) caspase 3. Caspase 3 in EMP was studied using Western blot (n= 6) and flow cytometry (n= 6). Plasma from healthy subjects and SLE 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 present in the detached cell fraction and in EMP from both control and IL-1a-treated cultures, but not in adherent cell fractions. Procaspase 3 was present only in the adherent cell fraction. 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.