Release of annexin V-binding membrane microparticles from cultured human umbilical vein endothelial cells after treatment with camptothecin -: art. no. 11

Background: Elevated plasma counts of endothelial micoparticles (MP) have been demonstrated in various diseases with a vascular injury component. We used flow cytometry to study the MP-release from cultured human umbilical vein endothelial cells HUVEC) stimulated by various agonists. MP-release by a topoisomerase I inhibitor camptothecin has been studied in detail. Results: Overnight stimulation of HUVEC with either LPS or TNFalpha, or 30 min stimulation with thrombin, phorbol-myristate-acetate, tissue plasminogen activator, or angiotensin-II did not cause a significant release of annexin V-binding MP. In contrast, induction of apoptosis with 5 muM camptothecin, documented by 60-70% desquamation of HUVEC culture, annexin V-binding to the cells and DNA-fragmentation, led to a release of annexin V-binding MP similar to80,000 MP/10(3) cells). This MP-release was prevented by Z-Val-Ala-Asp-fluoromethyl-ketone ZVAD). Lowe concentration of camptothecin 500 nM) induced comparable MP-release without loss of the culture confluence and without increase in annexin V-binding to the cells or DNA-fragmentation. Analyzed MP were free of nucleic acids and 95% of MP were 0.3-1 mum in size. Double-labeling flow cytometry assay showed that all annexin V-binding MP expressed CD59 but only approximately 50% of these also expressed CD105. Conclusions: Camptothecin treated HUVEC released different populations of annexin V-binding membrane MP at early stage after proapoptotic stimulation before detection of phosphatidyl serine exposure on the cells of DNA fragmentation. The MP-release was ZVAD sensitive but was not enhanced at the executive phase of apoptosis. These observations offer a new insight into MP-release as a make of endothelial stimulation and injury.