Release of annexin V-binding membrane microparticles from cultured human umbilical vein endothelial cells after treatment with camptothecin Export

@article{citeulike:1059755, abstract = {BACKGROUND:Elevated plasma counts of endothelial microparticles (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 microparticles (\~{}80,000 MP/103 cells). This microparticle-release was prevented by Z-Val-Ala-Asp-fluoromethyl-ketone (ZVAD). Lower concentration of camptothecin (500 nM) induced comparable microparticle-release without loss of the culture confluence and without increase in annexin V-binding to the cells or DNA-fragmentation. Analyzed microparticles were free of nucleic acids and 95 of microparticles were 0.3-1 mum in size. Double-labeling flow cytometry assay showed that all annexin V-binding Microparticles expressed CD59 but only approximately 50\% of these also expressed CD105.CONCLUSIONS:Camptothecin treated HUVEC released different populations of annexin V-binding membrane microparticles at early stage after proapoptotic stimulation before detection of phosphatidylserine exposure on the cells or DNA fragmentation. The microparticle-release was ZVAD sensitive but was not enhanced at the executive phase of apoptosis. These observations offer a new insight into microparticle-release as a marker of endothelial stimulation and injury.}, author = {Simak, Jan and Holada, Karel and Vostal, Jaroslav}, citeulike-article-id = {1059755}, citeulike-linkout-0 = {http://dx.doi.org/10.1186/1471-2121-3-11}, citeulike-linkout-1 = {http://view.ncbi.nlm.nih.gov/pubmed/12052248}, citeulike-linkout-2 = {http://www.hubmed.org/display.cgi?uids=12052248}, doi = {10.1186/1471-2121-3-11}, journal = {BMC Cell Biology}, keywords = {annexin\_v, apoptosis, facs, flow\_cytometry, huvec, microparticles}, number = {1}, posted-at = {2007-01-22 13:15:24}, priority = {0}, title = {Release of annexin V-binding membrane microparticles from cultured human umbilical vein endothelial cells after treatment with camptothecin}, url = {http://dx.doi.org/10.1186/1471-2121-3-11}, volume = {3}, year = {2002} }

TY - JOUR ID - citeulike:1059755 L3 - citeulike-article-id:1059755 N2 - BACKGROUND:Elevated plasma counts of endothelial microparticles (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 microparticles (~80,000 MP/103 cells). This microparticle-release was prevented by Z-Val-Ala-Asp-fluoromethyl-ketone (ZVAD). Lower concentration of camptothecin (500 nM) induced comparable microparticle-release without loss of the culture confluence and without increase in annexin V-binding to the cells or DNA-fragmentation. Analyzed microparticles were free of nucleic acids and 95 of microparticles were 0.3-1 mum in size. Double-labeling flow cytometry assay showed that all annexin V-binding Microparticles expressed CD59 but only approximately 50% of these also expressed CD105.CONCLUSIONS:Camptothecin treated HUVEC released different populations of annexin V-binding membrane microparticles at early stage after proapoptotic stimulation before detection of phosphatidylserine exposure on the cells or DNA fragmentation. The microparticle-release was ZVAD sensitive but was not enhanced at the executive phase of apoptosis. These observations offer a new insight into microparticle-release as a marker of endothelial stimulation and injury. IS - 1 JF - BMC Cell Biology TI - Release of annexin V-binding membrane microparticles from cultured human umbilical vein endothelial cells after treatment with camptothecin VL - 3 KW - annexin_v KW - apoptosis KW - facs KW - flow_cytometry KW - huvec KW - microparticles AU - Simak, Jan AU - Holada, Karel AU - Vostal, Jaroslav PY - 2002/// UR - http://dx.doi.org/10.1186/1471-2121-3-11 ER -