Release of annexin V-binding membrane microparticles from cultured human umbilical vein endothelial cells after treatment with camptothecin.
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 microM 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 (approximately 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 microm 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.
Simák
Jan
J
Laboratory of Cellular Hematology, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892, USA. simak@cber.fda.gov
Holada
Karel
K
Vostal
Jaroslav G
JG
D017304:Annexin A5 / Q000378:metabolism*; D015703:Antigens, CD; D017209:Apoptosis / Q000187:drug effects; D018957:CD59 Antigens / Q000096:biosynthesis; D000001:Calcimycin / Q000494:pharmacology; D002166:Camptothecin / Q000494:pharmacology*; D002457:Cell Extracts / Q000737:chemistry; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D000071063:Endoglin; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D007249:Inflammation / Q000209:etiology / Q000378:metabolism; D007476:Ionophores / Q000494:pharmacology; D011485:Protein Binding; D011956:Receptors, Cell Surface; D014471:Umbilical Veins; D019010:Vascular Cell Adhesion Molecule-1 / Q000096:biosynthesis
D017304:Annexin A5; D015703:Antigens, CD; D018957:CD59 Antigens; D002457:Cell Extracts; C064206:ENG protein, human; D000071063:Endoglin; D007476:Ionophores; D011956:Receptors, Cell Surface; D019010:Vascular Cell Adhesion Molecule-1; D000001:Calcimycin; D002166:Camptothecin