Verocytotoxin-induced apoptosis of human microvascular endothelial cells.
The pathogenesis of the epidemic form of hemolytic uremic syndrome is characterized by endothelial cell damage. In this study, the role of apoptosis in verocytotoxin (VT)-mediated endothelial cell death in human glomerular microvascular endothelial cells (GMVEC), human umbilical vein endothelial cells, and foreskin microvascular endothelial cells (FMVEC) was investigated. VT induced apoptosis in GMVEC and human umbilical vein endothelial cells when the cells were prestimulated with the inflammatory mediator tumor necrosis factor-alpha (TNF-alpha). FMVEC displayed strong binding of VT and high susceptibility to VT under basal conditions, which made them suitable for the study of VT-induced apoptosis without TNF-alpha interference. On the basis of functional (flow cytometry and immunofluorescence microscopy using FITC-conjugated annexin V and propidium iodide), morphologic (transmission electron microscopy), and molecular (agarose gel electrophoresis of cellular DNA fragments) criteria, it was documented that VT induced programmed cell death in microvascular endothelial cells in a dose- and time-dependent manner. Furthermore, whereas partial inhibition of protein synthesis by VT was associated with a considerable number of apoptotic cells, comparable inhibition of protein synthesis by cycloheximide was not. This suggests that additional pathways, independent of protein synthesis inhibition, may be involved in VT-mediated apoptosis in microvascular endothelial cells. Specific inhibition of caspases by Ac-Asp-Glu-Val-Asp-CHO, but not by Ac-Tyr-Val-Ala-Asp-CHO, was accompanied by inhibition of VT-induced apoptosis in FMVEC and TNF-alpha-treated GMVEC. These data indicate that VT can induce apoptosis in human microvascular endothelial cells.
Department of Pediatrics, University Hospital, Nijmegen, The Netherlands.
Antonetta H J M
AHJM
Pijpers
Department of Pediatrics, University Hospital, Nijmegen, The Netherlands.|Gaubius Laboratory TNO Prevention and Health, Leiden, The Netherlands.
Petra A VAN
PAV
Setten
Department of Pediatrics, University Hospital, Nijmegen, The Netherlands.
Lambertus P W J VAN DEN
LPWJVD
Heuvel
Department of Pathology, University Hospital, Nijmegen, The Netherlands.
Karel J M
KJM
Assmann
Department of Pathology, University Hospital, Nijmegen, The Netherlands.
Hendrikus B P M
HBPM
Dijkman
Department of Hematology, University Hospital, Nijmegen, The Netherlands.
Arie H M
AHM
Pennings
Department of Pediatrics, University Hospital, Nijmegen, The Netherlands.
Leo A H
LAH
Monnens
Gaubius Laboratory TNO Prevention and Health, Leiden, The Netherlands.
Victor W M VAN
VWMV
Hinsbergh
D017209:Apoptosis / Q000187:drug effects; D061945:Caspase Inhibitors; D003513:Cycloheximide / Q000494:pharmacology; D053938:DNA Fragmentation; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects* / Q000502:physiology* / Q000648:ultrastructure; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007678:Kidney Glomerulus / Q000098:blood supply; D008833:Microcirculation / Q000187:drug effects; D008854:Microscopy, Electron; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D022622:Shiga Toxin 1 / Q000494:pharmacology*; D012867:Skin / Q000098:blood supply
D061945:Caspase Inhibitors; D004791:Enzyme Inhibitors; D011500:Protein Synthesis Inhibitors; D022622:Shiga Toxin 1; D003513:Cycloheximide