Verocytotoxin-induced apoptosis of human microvascular endothelial cells

The pathogenesis of the epidemic form of hemolytic uremic syndrome is chara cterized by endothelial cell damage. In this study, the role of apoptosis i n verocytotoxin (VT)mediated endothelial cell death in human glomerular mic rovascular endothelial cells (GMVEC), human umbilical vein endothelial cell s, and foreskin microvascular endothelial cells (FMVEC) was investigated. V T induced apoptosis in GMVEC and human umbilical vein endothelial cells whe n the cells were prestimulated with the inflammatory mediator tumor necrosi s factor-alpha (TNF-alpha). FMVEC displayed strong binding of VT and high s usceptibility to VT under basal conditions, which made them suitable for th e study of VT-induced apoptosis without TNF-alpha interference. On the basi s of functional (flow cytometry and immunofluorescence microscopy using FIT C-conjugated annexin V and propidium iodide), morphologic (transmission ele ctron microscopy), and molecular (agarose gel electrophoresis of cellular D NA fragments) criteria, it was documented that VT induced programmed cell d eath in microvascular endothelial cells in a dose- and time-dependent manne r. Furthermore, whereas partial inhibition of protein synthesis by VT was a ssociated with a considerable number of apoptotic cells, comparable inhibit ion of protein synthesis by cycloheximide was not. This suggests that addit ional pathways, independent of protein synthesis inhibition, may be involve d in VT-mediated apoptosis in microvascular endothelial cells. Specific inh ibition of caspases by Ac-Asp-Glu-Val-Asp-CHO, but not by Ac-Tyr-Val-Ala-As p-CHO, was accompanied by inhibition of VT-induced apoptosis in FMVEC and T NF-alpha -treated GMVEC. These data indicate that VT can induce apoptosis i n human microvascular endothelial cells.