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.

Detection of verocytotoxin bound to circulating polymorphonuclear leukocytes of patients with hemolytic uremic syndrome.

The epidemic form of hemolytic uremic syndrome (HUS) is the most common cause of acute renal failure in children and is characterized by a prodromal phase of sometimes bloody diarrhea. The role of verocytotoxin (VT)-producing Escherichia coli has been strongly implicated. Although antibodies against VT have been detected in the serum of patients with HUS, VT itself has never been detected in circulating blood. In this study, VT-2 was detected in the systemic circulation in 9 of 10 patients with the epidemic form of HUS. In those cases, VT-2 was bound exclusively to polymorphonuclear leukocytes (PMN). The detection of VT-2 bound to PMN was associated with the presence of diarrhea at the time the blood samples were obtained. The one patient for whom VT was not detected presented with atypical HUS. For 5 of the 10 patients with HUS who were studied, the time course of VT binding was analyzed; binding decreased in four patients. The finding of VT bound to PMN in the systemic circulation of patients with HUS is important for a clearer understanding of the pathogenesis of HUS and suggests new approaches for treatment in the future.