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Abstract: FR-PO1066

Patient Derived Endothelial Cells as Tool to Study the Effects of Shiga Toxin on the Vascular Endothelium: The BO(ST)EC-Study

Session Information

Category: Glomerular Diseases

  • 1202 Glomerular Diseases: Immunology and Inflammation

Authors

  • Feitz, Wouter J.c., The Hospital for Sick Children, Toronto, Ontario, Canada
  • Van De Kar, Nicole, UMC St. Radboud Nymegen, Nijmegen, Netherlands
  • Licht, Christoph, The Hospital for Sick Children, Toronto, Ontario, Canada
Background

Hemolytic Uremic Syndrome (HUS) is a form of thrombotic microangiopathy (TMA), which is characterized by hemolytic anemia, thrombocytopenia and acute renal failure. HUS is caused by infections with Shiga toxin (Stx) producing Escherichia coli with subsequent damage of the vascular endothelium (STEC-HUS or eHUS). We have established an in vitrosystem to screen for endothelial susceptibility factors: using Blood Outgrowth Endothelial Cells (BOECs) we can study the function of the endothelium of individual eHUS patients. We hypothesize that endothelial characteristics determine the response to Stx and thus the manifestation of eHUS.

Methods

BOECs from a healthy donor were used (male, 50 years old). Experiments were done in threefold (N=3) or as indicated in the figure. Previous literature has shown that Stx preferentially binds to the endothelial Gb3 receptor. In our study, Vero cells (positive control) and BOECs were cultured. Immunofluorescence imaging of live cells was used to confirm presence or absence of the Gb3 receptor on the cell surface. Using confocal immunofluorescence microscopy, binding of Stx to the cell surface was determined. A lactate dehydrogenase (LDH) cytotoxicity assay was used to assess for cytotoxicity of Stx and to standardize Stx concentrations for future experiments aiming at a level of cell damage of 50%. With flow cytometry, using antibodies for Annexin V and PI, necrotic vs. apoptotic cell death in response to Stx incubation was quantified. Finally, the effect of Stx on endothelial cell motility was determined in a scratch wound assay.

Results

We found that: 1) the Gb3 receptor is present on the cell surface of BOECs; 2) there is an expected positive dose-response relationship, wherein a higher concentration of Stx resulted in increased cell damage. Pre-treatment with TNF-alpha 10 ng/ml for 24 hours and treatment with Stx type 2 10-5 ug/ml resulted in 50% cell death; 3) incubation of BOECs with Stx resulted in more apoptotic than necrotic cell death; and 4) endothelial wound healing was impaired when cells were incubated with Stx.

Conclusion

BOECs are a promising tool with high translational potential to study effects of Stx on the vascular endothelium.

Funding

  • Government Support - Non-U.S.