Development of an in vitro model for vascular injury with human endothelial cells.
The aim of the present work was to establish an in vitro screening assay for drug candidates using human endothelial cells as a model for vascular injury after intravenous application. Different endpoints for viability and functionality of endothelial cells were investigated in human umbilical vein endothelial cells (HUVEC) and in immortalised human endothelial cells (IVEC). Cellular viability was determined by measuring ATP content and by the AlamarBlue assay. For comparison, the toxicity of the selected compounds was also tested in a murine fibroblast cell line (3T3 cells). Selected endpoints for endothelial cell-specific function were vascular permeability, determined by measurement of the transendothelial resistance and the diffusion of tracer molecules (FITC-dextran), and the release of prostaglandin and thromboxane as indicators for prothrombotic or vasoconstrictory action. Five compounds (cyclosporin A, mitomycin C, menadione, amrinone and rolipram) were selected due to their known effects on the vasculature. The cytotoxicity of all compounds was similar in endothelial and 3T3 cells. ATP content and AlamarBlue metabolism did not differ significantly except for amrinone. A dose-dependent decrease of transendothelial resistance and an increase in FITC-dextran permeability could be measured in HUVEC cells for the tested compounds, but the sensitivity was not higher than that of the cytotoxicity assays. Increased prostaglandin or thromboxane release was detected for all compounds at cytotoxic concentrations and for rolipram also at non-toxic concentrations. In conclusion, for a first ranking of drug candidates, cytotoxicity assays on any of the three cell types used are appropriate. For a more detailed characterisation of individual compounds, functional assays on HUVEC cells are proposed.
Schleger
Claudia
C
Boehringer Ingelheim Pharma GmbH & Co. KG, Department of Non-clinical Drug Safety, D-Biberach, Germany. claudia.schleger@bc.boehringeringelheim.com
Platz
Stefan J
SJ
Deschl
Ulrich
U
D016475:3T3 Cells; D000676:Amrinone / Q000633:toxicity; D000826:Animal Testing Alternatives; D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000633:toxicity; D000933:Antifibrinolytic Agents / Q000633:toxicity; D002463:Cell Membrane Permeability / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D016572:Cyclosporine / Q000633:toxicity; D004305:Dose-Response Relationship, Drug; D017097:Electric Impedance; D042783:Endothelial Cells / Q000187:drug effects*; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects*; D011464:Epoprostenol / Q000378:metabolism; D006801:Humans; D007166:Immunosuppressive Agents / Q000633:toxicity; D007275:Injections, Intravenous / Q000009:adverse effects; D051379:Mice; D016685:Mitomycin / Q000633:toxicity; D008954:Models, Biological; D010726:Phosphodiesterase Inhibitors / Q000633:toxicity; D020889:Rolipram / Q000633:toxicity; D013928:Thromboxane A2 / Q000378:metabolism; D014471:Umbilical Veins / Q000166:cytology / Q000187:drug effects; D024483:Vitamin K 3 / Q000633:toxicity
D000903:Antibiotics, Antineoplastic; D000933:Antifibrinolytic Agents; D007166:Immunosuppressive Agents; D010726:Phosphodiesterase Inhibitors; D016685:Mitomycin; D013928:Thromboxane A2; D024483:Vitamin K 3; D016572:Cyclosporine; D011464:Epoprostenol; D000676:Amrinone; D020889:Rolipram