A Simplified in vitro Model of Oxidant Injury Using Vascular Endothelial Cells

• Published in: In Vitro Cellular & Developmental Biology - Animal 1993-07, Vol.29A (7), p.531-536
• Main Authors: Li, Lin, Benjamin H. S. Lau
• Format: Article
• Language: English
• Subjects: Animals; Antioxidants; Biological and medical sciences; Cattle; Cell Membrane - metabolism; Cell Membrane - physiology; Cell Membrane - ultrastructure; Cell membranes; Cell Survival - drug effects; Cells, Cultured; Cellular and Molecular Toxicology; Colorimetry; Cultured cells; Diverse techniques; Dose-Response Relationship, Drug; Endothelial cells; Endothelium, Vascular - cytology; Endothelium, Vascular - drug effects; Endothelium, Vascular - ultrastructure; Flasks; Fluorometry; Fundamental and applied biological sciences. Psychology; Hydrogen; Hydrogen Peroxide - pharmacology; L-Lactate Dehydrogenase - analysis; L-Lactate Dehydrogenase - metabolism; Lipid Peroxidation; Lipids; Malondialdehyde - analysis; Malondialdehyde - metabolism; Models, Biological; Molecular and cellular biology; Oxidants - pharmacology; Physical trauma; Pulmonary Artery - cytology; Tetrazolium Salts; Ungulates; Viability
• ISSN: 1071-2690; 0883-8364; 1543-706X; 2327-431X
• DOI: 10.1007/BF02634146

Oxidant injury of the vascular endothelium is considered an early event in the pathogenesis of atherosclerosis. The model of oxidant injury is crucial to the investigation of antioxidants. In the present study, a convenient in vitro model of oxidant injury induced by hydrogen peroxide (H2O 2) was developed using bovine pulmonary artery endothelial cells (PAEC). Viability of PAEC grown in 96-well culture plates was determined with methylthiazol tetrazolium (MTT) colorimetric assay. Cell membrane integrity was measured by lactate dehydrogenase (LDH) release from PAEC grown in 24-well plates. Malondialdehyde (MDA, a product of lipid peroxidation) in PAEC grown in 6-well plates was detected by a thiobarbituric acid fluorometric assay. Incubation of H2O 2with PAEC caused a dose-dependent decrease of cell viability, an increase of LDH release, and an elevation of MDA production. MTT assay was convenient, quantitative, non-radioactive, and suitable for testing a large number of samples. The fluorometric assay for measuring MDA production in endothelial cells used 6-well plates instead of$80-cm^2$flasks employed by previous investigators. The use of multiwell culture plates in these assays made it possible for more samples to be tested in any single experiment. The three assays are reproducible with low intraplate and interplate coefficients of variation. This in vitro model is suitable for screening antioxidants and for studying pharmacodynamics at the cellular level.