A SIMPLIFIED IN-VITRO MODEL OF OXIDANT INJURY USING VASCULAR ENDOTHELIAL-CELLS

Oxidant injury of the vascular endothelium is considered an early even t 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 per oxide (H2O2) was developed using bovine pulmonary artery endothelial c ells (PAEC). Viability of PAEC grown in 96-well culture plates was det ermined 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 H2O2 with PAEC c aused 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 numbe r of samples. The fluorometric assay for measuring MDA production in e ndothelial cells used 6-well plates instead of 80-cm2 flasks employed by previous investigators. The use of multiwell culture plates in thes e 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 ce llular level.