Oxidative stress mediates monocrotaline-induced alterations in tenascin expression in pulmonary artery endothelial...

Oxidative stress may be involved in monocrotaline (MCT)-induced endothelial cell injury and upregulation of extracellular matrix proteins in the pulmonary vasculature. To test this hypothesis, cytotoxicity, expression and distribution of tenascin (TN) as well as cellular oxidation were determined in porcine pulmonary artery endothelial cells (PAECs) exposed to MCT and/or to an oxygen radical scavenger, dimethylthiourea (DMTU). Relative to controls, treatment with 2.5 mM MCT for 24 hr produced cytotoxicity as evidenced by changes in cellular morphology, cell detachment, hypertrophy, reduction in cellular proliferation and severe cytoplasmic vacuolization. Parallel studies showed that MCT markedly altered the expression and distribution of TN in PAEC as determined by immunocytochemistry. Western analysis showed that MCT increased cellular TN content and promoted the appearance of an additional, smaller TN isoform. Northern analysis demonstrated an increase in the steady-state level of TN-specific mRNA in response to MCT treatment. Exposure to MCT also increased the synthesis of cell-associated and media-associated TN as determined by immunoprecipitation. In addition, MCT increased the intensity of cellular oxidative stress as measured by 2,7-dichlorofluorescein fluorescence. Co-treatment with DMTU prevented MCT-induced cytotoxicity, alterations in TN distribution and content, and reduced the increase in DCF fluorescence. These results suggest that MCT-induced cytotoxicity and upregulation of TN are mediated, at least in part, by induction of cellular oxidative stress.