Methotrexate causes apoptosis in postmitotic endothelial cells

Methotrexate (MTX) is a commonly used chemotherapy agent for a variety of cancers. However, therapeutic levels are associated with numerous untoward effects such as central nervous system damage in children with acute lymphoblastic leukemia. The purpose of this study was to determine if MTX caused injury to endothelial cells using cultured bovine pulmonary artery endothelial cells as a model. Light microscopy showed gaps between cells and reduced numbers of endothelial cells after exposure to MTX (10(-9) to 10(-5) M), a range consistent with therapeutic drug levels. Proliferation and viability of subconfluent and confluent MTX-treated endothelial cells were measured by colorimetric (MTS) assay. There was a significant decline in cell numbers in MTX-treated subconfluent (growing) cells cultured after 4 days of MTX exposure compared to controls, as expected. However, there was also an unexpected decline in cell numbers in MTX-treated postmitotic endothelial cells after 1, 3, and 4 days of drug exposure. This suggested that MTX induced endothelial cell death. Fluorescent ApoAlert Enhanced Annexin-V binding demonstrated apoptosis in endothelial cells after 1 day of MTX exposure. Apoptosis was confirmed by a DNA fragment assay. This is apparently the first report of MTX-induced apoptosis of postmitotic, cultured endothelial cells. The findings suggest that apoptosis may be one mechanism of MTX-induced injury to endothelial cells.