Endothelial injury, an intriguing effect of methotrexate and cyclophosphamide during hematopoietic stem cell transplantation in mice.

OBJECTIVE: Elevated circulating endothelial cells (EC) in peripheral blood are an important indicator of endothelial damage and graft-versus-host disease (GVHD). However, the injured endothelial vasculature may in turn promote GVHD. In this study, we investigated whether methotrexate or cyclophosphamide, two conventional chemotherapeutic agents used in hematopoietic stem cell transplantation, caused endothelial injury and what were the functional consequences of this injury on GVHD.
METHODS: Six to 8-week-old female mice were randomly separated into three groups, including methotrexate (15 mg/m2 on day 1 and 10 mg/m2 on days 3, 6, and 11), cyclophosphamide (60 mg/kg; days 1, 2), and PBS saline alone (control). Circulating EC (CD31+CD133(-)CD45low) and the CD4+/CD8+ T lymphocytes in the peripheral blood were estimated by flow cytometry on days 1, 3, 5, 7, 9, 11, 16, 21, and 26. The morphologic changes of the endothelium were examined by phase contrast light microscopy to determine the integrity of the endothelial vasculature.
RESULTS: Elevated EC were detected at day 1 or 3 in mice receiving cyclophosphamide or methotrexate, respectively, with a peak increase at day 5 or day 3, respectively. The ratio of CD4+/CD8+ T lymphocytes showed a delayed increase in the peak to day 11 for both groups. In the cyclophosphamide group, there was significant apomorphosis with necrosis/thrombosis under light microscopy, whereas only apomorphosis was noticed in the methotrexate group. In both groups, EC showed hydropsia and cytomembrane damage.
CONCLUSIONS: Circulating EC increase during the early phases of cyclophosphamide or methotrexate conditioning, suggesting that both chemotherapeutic drugs induce endothelial damage, which occurs a little earlier than suppression of the immune system.