Loss of cyclin A and G1-cell cycle arrest are a prerequisite of ceramide-induced toxicity in human arterial endothelial cells

Background: Ceramide is an important messenger of TNF- and lipid-induced ap optosis. We previously demonstrated the adverse effect of TNF in the proces s of reendothelialization as well as the dependence of its effect on cell-c ycle regulation. The current study was designed to investigate the linkage between ceramide induced toxicity and growth arrest in human endothelial ce lls. Methods and results: Cultured human arterial endothelial cells (HAEC) served as an in-vitro model to test the cellular effects of C2-ceramide (C2 ). C2-induced cell death in HAECs occurred time- and dose-dependently. The LD50 in subconfluent cells was three times lower than in confluent cell lay ers (25 vs. 75 muM). C2 caused up to 70% inhibition of BrdU and [H-3]thymid ine incorporation at non-toxic concentrations as a result of G1 cell-cycle arrest. Downregulation of cyclin A and p21(Cip1/Waf1) protein expression wa s observed independently of C2-toxicity, while expression of other cell-cyc le regulatory genes was not affected. Inhibition of cyclin A protein expres sion by sequence-specific antisense-oligonucleotides was paralleled by sign ificant growth-inhibition. The protein phosphatase inhibitor okadaic acid i nduced endothelial cell proliferation, which was completely abrogated by C2 . In contrast, aphidicolin-synchronized endothelial cells demonstrated elev ated cyclin A levels along with 30% higher BrdU-incorporation and 70% less C2-toxicity. G1-arrested cells, however, showed significantly enhanced C2-t oxicity, lack of cyclin A expression and induction of uncleaved caspase-3 ( CPP32). Conclusions: Ceramide abrogates endothelial cell proliferation inde pendently of apoptosis or necrosis at low concentrations (less than or equa l to 10 muM) through loss of cyclin A expression with subsequent G1 cell-cy cle arrest. Synchronization of HAECs in S-phase with aphidicolin overcomes C2-induced G1-arrest and partially blocks ceramide toxicity. These findings demonstrate the dependence of ceramide toxicity on cell cycle regulation, suggesting a strong bidirectional relationship between cell-cycle control a nd cell death in vessel biology. (C) 2001 Elsevier Science B.V. All rights reserved.