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Cardiovascular Research 2001 50(1):97-107; doi:10.1016/S0008-6363(01)00196-1
© 2001 by European Society of Cardiology
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Copyright © 2001, European Society of Cardiology

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

Ioakim Spyridopoulosa,*, Petra Mayera, Kerida S. Shooka, Dorothea I. Axela, Richard Viebahnb and Karl R. Karscha

aDepartment of Cardiology and Cardiovascular Research, Medizinische Klinik III, Otfried-Mueller-Str. 10, 72076 Tübingen, Germany
bDepartment of Surgery, University of Tübingen, Tübingen, Germany

* Corresponding author. Tel.: +49-7071-298-2711; fax: +49-7071-293-169 ioakim_s{at}hotmail.com

Background: Ceramide is an important messenger of TNF- and lipid-induced apoptosis. We previously demonstrated the adverse effect of TNF in the process of reendothelialization as well as the dependence of its effect on cell-cycle regulation. The current study was designed to investigate the linkage between ceramide induced toxicity and growth arrest in human endothelial cells. 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 layers (25 vs. 75 µM). C2 caused up to 70% inhibition of BrdU and [3H]thymidine incorporation at non-toxic concentrations as a result of G1 cell-cycle arrest. Downregulation of cyclin A and p21Cip1/Waf1 protein expression was observed independently of C2-toxicity, while expression of other cell-cycle regulatory genes was not affected. Inhibition of cyclin A protein expression by sequence-specific antisense-oligonucleotides was paralleled by significant growth-inhibition. The protein phosphatase inhibitor okadaic acid induced endothelial cell proliferation, which was completely abrogated by C2. In contrast, aphidicolin-synchronized endothelial cells demonstrated elevated cyclin A levels along with 30% higher BrdU-incorporation and 70% less C2-toxicity. G1-arrested cells, however, showed significantly enhanced C2-toxicity, lack of cyclin A expression and induction of uncleaved caspase-3 (CPP32). Conclusions: Ceramide abrogates endothelial cell proliferation independently of apoptosis or necrosis at low concentrations (≤10 µM) through loss of cyclin A expression with subsequent G1 cell-cycle 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 and cell death in vessel biology.

KEYWORDS C2, C2-ceramide (D-erythro-N-acetylsphingosine); C6, C6-ceramide (D-erythro-N-hexanoylsphingosine); HAEC, human arterial endothelial cells; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazolium; OA, okadaic acid; PI, propidium iodide; PMA, phorbol 12-myristate 13-acetate; PP2A, protein phosphatase 2A; SMase, sphingomyelinase; TNF, tumor-necrosis-factor {alpha}


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