Cardiovascular Research Advance Access [Accepted Manuscript] published online on February 4, 2008
Cardiovascular Research, doi:10.1093/cvr/cvn024
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IN HUMAN ENDOTHELIAL CELLS RAPAMYCIN CAUSES mTORC2 INHIBITION AND IMPAIRS CELL VIABILITY AND FUNCTION
1 Department of Experimental Medicine, Unit of General and Clinical Pathology, University of Parma, Italy
2 Department of Heart Surgery, University of Milano, Centro Cardiologico Fondazione Monzino I.R.C.C.S., Milano, Italy
3 Laboratory of Hematology and BMT Center, University of Parma, Italy
4 Department of Pharmacology, Johannes Gutenberg University, Mainz, Germany
Corresponding author: Dr. Valeria Dall'Asta Dipartimento di Medicina Sperimentale Università di Parma Via Volturno, 39 43100 Parma Italy Tel. +39 0521 033784 Fax +39 0521 033742 e-mail valeria.dallasta{at}unipr.it
Aim: Drug-eluting stents are widely used to prevent restenosis but are associated with late endothelial damage. To understand the basis for this effect, we have studied the consequences of a prolonged incubation with rapamycin on the viability and functions of endothelial cells.
Methods and Results: Human umbilical vein (HUVECs) or aorta (HAECs) endothelial cells were exposed to rapamycin in the absence or in the presence of tumour necrosis factor 
(TNF). After a 24h-incubation, rapamycin (100 nM) caused a significant cell loss associated with the increase of both apoptosis and necrosis, as quantified by propidium iodide staining, caspase 3 activity and lactate dehydrogenase (LDH) release. Rapamycin also impaired cell mobility, as assessed by a wound test, and promoted the formation of actin stress fibres, as determined with confocal microscopy. Moreover, the inhibitor prolonged TNF-dependent E-selectin induction, inhibited endothelial nitric oxide synthase (eNOS) expression at both mRNA (qRT-PCR) and protein level (ELISA and Western blot), and lowered bioactive nitric oxide output (RFL-6 reporter cell assay). Under the conditions adopted, rapamycin inhibited both mammalian target-of-rapamycin complexes (mTORC1 and mTORC2), as indicated by the reduced amount of raptor and rictor bound to mTOR in immunoprecipitates and by the marked hypophosphorylation of protein S6 kinase I (p70S6K) and Akt, determined by Western blotting. The selective inhibition of mTORC1 by AICAR did not affect endothelial viability.
Conclusions: A prolonged treatment with rapamycin impairs endothelial function and hinders cell viability. Endothelial damage seems dependent on mTORC2 inhibition.
KEYWORDS mTOR; apoptosis; TNFalpha; nitric oxide; restenosis
Time for primary review: 53
* The two first authors (AB and RV) contributed equally
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