In human endothelial cells rapamycin causes mTORC2 inhibition and impairs cell viability and function

Amelia Barilli, Rossana Visigalli, Roberto Sala, Gian C. Gazzola, Alessandro Parolari, Elena Tremoli, Sabrina Bonomini, Alexandra Simon, Ellen I. Closs, Valeria Dall'Asta, Ovidio Bussolati

Research output: Contribution to journalArticle

Abstract

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 or aorta endothelial cells were exposed to rapamycin in the absence or in the presence of tumour necrosis factor α (TNFα). After a 24 h-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 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 TNFα-dependent E-selectin induction, inhibited endothelial nitric oxide synthase expression at both mRNA (quantitative real-time polymerase chain reaction) and protein level (enzyme-linked immunosorbent assay 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. Conclusion: A prolonged treatment with rapamycin impairs endothelial function and hinders cell viability. Endothelial damage seems dependent on mTORC2 inhibition.

Original languageEnglish
Pages (from-to)563-571
Number of pages9
JournalCardiovascular Research
Volume78
Issue number3
DOIs
Publication statusPublished - Jun 2008

Keywords

  • Apoptosis
  • mTOR
  • Nitric oxide
  • Restenosis
  • TNFalpha

ASJC Scopus subject areas

  • Cardiology and Cardiovascular Medicine

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    Barilli, A., Visigalli, R., Sala, R., Gazzola, G. C., Parolari, A., Tremoli, E., Bonomini, S., Simon, A., Closs, E. I., Dall'Asta, V., & Bussolati, O. (2008). In human endothelial cells rapamycin causes mTORC2 inhibition and impairs cell viability and function. Cardiovascular Research, 78(3), 563-571. https://doi.org/10.1093/cvr/cvn024