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Canadian Journal of Cardiology

The Mammalian Target of Rapamycin Signalling Pathway Is Involved in Osteoblastic Differentiation of Vascular Smooth Muscle Cells

Published:November 11, 2013DOI:https://doi.org/10.1016/j.cjca.2013.11.005

      Abstract

      Background

      Vascular calcification is a major risk factor for cardiovascular diseases. Osteoblastic differentiation of vascular smooth muscle cells (VSMCs) is a key step in vascular calcification, but the molecular mechanisms driving the differentiation remain elusive. In this study, the involvement of mammalian target of rapamycin (mTOR) signalling in osteoblastic differentiation of VSMCs is investigated.

      Methods

      Calcification of VSMCs was induced in vitro using β-glycerophosphate (β-GP). Real-time polymerase chain reaction was used to measure messenger RNA (mRNA) expression, and Western blot was used to detect protein expression. Inhibition of mTOR expression was established by small interfering RNA (siRNA) and mTOR inhibitors.

      Results

      The model for osteoblastic differentiation of VSMCs was established in vitro by treating mouse VSMCs with 10 mM β-GP for 3-15 days. Overexpression of mTOR was observed in differentiated VSMCs. Downregulation of mTOR by siRNA or rapamycin significantly inhibited osteoblastic differentiation of VSMCs and decreased the expression and phosphorylation of mTOR and P70 ribosomal S6 kinase in a time- and concentration-dependent manner. Furthermore, adiponectin inhibited the mRNA and protein expression of mTOR in β-GP-treated VSMCs in a time- and concentration-dependent manner.

      Conclusions

      mTOR signalling plays a crucial role in the osteoblastic differentiation of VSMCs. Rapamycin and adiponectin might inhibit vascular calcification through regulation of the mTOR pathway.

      Résumé

      Introduction

      La calcification vasculaire est un facteur de risque majeur de maladies cardiovasculaires. La différenciation ostéoblastique des cellules musculaires lisses vasculaires (CMLV) est une étape essentielle à la calcification vasculaire, mais les mécanismes moléculaires soutenant la différenciation demeurent imprécis. Dans cette étude, la participation de la voie de signalisation de la cible de la rapamycine chez les mammifères (mTOR : mammalian target of rapamycin) à la différenciation ostéoblastique des CMLV est examinée.

      Méthodes

      La calcification des CMLV a été induite in vitro par la β-glycérophosphate (β-GP). La réaction en chaîne de la polymérase en temps réel a été utilisée pour mesurer l’expression de l’ARN messager (ARNm) et le buvardage de Western a été utilisé pour détecter l’expression de protéines. L’inhibition de l’expression de la mTOR a été établie par les inhibiteurs de l'ARN de petite taille (ARNsi) et de la mTOR.

      Résultats

      Le modèle de différenciation ostéoblastique des CMLV a été établi in vitro en traitant les CMLV de souris par 10 mmol de β-GP durant 3 à 15 jours. La surexpression de la mTOR a été observée dans les CMLV différenciées. La régulation à la baisse de la mTOR par l’ARNsi ou la rapamycine a significativement inhibé la différenciation ostéoblastique des CMLV, et a diminué l’expression et la phosphorylation de la mTOR et de la protéine kinase S6 ribosomique P70 selon le temps et la concentration. De plus, l’adiponectine a inhibé l’ARNm et l’expression de la protéine de la mTOR dans les CMLV traitées par la β-GP selon le temps et la concentration.

      Conclusions

      La voie de signalisation de la mTOR joue un rôle crucial dans la différenciation des CMLV. La rapamycine et l’adiponectine inhiberaient la calcification vasculaire par la régulation de la voie de la mTOR.
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