Rapamycin promotes endothelial\u2013mesenchymal transition during stress-induced premature senescence through the activation of autophagy

Norihiko Sasaki,Yoko Itakura,Masashi Toyoda

doi:10.1186/s12964-020-00533-w

Norihiko Sasaki, Yoko Itakura + Show 1 more

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https://doi.org/10.1186/s12964-020-00533-w

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Abstract

BackgroundRapamycin is known to be effective in suppressing senescence and the senescence-associated secretory phenotype (SASP). Therefore, it is highly expected to represent an anti-aging drug. Its anti-aging effect has been demonstrated at the mouse individual level. However, there are not many clinical findings with respect to its activity in humans. Here, we aimed to clarify the effect of rapamycin on human endothelial cells (ECs) as an in vitro model of human blood vessels.MethodsOver the course of oxidative stress-induced senescence using hydrogen peroxide, we examined the effect of rapamycin on human coronary artery ECs (HCAECs). Senescence was evaluated by detecting senescence-associated β-galactosidase (SA-β-Gal) activity and the real-time PCR analysis of p16INK4a. Furthermore, expression levels of SASP factors were examined by real-time PCR and the expression of senescence-related antigens, such as intercellular adhesion molecule-1 (ICAM-1) and ganglioside GM1, were examined by fluorescence-activated cell sorting analysis and immunostaining. The inhibitory effect of rapamycin on mTOR signaling was examined by immunoblotting. The adhesion of leukocytes to HCAECs was evaluated by adhesion assays. Endothelial–mesenchymal transition (EndMT) induced by rapamycin treatment was evaluated by real-time PCR analysis and immunostaining for EndMT markers. Finally, we checked the activation of autophagy by immunoblotting and examined its contribution to EndMT by using a specific inhibitor. Furthermore, we examined how the activation of autophagy influences TGF-β signaling by immunoblotting for Smad2/3 and Smad7.ResultsA decrease in SA-β-Gal activity and the suppression of SASP factors were observed in HCAECs undergoing stress-induced premature senescence (SIPS) after rapamycin treatment. In contrast, ICAM-1 and ganglioside GM1 were upregulated by rapamycin treatment. In addition, leukocyte adhesion to HCAECs was promoted by this treatment. In rapamycin-treated HCAECs, morphological changes and the promotion of EndMT were also observed. Furthermore, we found that autophagy activation induced by rapamycin treatment, which led to activation of the TGF-β pathway, contributed to EndMT induction.ConclusionsWe revealed that although rapamycin functions to inhibit senescence and suppress SASP in HCAECs undergoing SIPS, EndMT is induced due to the activation of autophagy.9enZn1zW9q18ZYaMrTcU4uVideo abstract

Highlights

Rapamycin is known to be effective in suppressing senescence and the senescence-associated secretory phenotype (SASP)
We reported that senescent Endothelial cell (EC) exhibit the increased expression of ganglioside GM1, a glycolipid that is localized to the cell membrane, and that increased GM1 contributes to vascular insulin resistance, which is considered to play an important role in the pathogenesis of vascular-related diseases including atherosclerosis [8,9,10]
SA-β-gal activity is inhibited by rapamycin in ECs undergoing stress-induced premature senescence (SIPS) The inhibition of mTOR complex 1 (mTORC1) by rapamycin occurs within minutes, whereas the inhibition of mTOR complex 2 (mTORC2) occurs after prolonged (> 24 h) treatment [35]

Summary

Introduction

Rapamycin is known to be effective in suppressing senescence and the senescence-associated secretory phenotype (SASP). It is important to clarify the mechanisms underlying senescence-associated diseases to lower the risk for vascular disease and extend healthy life expectancy. We reported that senescent ECs exhibit the increased expression of ganglioside GM1, a glycolipid that is localized to the cell membrane, and that increased GM1 contributes to vascular insulin resistance, which is considered to play an important role in the pathogenesis of vascular-related diseases including atherosclerosis [8,9,10]. It is considered that inhibiting the SASP in senescent ECs, which is effective for all vascular-related diseases in the body, is important

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