Rapamycin promotes endothelial–mesenchymal transition during stress-induced premature senescence through the activation of autophagy

Abstract

Background: Rapamycin 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. Methods: Over 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 senescenceassociated β-galactosidase (SA-β-Gal) activity and the real-time PCR analysis of p16 INK4a. 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. Endothelialmesenchymal 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. Results: A 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.

Impaired ATG16L-Dependent Autophagy Promotes Renal Interstitial Fibrosis in Chronic Renal Graft Dysfunction Through Inducing EndMT by NF-κB Signal Pathway

“…Several studies suggested that autophagy inhibition could induce EMT and fibrosis by affecting the aberrant epithelial–fibroblast crosstalk in idiopathic pulmonary fibrosis ( 21 ). Others showed that Rapamycin promotes EndMT through the activation of autophagy during premature senescence ( 22 ). ATG16L gene mainly includes ATG16L1 and ATG16L2.…”

Sirtuin 3 governs autophagy‐dependent glycolysis during Angiotensin II‐induced endothelial‐to‐mesenchymal transition

“…10 Pharmacological inhibition of autophagy or small interfering RNA (siRNA) for ATG5-induced EndoMT. 4,11 Metabolic changes that rely on autophagy impairment drive endothelial phenotypic differentiation. In general, ECs tend to utilize aerobic glycolysis metabolism to survive, even under ample oxygen conditions.…”

“…Functionally, cells acquire myofibroblast‐like characteristics with contractile function, enhanced migratory phenotypes, and increased extracellular matrix production 10 . Pharmacological inhibition of autophagy or small interfering RNA (siRNA) for ATG5‐induced EndoMT 4,11 . Metabolic changes that rely on autophagy impairment drive endothelial phenotypic differentiation.…”

Cellular Senescence Affects Cardiac Regeneration and Repair in Ischemic Heart Disease

“…Rapamycin inhibits the SASP by downregulating IL-6, IL-8, monocytes, chemotactic proteins, etc. [ 74 , 169 ]. Flavonoid 4,4′dimethoxychalcone (DMC) is considered an antiaging compound that has regulate the SASPs effects in mice and promotes autophagy through a pathway that involves specific GATA transcription factors [ 170 ].…”