TGF-β induced PI3K/AKT/mTOR pathway controls myofibroblast differentiation and secretory phenotype of valvular interstitial cells through the modulation of cellular senescence in a naturally occurring in vitro canine model of myxomatous mitral valve disease

Objectives: PI3K/AKT/mTOR signaling contributes to several cardiovascular
disorders. The aim of this study was to examine the PI3K/AKT/mTORpathwayin myxomatous mitral valve disease (MMVD).
Methods: Double-immunofluorescence examined expression of PI3K and TGF-β1in canine valves. Valve interstitial cells (VICs) from healthy or MMVDdogs were isolated and characterised. Healthy VICs were treated with TGF-β1 and SC-79 to induce activated myofibroblast phenotypes (aVICs). Diseased valve-derived aVICs
were treated with PI3K antagonists and expression of RPS6KB1 (encoding p70 S6K)
was modulated using siRNA and gene overexpression. SA-β-gal and TUNELstaining were used to identify cell senescence and apoptosis, and qPCR and ELISAto examine for senescence associated secretory phenotype (SASP). Protein immunoblotting was used to examine expression of phosphorylated and total proteins.
Results: TGF-β1 and PI3K are highly expressed in mitral valve tissues. Activationof
34 PI3K/AKT/mTOR and increased expression of TGF-β are found in aVICs. TGF-β35 transitions qVICs to aVICs by up-regulation of PI3K/AKT/mTOR. Antagonism of PI3K/AKT/mTOR reverses aVIC myofibroblast transition by inhibiting senescence and promoting autophagy. Up-regulation of mTOR/S6K induces transformation of senescent aVICs, with reduced capacity for apoptosis and autophagy. Selective knockdown of p70 S6K reverses cell transition by attenuating cell senescence, inhibiting apoptosis and improving autophagy.
Conclusions: TGF-β induced PI3K/AKT/mTOR signaling contributes to MMVD pathogenesis and plays crucial roles in regulation of myofibroblast differentiation, apoptosis, autophagy and senescence in MMVD.