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Regulatory Role of Ceramide/mTOR Signaling in Exosome Secretion from Smooth Muscle Cells during Arterial Stiffening and Medial Calcification
Author(s) -
Bhat Owais M.,
Yuan Xinxu,
Li Guangbi,
Camus Sarah,
Kukreja Rakesh C.,
Gulbins Erich,
Li Pin-Lan
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.06477
Subject(s) - colocalization , osteopontin , lysosome , exosome , pi3k/akt/mtor pathway , microbiology and biotechnology , calcification , chemistry , endocrinology , medicine , microvesicles , biology , signal transduction , biochemistry , microrna , gene , enzyme
Increased secretion of exosomes from arterial smooth muscle cells (SMCs) has been shown to contribute to arterial medial calcification (AMC) associated with accumulation of calcium deposits in the arterial wall and osteoblastic differentiation of SMCs. In this study, we further explored the molecular mechanisms increasing exosome secretion during AMC, focusing on lysosomal‐ceramide/mTOR (mammalian target of rapamycin) signaling in the regulation of lysosome trafficking and exosome release. Smooth muscle‐specific lysosomal acid ceramidase (Ac) gene knock out ( Asah1 fl/fl /SM cre ) mice were treated with a high dose of Vit D (500,000 IU/kg/day) for 4 days. By Alizarin Red S and Von Kossa staining, we observed significantly increased aortic medial calcification with augmented expression of osteogenic markers like osteopontin and RUNX2 in Asah1 fl/fl /SM cre mice compared to their wild type littermates, which was markedly attenuated by pretreatment of these mice with Torin‐1, an mTOR inhibitor. Confocal microscopy showed Asah1 fl/fl /SM cre mice had markedly increased colocalization of mTORC1 with Lamp‐1(lysosome marker), but decreased colocalization of VPS16 (a multivesicular bodies (MVBs) marker) with Lamp‐1, suggesting mTOR activation and reduced MVBs interaction with lysosomes. However, mTOR inhibition by Torin‐1 significantly reduced the colocalization of mTOR vs Lamp‐1 which inturn increased lysosome‐MVBs interaction. Torin‐1 was also found to reduce accumulation of CD63 and annexin 2 (AnX2) (exosome markers) in the coronary arterial wall of Asah1 fl/fl /SM cre mice compared to wild type littermates. Using CASMCs, we observed that Ac gene deletion led to peripheral distribution of lysosomes and increased exosome secretion, which was blocked by Torin‐1. Functionally, Torin‐1 was found to significantly reduce arterial stiffening Asah1 fl/fl /SM cre mice as shown by restoration from increased pulse wave velocity (PWV). Altogether, these results suggest that lysosomal ceramide‐mTOR signaling is critical for the control of lysosome trafficking, exosome secretion and arterial stiffening or osteogenesis. Support or Funding Information Supported by NIH grants HL057244, HL075316 and DK120491