Regulatory Role of Ceramide/mTOR Signaling in Exosome Secretion from Smooth Muscle Cells during Arterial Stiffening and Medial Calcification

Abstract

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 (Asah1fl/fl/SMcre) 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 Asah1fl/fl/SMcre 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 Asah1fl/fl/SMcre 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 Asah1fl/fl/SMcre 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 Asah1fl/fl/SMcre 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

mTORsignalling pathway in stem cell bioactivities and angiogenesis potential

“…In an experiment, the inhibition of mTOR1 with a specific inhibitor, Torin‐1, increased MVBs interaction with lysosomal marker LAMP1 and Exo release. In line with these changes, the co‐localization of mTOR1 with VPS16 and LAMP1 is inhibited, suggesting a critical role of ceramide‐mTOR signalling in Exo secretion 103 . In addition to the modulatory effect of mTOR on the Exo abscission rate, emerging data have proved that Exos can also regulate the function of the mTOR axis after entry into the target cells 107 .…”

“…In addition to the differentiation capacity of stem cells towards ECs, the production and release of several angiogenic factors can promote the formation of de novo blood vessels in the target sites 102 . Emerging data have confirmed the close association between mTOR signalling and the release of factors via nano‐sized vesicles namely Exos 103 . Exos with endosomal origin range between 30 and 100 nm and are produced via the invagination of the vesicular membrane into the lumen of MVBs.…”

“… 102 Emerging data have confirmed the close association between mTOR signalling and the release of factors via nano‐sized vesicles namely Exos. 103 Exos with endosomal origin range between 30 and 100 nm and are produced via the invagination of the vesicular membrane into the lumen of MVBs. Molecular investigations revealed that Exos contains certain cargoes with several types of growth factors that are involved in inter‐cellular communication.…”

“…In line with these changes, the co‐localization of mTOR1 with VPS16 and LAMP1 is inhibited, suggesting a critical role of ceramide‐mTOR signalling in Exo secretion. 103 In addition to the modulatory effect of mTOR on the Exo abscission rate, emerging data have proved that Exos can also regulate the function of the mTOR axis after entry into the target cells. 107 It has been indicated that Exos isolated from miR‐26a‐expressing MSCs promoted axonal regeneration in a rat model of spinal cord injury via the modulation of the PTEN/AKT/mTOR axis.…”

Release and Actions of Inflammatory Exosomes in Pulmonary Emphysema: Potential Therapeutic Target of Acupuncture

“…Such exosomes containing NLRP3 inflammasome products are referred to as inflammatory exosomes in recent studies. [25][26][27] We have used different approaches to test this hypothesis in a wellestablished animal model of emphysema.…”