Abstract
Arterial medial calcification (AMC) involves an increased small extracellular vesicle (sEV) secretion and apatite calcium precipitation in the arterial wall. The mechanisms mediating AMC remain poorly understood. In the present study, smooth muscle-specific acid ceramidase (Ac) gene knockout mice (Asah1fl/fl/SMCre) were used to demonstrate the role of lysosomal ceramide signaling pathway in AMC. Asah1fl/fl/SMCre mice were found to have more severe AMC in both aorta and coronary arteries compared to their littermates (Asah1fl/fl/SMwt and WT/WT mice) after receiving a high dose vitamin D. These mice also had pronounced upregulation of osteopontin and RUNX2 (osteogenic markers), CD63, AnX2 (sEV markers) and ALP expression (mineralization marker) in the arterial media. In cultured coronary arterial smooth muscle cells (CASMCs) from Asah1fl/fl/SMCre mice, high dose of Pi led to a significantly increased calcium deposition, phenotypic change and sEV secretion compared to WT CASMCs, which was associated with reduced lysosome-multivesicular body (MVB) interaction. Also, GW4869, sEV release inhibitor decreased sEV secretion and calcification in these cells. Lysosomal transient receptor potential mucolipin 1 (TRPML1) channels regulating lysosome interaction with MVBs were found remarkably inhibited in Asah1fl/fl/SMCre CASMCs as shown by GCaMP3 Ca2+ imaging and Port-a-Patch patch clamping of lysosomes. Lysosomal Ac in SMCs controls sEV release by regulating lysosomal TRPML1 channel activity and lysosome-MVB interaction, which importantly contributes to phenotypic transition and AMC.
Highlights
Www.nature.com/scientificreports that increased serum Ca/Pi levels are correlated with the development and progression of calcification[13]
The present study revealed for the first time that lysosomal acid ceramidase (Ac) plays a key role in small extracellular vesicle (sEV) release, osteogenic phenotype transition and resulting in mineral deposition in arterial SMCs that contribute to Arterial medial calcification (AMC)
Our results show that lysosomal Ac and associated CER signaling pathway may control the fate of multivesicular body (MVB) via its regulatory action on transient receptor potential mucolipin 1 (TRPML1) channel activity and subsequent lyososome-MVB interaction, which leads to enhanced sEV release from SMCs that influence the AMC development
Summary
Www.nature.com/scientificreports that increased serum Ca/Pi levels are correlated with the development and progression of calcification[13]. In this regard, accumulated sphingomyelin inhibited its activity and reduced lysosomal Ca2+ release, leading to failure of lysosome trafficking and lysosomal storage disease as shown in Niemann-Pick disease[32,33] It remains unknown whether this lysosome TRPML1 channel-mediated Ca2+ release is involved in lysosome-MVB interaction and how this Ca2+ signaling pathway is regulating sphingolipids associated with sEV release. Our results demonstrate that Ac-associated sphingolipids importantly controls TRPML1 channel activity and regulates lysosome-MVB interaction, leading prolongation of MVB fate and consequent enhancement of sEV release
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