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Deficiency of Lysosomal Ceramide Hydrolysis Contributes to Enhanced Exosome Release and Calcification in Coronary Artery Myocytes
Author(s) -
Yuan Xinxu,
Bhat Owais M.,
Lohner Hannah,
Zhang Qinghua,
Zhang Yang,
Boini Krishna,
Li PinLan
Publication year - 2018
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.2018.32.1_supplement.676.9
Subject(s) - ceramide , chemistry , calcification , sphingomyelin , oil red o , sphingolipid , immunostaining , osteopontin , actinin , microbiology and biotechnology , exosome , medicine , endocrinology , biology , biochemistry , microvesicles , adipose tissue , cell , immunohistochemistry , apoptosis , cytoskeleton , membrane , adipogenesis , microrna , gene
Ceramide has been shown to contribute to vascular calcification under different pathological conditions. However, the precise mechanism mediating this process remains unknown. The present study investigated the role of lysosomal ceramide metabolism via acid ceramidase (AC) in the regulation of exosome excretion in coronary artery myocytes (CAMs) and the possible implication in the development of calcification. In cultured CAMs from smooth muscle (SM)‐specific Asah1 (mouse code of AC gene) knock‐out mice (Asah1 fl/fl /SM cre ), we observed that deletion of Asah1 gene increased lysosomal ceramide level compared to their control littermates, as detected by immunostaining of ceramide co‐localized with lysosome marker lamp‐1. Treatment of CAMs from wild type mice produced similar effects to reduce lysosomal ceramide given its effect to inhibit AC activity. Alizarin Red staining showed that more remarkable calcium deposition or calcification by 1.9 folds occurred in cultured CAMs from Asah1 fl/fl /SM cre mice compared to cells from their control littermates, if these cells were treated with 3 mM NaH 2 PO3 and atherogenic stimulator, 7‐ket. Correspondingly, CAMs from Asah1 fl/fl /SM cre mice were found to have significant phenotype transition as shown by Western blot analysis, increased expression of synthetic and osteogenic markers (more vimentin and osteopontin) and decreased contractile phenotypic marker (less SM22α), and this synthetic or osteogenic phenotype was enhanced by 7‐ket treatment. Using super‐resolution imaging with Structured Illumination Microscopy (SR‐SIM), we found that 7‐ket markedly reduced colocalization of Rab‐7a (multivesicular body (MVB) marker) with lamp‐1 in 7‐ket‐treated WT CAMs by 3 folds or CAMs from Asah1 fl/fl /SM cre mice by 2.5 folds, suggesting inhibition of MVB‐lysosome encountering or fusion during inhibition of AC expression and activity. Using nanoparticle tracking analysis, 7‐ket‐induced excretion of exosomes (80–140 nm in size) was found to be significantly increased in CAMs of Asah1 fl/fl /SM cre mice by 2.1 folds compared to CAMs from their control littermates. These results suggest that AC‐mediated ceramide hydrolysis in lysosomes critically controls MVB fate and exosome release and that insufficient AC expression and activity may lead to enhancement of exosome release, contributing to calcium deposition or calcification in CAMs or arterial media. Support or Funding Information (supported by NIH grants HL057244, HL075316 and HL122937). This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .