Vascular Smooth Muscle Cell Calcification Is Mediated by Regulated Exosome Secretion

Abstract

Rationale: Matrix vesicles (MVs), secreted by vascular smooth muscle cells (VSMCs), form the first nidus for mineralization and fetuin-A, a potent circulating inhibitor of calcification, is specifically loaded into MVs. However, the processes of fetuin-A intracellular trafficking and MV biogenesis are poorly understood. Objective: The objective of this study is to investigate the regulation, and role, of MV biogenesis in VSMC calcification. Methods and Results: Alexa488-labeled fetuin-A was internalized by human VSMCs, trafficked via the endosomal system, and exocytosed from multivesicular bodies via exosome release. VSMC-derived exosomes were enriched with the tetraspanins CD9, CD63, and CD81, and their release was regulated by sphingomyelin phosphodiesterase 3. Comparative proteomics showed that VSMC-derived exosomes were compositionally similar to exosomes from other cell sources but also shared components with osteoblast-derived MVs including calcium-binding and extracellular matrix proteins. Elevated extracellular calcium was found to induce sphingomyelin phosphodiesterase 3 expression and the secretion of calcifying exosomes from VSMCs in vitro, and chemical inhibition of sphingomyelin phosphodiesterase 3 prevented VSMC calcification. In vivo, multivesicular bodies containing exosomes were observed in vessels from chronic kidney disease patients on dialysis, and CD63 was found to colocalize with calcification. Importantly, factors such as tumor necrosis factor-α and platelet derived growth factor-BB were also found to increase exosome production, leading to increased calcification of VSMCs in response to calcifying conditions. Conclusions: This study identifies MVs as exosomes and shows that factors that can increase exosome release can promote vascular calcification in response to environmental calcium stress. Modulation of the exosome release pathway may be as a novel therapeutic target for prevention.

Sortilin mediates vascular calcification via its recruitment into extracellular vesicles

“…Loss-offunction and gain-of-function studies, however, did not reveal a sortilin-dependent change in number and size of EVs from calcifying SMCs. Whether the release of sortilin-enriched calcifying EVs occurs via exosomal mechanisms through multivesicular bodies (37) or via budding of vesicles directly from the plasma membrane and the characteristics of sortilin/TNAP-containing EVs remains unknown. Thus, sortilin operates via a mechanism involving direct modulation of the calcification potential of EVs by facilitating the intracellular trafficking and loading of TNAP into the EVs.…”

Biophysical aspects of biomineralization

“…PHOSPHO1 not only regulates MVmediated initiation of mineralization, but also MV formation. SMPD3 has also been implicated in the biogenesis of vesicles by vascular smooth muscles cells (VSMCs) (Kapustin et al 2015).…”

Medial Arterial Calcification

“…22 Recent evidence in VSMCs suggests that an exosomal pathway is the major source of matrix vesicles in the vessel wall and that this pathway enables the loading of these exosomes with specific cargoes that act as inhibitors to block calcification, including matrix Gla protein and fetuin-A. 23 However, prolonged exposure to procalcific conditions, such as mineral imbalance and dysregulation of calcium/phosphate homeostasis as occurs in CKD, leads to loss of inhibitors from these exosomes and exposure of phosphatidylserine and annexin on their surface to initiate mineralization.…”