Sortilin mediates vascular calcification via its recruitment into extracellular vesicles
Claudia Goettsch, Joshua D. Hutcheson, Masanori Aikawa, Hiroshi Iwata, Tan Pham, Anders Nykjaer, Mads Kjolby, Maximillian Rogers, Thomas Michel, Manabu Shibasaki, Sumihiko Hagita, Rafael Kramann, Daniel J. Rader, Peter Libby, Sasha A. Singh, Elena Aikawa
Claudia Goettsch, Joshua D. Hutcheson, Masanori Aikawa, Hiroshi Iwata, Tan Pham, Anders Nykjaer, Mads Kjolby, Maximillian Rogers, Thomas Michel, Manabu Shibasaki, Sumihiko Hagita, Rafael Kramann, Daniel J. Rader, Peter Libby, Sasha A. Singh, Elena Aikawa
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Research Article Vascular biology

Sortilin mediates vascular calcification via its recruitment into extracellular vesicles

Abstract

Vascular calcification is a common feature of major cardiovascular diseases. Extracellular vesicles participate in the formation of microcalcifications that are implicated in atherosclerotic plaque rupture; however, the mechanisms that regulate formation of calcifying extracellular vesicles remain obscure. Here, we have demonstrated that sortilin is a key regulator of smooth muscle cell (SMC) calcification via its recruitment to extracellular vesicles. Sortilin localized to calcifying vessels in human and mouse atheromata and participated in formation of microcalcifications in SMC culture. Sortilin regulated the loading of the calcification protein tissue nonspecific alkaline phosphatase (TNAP) into extracellular vesicles, thereby conferring its calcification potential. Furthermore, SMC calcification required Rab11-dependent trafficking and FAM20C/casein kinase 2–dependent C-terminal phosphorylation of sortilin. In a murine model, Sort1-deficiency reduced arterial calcification but did not affect bone mineralization. Additionally, transfer of sortilin-deficient BM cells to irradiated atherosclerotic mice did not affect vascular calcification, indicating a primary role of SMC-derived sortilin. Together, the results of this study identify sortilin phosphorylation as a potential therapeutic target for ectopic calcification/microcalcification and may clarify the mechanism that underlies the genetic association between the SORT1 gene locus and coronary artery calcification.

Authors

Claudia Goettsch, Joshua D. Hutcheson, Masanori Aikawa, Hiroshi Iwata, Tan Pham, Anders Nykjaer, Mads Kjolby, Maximillian Rogers, Thomas Michel, Manabu Shibasaki, Sumihiko Hagita, Rafael Kramann, Daniel J. Rader, Peter Libby, Sasha A. Singh, Elena Aikawa

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Figure 7

Phosphorylation of the sortilin C-terminus accelerates calcification.

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Phosphorylation of the sortilin C-terminus accelerates calcification. (A...
(A) Strategy for mutation of the C-terminal peptide. The numbers indicate amino acid positions. ECD, extracellular domain; TM, transmembrane domain; ICD, intracellular domain. (B–F) hSMCs were transduced with adenoviral vectors to overexpress sortilin (AdSORT1) or the different mutated forms (phosphorylation-null S825A; phosphorylation-mimetic S825D). Adenovirus LacZ served as control (AdLacZ). (B) Representative sortilin Western blot of calcifying hSMCs with transduced mutated constructs. β-Actin served as loading control. (C) TNAP activity. n = 4. (D) Calcification. Top: representative alizarin red S staining. Bottom: Quantification of eluated alizarin red S, n = 4. (E) TNAP activity in EVs. TNAP activity was normalized to EV protein content. n = 3. *P < 0.05, ***P < 0.005 vs. AdSORT1 by t test for all experiments. Error bars indicate ±SD. (F) Immunofluorescence using super-resolution microscopy (insets: confocal microscopy) shows the expression of sortilin (green) and TGN (GALTN2, red) or lysosome (LAMP1, red) in hSMCs overexpressing sortilin S825A or S825D. Arrows indicate coexpression. Scale bars: 2 μm; inset scale bars: 20 μm. Each n indicates an independent hSMC donor. Representative of 3 independent experiments. (G) Schematic representation of the mechanism of sortilin-induced calcification via C-terminus phosphorilation (yellow), Rab1-dependent trafficking, and TNAP loading into EV.