Sortilin mediates vascular calcification via its recruitment into extracellular vesicles
Claudia Goettsch, … , Sasha A. Singh, Elena Aikawa
Claudia Goettsch, … , Sasha A. Singh, Elena Aikawa
Published April 1, 2016; First published March 7, 2016
Citation Information: J Clin Invest. 2016;126(4):1323-1336. https://doi.org/10.1172/JCI80851.
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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 1

Sortilin localizes to calcified tissue from humans and mice with CRD.

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Sortilin localizes to calcified tissue from humans and mice with CRD. (A...
(A) von Kossa staining indicates calcified areas (red arrow). Calcified regions of human atherosclerotic carotid arteries contain sortilin. SMCs are stained with αSMA and macrophages with CD68. Scale bars 100 μm. (B) Immunofluorescence double staining of sortilin (green)/αSMA (red) or CD68 (red). One representative sample of 20 patients. Scale bars: 5 μm. (C) Immunofluorescent visualization of sortilin and microcalcifications using near infrared fluorescence (NIRF) calcium tracer in human atherosclerotic carotid arteries. Scale bars: 20 μm. Graph: data points represent correlation between sortilin and tracer signal from 10 patients. Each image was separated into 250 smaller images for quantification using a total of 2,500 measurements. (D) Immunohistochemical staining of sortilin (brown reaction product, red arrows) in aorta of Apoe–/– mice without and with CRD. TNAP activity detects calcification-prone tissue. One of 4 animals per group is shown. Scale bars: 100 μm. (E) Quantification of sortilin-positive cells in atherosclerotic lesions and media. n = 3, WT, CRD; n = 4, Apoe–/–. n.d., not detected for WT. *P < 0.05, ANOVA. Error bars indicate ±SD. (F) Acta2 and Alpl mRNA expression from sortilin negative (–) and positive (+) SMCs (laser capture microdissection of Apoe–/– aorta). n = 7. *P < 0.05, Wilcoxon matched-pairs signed rank test. Error bars indicate ±SEM.