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 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 5

TNAP interacts with sortilin in calcifying SMCs.

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TNAP interacts with sortilin in calcifying SMCs. Rab11 controls sortilin...
Rab11 controls sortilin trafficking. (A) Number of peptide spectrum matches (PSM) from caveolin-1 (Cav-1) and TNAP identified in sortilin immoprecipitation experiments. n = 3. (B) Western blot of sortilin, Cav-1, and TNAP before (input) and after IP of sortilin. One of 3 hSMC donors is shown. (C) Transmission electron microscopy of aorta sections of CRD Apoe–/– mice. HA, hydroxyapatite. Scale bar: 2 μm. Inset shows a higher magnification depicting caveolae (red arrows). Scale bar: 200 nm. (D) hSMCs were cultured for 21 days in control medium (CM) or osteogenic medium (OM). CEM was resolved from other cellular constituents (nCEM). Representative Western blot, n = 3. (E and F) hSMCs were cultured for 14 days (E, TNAP activity) or 21 days (F, calcium eluted from the matrix) in OM. Cav-1 was silenced by siRNA (SiCav-1 or scramble control, Scr). n = 4. ***P < 0.005, t test. (G) Immunofluorescence staining of sortilin (red) and the TGN (GALTN2, green) in control and calcifying hSMCs. Scale bar: 10 μm. (H–L) Rab11a/b were silenced by siRNA (SiRab11). (H) Representative Western blot of sortilin and Rab11. β-Actin served as loading control. (I) Quantification of H. n = 3. *P < 0.005, t test. (J) TNAP activity at day 14. (K) Calcification quantified by measuring calcium eluted from the matrix, n = 3. ***P < 0.005, t test. (L) Sortilin was overexpressed by adenovirus (AdSORT1). LacZ served as control (AdLacZ). n = 3. *P < 0.05, **P < 0.01, ANOVA. Error bars indicate ±SD. Each n indicates an independent hSMC donor.