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 6

Ser825 of sortilin C-terminus is phosphorylated in calcifying hSMCs and in vivo.

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Ser825 of sortilin C-terminus is phosphorylated in calcifying hSMCs and ...
(A) MS/MS spectrum for SGYHDDSDEDLLE ([M+2H]2+ = 747.80), and phospho-peptide, pS825 ([M+2H]2+ = 787.78). (B–G) The underlined fragment ions were used to determine the percent of phospho-peptide in samples using parallel reaction monitoring (PRM). (B) Relative ratio of phosphorylated (bottom) and unphosphorylated (top) form. Fold-increase over control medium (CM) is plotted. (C) pS825 in calcified and noncalcified artery from Apoe–/– mice. n = 3. Each n correspond to pooled artery from 3 mice. *P < 0.05, t test. (D) pS825 in human calcified carotid arteries from endarterectomy and noncalcified arteries from autopsy. n = 4. *P < 0.05, t test. (E) Kinase assay using peptide standard (SGYHDDSDEDLLE). Time-dependent phosphorylation by Fam20C or catalytically inactive form D478A. (F) Kinase assay using membrane protein isolated from sortilin-overexpressing HEK293 cells. Membrane protein without enzyme incubation served as control. n = 3. *P < 0.05, t test. (G) pS825 in calcifying hSMCs overexpressing Fam20C. Empty vector served as control (Mock). Top: Fam20C Western blot. β-Actin served as loading control. n = 3. *P < 0.05, paired t test. (H) TNAP activity after Fam20C overexpression and sortilin silencing (SiSORT1). n = 3. **P < 0.01, *P < 0.05, ANOVA. (I) Time-dependent phosphorylation by Fam20C, CK1, and CK2. (J) Kinase assay using peptide standard. Cell lysates from calcifying hSMCs were used as a kinase source. Fam20C was silenced. TBB (10 μM) was used to inhibit CK2. DMSO served as solvent control. (I and J) Quantification was done using the MS1 ion signals. n = 4. *P < 0.05, ANOVA. (K) TNAP activity. *P < 0.05, ANOVA. Error bars indicate ±SD. Each n indicates an independent hSMC donor for G, H, J, and K. OM, osteogenic medium.