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.
View: Text | PDF
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

×

Figure 4

Sortilin promotes alkaline phosphatase activity and mineralization in calcifying hSMCs and EVs.

Options: View larger image (or click on image) Download as PowerPoint
Sortilin promotes alkaline phosphatase activity and mineralization in ca...
hSMCs were cultured up to 21 days in control medium (CM) or osteogenic medium (OM). (A) Relative sortilin protein abundance. Representative Western blot (n = 3). (B) Immunofluorescence microscopy of sortilin (blue, arrow) in areas of microcalcifications (red) at day 21. Collagen, green; nuclei, gray. Scale bar: 20 μm. (C and D) Sortilin was silenced by siRNA (siSORT1 or scramble control, Scr). n = 4. **P < 0.01, ANOVA. (C) TNAP activity at day 14. (D) Calcification at day 21. Top: representative images of calcification detected by alizarin red S–stained mineralized matrix. Bottom: quantification of eluted calcium. (E) EVs of different sizes and appearance (red arrows) including small loci of microcalcifications. Calcified plaque of CRD mouse. Scale bar: 1 μm. (F) Transmission electron microscopy–based immunogold staining of sortilin (red arrows) in EVs from aorta of CRD mouse. One of 3 animals is shown. Scale bar: 200 nm. (G) Endogenous sortilin and TNAP levels in EVs isolated from hSMC supernatant. n = 3. (H and I) TNAP activity in EVs isolated from hSMCs cultured for 14 days in CM or OM. Sortilin was silenced by siRNA (siSORT1 or scramble control, Scr) or overexpressed by adenovirus (AdSORT1 or LacZ control). n = 3, **P < 0.01, ANOVA (H) and n = 4, *P < 0.05, t test (I). Error bars indicate ±SD. Each n indicates an independent hSMC donor.