Abstract 51: Sortilin 1 Promotes Matrix Vesicle-Mediated Vascular Microcalcification via Rab11 Trafficking and C-Terminal Phosphorylation

Abstract

Background: Arterial microcalcification predicts cardiovascular events and contributes to plaque rupture. We recently demonstrated that matrix vesicles (MVs) participate in the formation of microcalcifications. Yet, the precise mechanisms by which MVs regulate calcification in atheroma remain unknown. We hypothesized that sortilin 1, a protein associated with arterial calcification in GWAS, promotes calcification via its phosphorylation and recruitment to MVs. Results: We detected sortilin 1 in calcified MVs of human plaques using transmission electron microscopy-based immunogold labeling. Sortilin 1 loading into MVs increased in sortilin 1-overexpressing smooth muscle cells (SMCs), and rose further in MVs derived from calcifying SMCs. Nanoparticle tracking analysis indicated no alteration in MV release. Silencing sortilin 1 reduced tissue non-specific alkaline phosphatase (TNAP) activity within MVs (-45%; p<0.01), whereas sortilin 1 overexpression promoted TNAP activity within MVs twofold (p<0.05). Silencing Rab11 reduced sortilin 1 protein (-44%, p<0.01) as well as TNAP activity (-34%, p<0.01) and mineralization (-36%, p<0.01). Further, silencing Rab11 abolished the induction of TNAP activity by sortilin 1 overexpression. We used a mass spectrometry-based strategy to monitor sortilin 1 post-translational modifications in calcifying SMCs and identified a calcification-dependent phosphorylation within the C-terminal domain. Mutations on Ser825 and Ser819, which prevented sortilin 1 phosphorylation, 1) abolished SMC TNAP activity (-43%; p<0.05) and calcification (-72%; p<0.05), 2) mitigated TNAP activity (-36%; p<0.05) and hydroxyapatite content (-34%; p<0.05) in MVs, and 3) prevented loading of sortilin 1 into MVs. In contrast, mutations on Ser825 and Ser819 that mimic a constitutive active phosphorylation enhanced TNAP activity without affecting sortilin 1 expression. Conclusion: Sortilin 1 promotes MV-mediated vascular microcalcification via Rab11 trafficking and C-terminal phosphorylation. Our data provide mechanistic insight into the correlation between sortilin 1 and calcification and identifies sortilin 1 as a potential therapeutic target to modulate the formation of microcalcifications.