Matrix Vesicles from Osteoblasts Promote Atherosclerotic Calcification

Backgrounds Vascular calcification often occurs with osteoporosis, a contradictory association known as “the calcification paradox”. Osteoblast-derived matrix vesicles (Ost-MVs) have been implicated in bone mineralization, and also have a potential role in ectopic vascular calcification. Herein, we aim to investigate the contributions that Ost-MVs make to the bone–vascular calcification paradox and the underlying mechanisms. Methods and Results Hyperlipidemia-induced atherosclerotic calcification in mice was accompanied with bone mineral loss, as evidenced by reduced deposition of Ost-MVs in the bone matrix and increased release of Ost-MVs into the circulation. Intravenous injection of fluorescent DiІ-labeled Ost-MVs revealed a marked fluorescence accumulation in the aorta of atherogenic mice, whereas no fluorescence signals were observed in normal controls. Using proteomics to analyze proteins in non-matrix bound Ost-MVs and mineralized SMC-derived MVs (SMC-MVs), we found Lamp1 was specifically expressed in SMC-MVs, and Nid2 was exclusively expressed in Ost-MVs. We further demonstrated that both Lamp1 and Nid2 were co-localized with Collagen І within calcific plaques, indicating the involvement of both Ost-MVs and SMC-MVs in atherosclerotic calcification. Mechanistically, LPS-induced vascular injury facilitated the transendothelial transport of Ost-MVs. The recruitment of circulating Ost-MVs was regulated by remodeled Collagen І during calcification progression. Furthermore, the phenotypic transition of SMCs determined the endocytosis of Ost-MVs. Finally, we demonstrated that either recruited Ost-MVs or resident SMC-MVs accelerated atherosclerotic calcification, depending on the Ras-Raf-ERK signaling. Conclusion Atherosclerotic calcification-induced Ost-MVs are released into circulation, facilitating the transport from bone to plaque lesions and exacerbating artery calcification progression. The mechanisms of Ost-MVs recruitment include vascular injury allowing transendothelial transport of Ost-MVs, collagen І remodeling promoting Ost-MVs aggregation, and SMC phenotypic switch to facilitate Ost-MVs uptake. Our results further revealed that both recruited Ost-MVs and calcifying SMC-MVs aggravate calcification through the Ras-Raf-ERK pathway. ### Competing Interest Statement The authors have declared no competing interest. * ### Abbreviations MVs : matrix vesicles SMCs : smooth muscle cells Ost-MVs : osteoblast-derived MVs SMC-MVs : calcifying SMC-derived MVs ApoE−/− mice : apolipoprotein E-deficient mice WT : wild-type HFD : high-fat diet ND : normal diet BMD : bone mineral density BV/TV : Bone volume/tissue volume Tb.N : trabecular number Tb.Th : trabecular thickness Tb.Sp : trabecular separation NM : normal medium OM : osteogenic medium ANX2/5/6 : annexins 2, 5 and 6 Lamp1 : Lysosome associated membrane protein 1 Nid2 : Nidogen 2 LPS : lipopolysaccharide α-SMA : α-smooth muscle actin SM22α : smooth muscle 22α CNN1 : Calponin 1 S100A4 : S100 calcium binding protein A4 MMP2 : matrix metallopeptidase 2 ALP : alkaline phosphatase MAPK : mitogen-activated protein kinase ERK : extracellular regulated protein kinases DiІ : 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorate DiO : 3,3’-dioctadecyloxacarbocyanine perchlorate PDGF-BB : platelet derived growth factor subunit B NTA : nanoparticle tracking analysis TEM : transmission electron microscope GSEA : gene set enrichment analysis