Regulatory Pathways in Growth Plate Chondrocytes that Are Impacted by Matrix Vesicle microRNA Identified by Targeted RISC Pulldown and Sequencing of the Resulting Transcriptome.

During endochondral bone formation, growth plate chondrocytes are differentially regulated by various factors and hormones. As the cellular phenotype changes, the composition of the extracellular matrix is altered, including the production and composition of matrix vesicles (MV) and their cargo of microRNA. The regulatory functions of these MV microRNA in the growth plate are still largely unknown. To address this question, we undertook a targeted bioinformatics approach. A subset of five MV microRNA was selected for analysis based on their specific enrichment in these extracellular vesicles compared to the parent cells (miR-1-3p, miR-22-3p, miR-30c-5p, miR-122-5p, and miR-133a-3p). Synthetic biotinylated versions of the microRNA were produced using locked nucleic acid (LNA) and were transfected into rat growth plate chondrocytes. The resulting LNA to mRNA complexes were pulled down and sequenced, and the transcriptomic data were used to run pathway analysis pipelines. Bone and musculoskeletal pathways were discovered to be regulated by the specific microRNA, notably those associated with transforming growth factor beta (TGFß) and Wnt pathways, cell differentiation and proliferation, and regulation of vesicles and calcium transport. These results can help with understanding the maturation of the growth plate and the regulatory role of microRNA in MV.

Autophagy reduces aortic calcification in diabetic mice by reducing matrix vesicle body-mediated IL-1ß release.

Vascular calcification (VC) is a common pathological process of cardiovascular disease that occurs in patients with type 2 diabetes mellitus (T2DM). However, the molecular basis of VC progression remains unknown. A GEO dataset (GSE146638) was analyzed to show that microbodies and IL-1ß may play important roles in the pathophysiology of VC. The release of matrix vesicle bodies (MVBs) and IL-1ß and the colocalization of IL-1ß with MVBs or autophagosomes were studied by immunofluorescence in an in vivo diabetes mouse model with aortic calcification and an in vitro high glucose cell calcification model. MVB numbers, IL-1ß levels and autophagy were increased in calcified mouse aortas and calcified vascular smooth muscle cells (VSMCs). IL-1ß colocalized with MVBs and autophagosomes. The MVBs from calcified VSMCs induced the calcification of normal recipient VSMCs, and this effect was alleviated by silencing IL-1ß. The autophagy inducer rapamycin reduced IL-1ß expression and calcification in VSMCs, while these processes were induced by the autophagy inhibitor chloroquine. In conclusion, our results suggested that MVBs could carry IL-1ß out of cells and induce VC in normal VSMCs, and these processes could be counteracted by autophagy. These results suggested that MVB-mediated IL-1ß release may be an effective target for treating vascular calcification.

Extracellular vesicles: From bone development to regenerative orthopedics.

Regenerative medicine aims to promote the replacement of tissues lost to damage or disease. While positive outcomes have been observed experimentally, challenges remain in their clinical translation. This has led to growing interest in applying extracellular vesicles (EVs) to augment or even replace existing approaches. Through the engineering of culture environments or direct/indirect manipulation of EVs themselves, multiple avenues have emerged to modulate EV production, targeting, and therapeutic potency. Drives to modulate release using material systems or functionalize implants for improved osseointegration have also led to outcomes that could have real-world impact. The purpose of this review is to highlight advantages in applying EVs for the treatment of skeletal defects, outlining the current state of the art in the field and emphasizing avenues for further investigation. Notably, the review identifies inconsistencies in EV nomenclature and outstanding challenges in defining a reproducible therapeutic dose. Challenges also remain in the scalable manufacture of a therapeutically potent and pure EV product, with a need to address scalable cell sources and optimal culture environments. Addressing these issues will be critical if we are to develop regenerative EV therapies that meet the demands of regulators and can be translated from bench to bedside.

Extracellular Vesicles: New Classification and Tumor Immunosuppression.

Extracellular vesicles (EVs) are cell-derived membrane-surrounded vesicles carrying various types of molecules. These EV cargoes are often used as pathophysiological biomarkers and delivered to recipient cells whose fates are often altered in local and distant tissues. Classical EVs are exosomes, microvesicles, and apoptotic bodies, while recent studies discovered autophagic EVs, stressed EVs, and matrix vesicles. Here, we classify classical and new EVs and non-EV nanoparticles. We also review EVs-mediated intercellular communication between cancer cells and various types of tumor-associated cells, such as cancer-associated fibroblasts, adipocytes, blood vessels, lymphatic vessels, and immune cells. Of note, cancer EVs play crucial roles in immunosuppression, immune evasion, and immunotherapy resistance. Thus, cancer EVs change hot tumors into cold ones. Moreover, cancer EVs affect nonimmune cells to promote cellular transformation, including epithelial-to-mesenchymal transition (EMT), chemoresistance, tumor matrix production, destruction of biological barriers, angiogenesis, lymphangiogenesis, and metastatic niche formation.

Shedding Light on the Role of Na,K-ATPase as a Phosphatase during Matrix-Vesicle-Mediated Mineralization.

Matrix vesicles (MVs) contain the whole machinery necessary to initiate apatite formation in their lumen. We suspected that, in addition to tissue-nonspecific alkaline phosphatase (TNAP), Na,K,-ATPase (NKA) could be involved in supplying phopshate (Pi) in the early stages of MV-mediated mineralization. MVs were extracted from the growth plate cartilage of chicken embryos. Their average mean diameters were determined by Dynamic Light Scattering (DLS) (212 ± 19 nm) and by Atomic Force Microcopy (AFM) (180 ± 85 nm). The MVs had a specific activity for TNAP of 9.2 ± 4.6 U·mg-1 confirming that the MVs were mineralization competent. The ability to hydrolyze ATP was assayed by a colorimetric method and by 31P NMR with and without Levamisole and SBI-425 (two TNAP inhibitors), ouabain (an NKA inhibitor), and ARL-67156 (an NTPDase1, NTPDase3 and Ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) competitive inhibitor). The mineralization profile served to monitor the formation of precipitated calcium phosphate complexes, while IR spectroscopy allowed the identification of apatite. Proteoliposomes containing NKA with either dipalmitoylphosphatidylcholine (DPPC) or a mixture of 1:1 of DPPC and dipalmitoylphosphatidylethanolamine (DPPE) served to verify if the proteoliposomes were able to initiate mineral formation. Around 69-72% of the total ATP hydrolysis by MVs was inhibited by 5 mM Levamisole, which indicated that TNAP was the main enzyme hydrolyzing ATP. The addition of 0.1 mM of ARL-67156 inhibited 8-13.7% of the total ATP hydrolysis in MVs, suggesting that NTPDase1, NTPDase3, and/or NPP1 could also participate in ATP hydrolysis. Ouabain (3 mM) inhibited 3-8% of the total ATP hydrolysis by MVs, suggesting that NKA contributed only a small percentage of the total ATP hydrolysis. MVs induced mineralization via ATP hydrolysis that was significantly inhibited by Levamisole and also by cleaving TNAP from MVs, confirming that TNAP is the main enzyme hydrolyzing this substrate, while the addition of either ARL-6715 or ouabain had a lesser effect on mineralization. DPPC:DPPE (1:1)-NKA liposome in the presence of a nucleator (PS-CPLX) was more efficient in mineralizing compared with a DPPC-NKA liposome due to a better orientation of the NKA active site. Both types of proteoliposomes were able to induce apatite formation, as evidenced by the presence of the 1040 cm-1 band. Taken together, the findings indicated that the hydrolysis of ATP was dominated by TNAP and other phosphatases present in MVs, while only 3-8% of the total hydrolysis of ATP could be attributed to NKA. It was hypothesized that the loss of Na/K asymmetry in MVs could be caused by a complete depletion of ATP inside MVs, impairing the maintenance of symmetry by NKA. Our study carried out on NKA-liposomes confirmed that NKA could contribute to mineral formation inside MVs, which might complement the known action of PHOSPHO1 in the MV lumen.

Matrix Vesicle-Mediated Mineralization and Osteocytic Regulation of Bone Mineralization.

Bone mineralization entails two mineralization phases: primary and secondary mineralization. Primary mineralization is achieved when matrix vesicles are secreted by osteoblasts, and thereafter, bone mineral density gradually increases during secondary mineralization. Nearby extracellular phosphate ions (PO43-) flow into the vesicles via membrane transporters and enzymes located on the vesicles' membranes, while calcium ions (Ca2+), abundant in the tissue fluid, are also transported into the vesicles. The accumulation of Ca2+ and PO43- in the matrix vesicles induces crystal nucleation and growth. The calcium phosphate crystals grow radially within the vesicle, penetrate the vesicle's membrane, and continue to grow outside the vesicle, ultimately forming mineralized nodules. The mineralized nodules then attach to collagen fibrils, mineralizing them from the contact sites (i.e., collagen mineralization). Afterward, the bone mineral density gradually increases during the secondary mineralization process. The mechanisms of this phenomenon remain unclear, but osteocytes may play a key role; it is assumed that osteocytes enable the transport of Ca2+ and PO43- through the canaliculi of the osteocyte network, as well as regulate the mineralization of the surrounding bone matrix via the Phex/SIBLINGs axis. Thus, bone mineralization is biologically regulated by osteoblasts and osteocytes.

Mineralization Profile of Annexin A6-Harbouring Proteoliposomes: Shedding Light on the Role of Annexin A6 on Matrix Vesicle-Mediated Mineralization.

The biochemical machinery involved in matrix vesicles-mediated bone mineralization involves a specific set of lipids, enzymes, and proteins. Annexins, among their many functions, have been described as responsible for the formation and stabilization of the matrix vesicles' nucleational core. However, the specific role of each member of the annexin family, especially in the presence of type-I collagen, remains to be clarified. To address this issue, in vitro mineralization was carried out using AnxA6 (in solution or associated to the proteoliposomes) in the presence or in the absence of type-I collagen, incubated with either amorphous calcium phosphate (ACP) or a phosphatidylserine-calcium phosphate complex (PS-CPLX) as nucleators. Proteoliposomes were composed of 1,2-dipalmitoylphosphatidylcholine (DPPC), 1,2-dipalmitoylphosphatidylcholine: 1,2-dipalmitoylphosphatidylserine (DPPC:DPPS), and DPPC:Cholesterol:DPPS to mimic the outer and the inner leaflet of the matrix vesicles membrane as well as to investigate the effect of the membrane fluidity. Kinetic parameters of mineralization were calculated from time-dependent turbidity curves of free Annexin A6 (AnxA6) and AnxA6-containing proteoliposomes dispersed in synthetic cartilage lymph. The chemical composition of the minerals formed was investigated by Fourier transform infrared spectroscopy (FTIR). Free AnxA6 and AnxA6-proteoliposomes in the presence of ACP were not able to propagate mineralization; however, poorly crystalline calcium phosphates were formed in the presence of PS-CPLX, supporting the role of annexin-calcium-phosphatidylserine complex in the formation and stabilization of the matrix vesicles' nucleational core. We found that AnxA6 lacks nucleation propagation capacity when incorporated into liposomes in the presence of PS-CPLX and type-I collagen. This suggests that AnxA6 may interact either with phospholipids, forming a nucleational core, or with type-I collagen, albeit less efficiently, to induce the nucleation process.

Inhibition of Sphingosine-1-Phosphate Receptor 2 by JTE013 Promoted Osteogenesis by Increasing Vesicle Trafficking, Wnt/Ca2+, and BMP/Smad Signaling.

Sphingosine-1-phosphate receptor 2 (S1PR2) is a G protein-coupled receptor that regulates various immune responses. Herein, we determine the effects of a S1PR2 antagonist (JTE013) or a S1PR2 shRNA on osteogenesis by culturing murine bone marrow stromal cells (BMSCs) in osteogenic media with JTE013, dimethylsulfoxide (DMSO), a S1PR2 shRNA, or a control shRNA. Treatment with JTE013 or the S1PR2 shRNA increased alkaline phosphatase and alizarin red s staining, and enhanced alkaline phosphatase, RUNX2, osteocalcin, and osterix mRNA levels in BMSCs compared with the controls. Protein analysis revealed that a high dose of JTE013 (4 or 8 µM) increased vesicle trafficking-associated proteins (F-actin, clathrin, Early Endosome Antigen 1 (EEA1), and syntaxin 6) and Wnt/Ca2+ signaling. On the other hand, a low dose of JTE013 (1 to 2 µM) increased BMP/Smad signaling. In contrast, the S1PR2 shRNA reduced vesicle trafficking-associated proteins and attenuated Wnts and BMP/Smad signaling, but enhanced p-CaMKII compared with the control, suggesting that the S1PR2 shRNA influenced osteogenesis via different signaling pathways. Moreover, inhibiting protein trafficking by brefeldin A in BMSCs suppressed Wnts and BMPRs expressions. These data supported that enhanced osteogenesis in JTE013-treated BMSCs is associated with increased vesicle trafficking, which promotes the synthesis and transport of osteogenic protein and matrix vesicles and enhances matrix mineralization.

Regulation of reactive oxygen species in the pathogenesis of matrix vesicles induced calcification of recipient vascular smooth muscle cells.

INTRODUCTION: Increased oxidative stress is associated with vascular calcification in patients with chronic kidney disease (CKD). We have previously demonstrated that cellular-derived matrix vesicles (MV), but not media-derived MV, are endocytosed in the presence of phosphorus by recipient normal rat vascular smooth muscle cells (VSMC) and induce calcification through ERK1/2 and [Ca2+]i signaling. We hypothesized that these changes were mediated by increased reactive oxygen species (ROS) production. METHODS: MV were co-cultured with recipient VSMC in the presence of high phosphorus and ROS production and cell signaling assessed. RESULTS: The results demonstrated MV endocytosis led to increased ROS production in recipient VSMC with no increase in mitochondrial oxygen consumption or oxidative phosphorylation (OXPHOS), indicating the ROS was not from the mitochondria. The use of inhibitors demonstrated that endocytosis of these MV by VSMC led to a signaling cascade in the cytoplasm beginning with ERK1/2 signaling, then increased [Ca2+]i and stimulation of ROS production, mediated by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX)1/4. Media-derived MV did not induce this cascade, indicating endocytosis itself was not a factor. Furthermore, inhibition of either ERK1/2 activation or [Ca2+]i reduced vascular calcification. CONCLUSION: We conclude that endocytosis of pro-mineralizing MV can induce a series of signaling events in normal VSMC that culminate in generation of ROS via activation of NOX1/4. Understanding these pathways will allow the development of future targeted therapeutics.

Ablation of Enpp6 Results in Transient Bone Hypomineralization.

Biomineralization is a fundamental process key to the development of the skeleton. The phosphatase orphan phosphatase 1 (PHOSPHO1), which likely functions within extracellular matrix vesicles, has emerged as a critical regulator of biomineralization. However, the biochemical pathways that generate intravesicular PHOSPHO1 substrates are currently unknown. We hypothesized that the enzyme ectonucleotide pyrophosphatase/phosphodiesterase 6 (ENPP6) is an upstream source of the PHOSPHO1 substrate. To test this, we characterized skeletal phenotypes of mice homozygous for a targeted deletion of Enpp6 (Enpp6 -/- ). Micro-computed tomography of the trabecular compartment revealed transient hypomineralization in Enpp6 -/- tibias (p < 0.05) that normalized by 12 weeks of age. Whole-bone cortical analysis also revealed significantly hypomineralized proximal bone in 4- but not 12-week-old Enpp6 -/- mice (p < 0.05) compared with WT animals. Back-scattered SEM revealed a failure in 4-week-old trabecular bone of mineralization foci to propagate. Static histomorphometry revealed increased osteoid volume (p > 0.01) and osteoid surface (p < 0.05), which recovered by 12 weeks but was not accompanied by changes in osteoblast or osteoclast number. This study is the first to characterize the skeletal phenotype of Enpp6 -/- mice, revealing transient hypomineralization in young animals compared with WT controls. These data suggest that ENPP6 is important for bone mineralization and may function upstream of PHOSPHO1 as a novel means of generating its substrates inside matrix vesicles. © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.

Effects of Sapindus mukorossi Seed Oil on Proliferation, Osteogenetic/Odontogenetic Differentiation and Matrix Vesicle Secretion of Human Dental Pulp Mesenchymal Stem Cells.

Stem cells have attracted great interest in the development of tissue engineering. However, the self-regeneration and multi-differentiation capabilities of stem cells are easily impaired during cell transplantation. Recent studies have demonstrated that Sapindus mukorossi (S. mukorossi) seed oil has various positive biological effects. However, it is not yet clear whether S. mukorossi seed oil can increase the growth and differentiation of dental pulp mesenchymal stem cells (DPSCs). The aim of this study is to investigate the effects of S. mukorossi seed oil on the proliferation and differentiation of DPSCs. DPSCs with and without S. mukorossi seed oil, respectively, were evaluated and compared. The viabilities of the cells were assessed by MTT tests. The osteogenetic and odontogenetic capacities of the DPSCs were tested using Alizarin red S staining and alkaline phosphatase (ALP) activity assays. In addition, real-time PCR was performed to examine the gene expression of ALP, BMP-2 and DMP-1. Finally, extracellular matrix vesicle secretion was detected via scanning electron microscopy. No significant difference was observed in the viabilities of the DPSCs with and without S. mukorossi seed oil, respectively. However, under osteogenic and odontogenic induction, S. mukorossi seed oil increased the secretion of mineralized nodules and the ALP activity of the DPSCs (p < 0.05). The ALP gene expression of the differentiation-induced DPSCs was also enhanced. Finally, a greater secretion of extracellular matrix vesicles was detected in the DPSCs following odontogenic induction complemented with S. mukorossi seed oil. Overall, the present results show that S. mukorossi seed oil promotes the osteogenic/odontogenic differentiation and matrix vesicle secretion of DPSCs.

Role of matrix vesicles and crystal ghosts in bio-mineralization.

Matrix vesicles (MVs) are extracellular membrane-bound vesicles of about ~ 50-200 nm in diameter that play a role in the bio-mineralization process of hard tissue formation. The present review is based on the empirical phenomenon of primary mineralization process via matrix vesicle-mediated mechanism with special reference to crystal ghosts as well as the mechanism on the organic-inorganic relationship between matrix vesicles and crystal ghosts, and the transformation that these structures undergo during bio-mineralization.

Static magnetic field-enhanced osteogenic differentiation of human umbilical cord-derived mesenchymal stem cells via matrix vesicle secretion.

METHODS: In methodology, WJMSCs were treated with a 0.4-T SMF. The cell viability was tested using the MTT assay. For the osteogenic analysis, the alkaline phosphatase activity assay and alizarin red S staining were performed. The osteogenic-related gene expression of ALP, BMP-2, and Runx2 was examined using real-time polymerase chain reaction. Scanning electron microscopy combined with energy-dispersive X-ray spectroscopy was used to analyze matrix vesicle secretion. RESULTS: The cell viability showed no significant difference between the SMF-treated group and the sham-exposed cells. However, the SMF-treated group exhibited significantly more mineralized nodule formation and higher ALP activity than their control counterparts (p < .05). The expressions of osteogenic-related markers, ALP, BMP-2, and Runx2, were also significantly higher in the SMF-treated WJMSCs. The scanning electron microscopy results showed much more matrix vesicle secretion in the SMF-treated cells than in the sham-treated cells. A mineralized sheath was noted in the SMF-treated cells, along with a sporadic accumulation of spherical mineralized deposits on the cell surface. CONCLUSIONS: The results suggest that 0.4-T SMF treatment enhances the osteogenesis of WJMSCs at the early-to-middle stage of osteogenic differentiation by increasing the matrix vesicle secretion and mineralization.

Lipid composition modulates ATP hydrolysis and calcium phosphate mineral propagation by TNAP-harboring proteoliposomes.

Bone biomineralization is mediated by a special class of extracellular vesicles, named matrix vesicles (MVs), released by osteogenic cells. The MV membrane is enriched in sphingomyelin (SM), cholesterol (Chol) and tissue non-specific alkaline phosphatase (TNAP) compared with the parent cells' plasma membrane. TNAP is an ATP phosphohydrolase bound to cell and MV membranes via a glycosylphosphatidylinositol (GPI) anchor. Previous studies have shown that the lipid microenvironment influences the catalytic activity of enzymes incorporated into lipid bilayers. However, there is a lack of information about how the lipid microenvironment controls the ability of MV membrane-bound enzymes to induce mineral precipitation. Herein, we used TNAP-harboring proteoliposomes made of either pure dimyristoylphosphatidylcholine (DMPC) or DMPC mixed with either Chol, SM or both of them as MV biomimetic systems to evaluate how the composition modulates the lipid microenvironment and, in turn, TNAP incorporation into the lipid bilayer by means of calorimetry. These results were correlated with the proteoliposomes' catalytic activity and ability to induce the precipitation of amorphous calcium phosphate (ACP) in vitro. DMPC:SM proteoliposomes displayed the highest efficiency of mineral propagation, apparent affinity for ATP and substrate hydrolysis efficiency, which correlated with their highest degree of membrane organization (highest ΔH), among the tested proteoliposomes. Results obtained from turbidimetry and Fourier transformed infrared (FTIR) spectroscopy showed that the tested proteoliposomes induced ACP precipitation with the order DMPC:SM>DMPC:Chol:SM≈DMPC:Chol>DMPC which correlated with the lipid organization and the presence of SM in the proteoliposome membrane. Our study arises important insights regarding the physical properties and role of lipid organization in MV-mediated mineralization.

Vascular Calcification-New Insights Into Its Mechanism.

Vascular calcification (VC), which is categorized by intimal and medial calcification, depending on the site(s) involved within the vessel, is closely related to cardiovascular disease. Specifically, medial calcification is prevalent in certain medical situations, including chronic kidney disease and diabetes. The past few decades have seen extensive research into VC, revealing that the mechanism of VC is not merely a consequence of a high-phosphorous and -calcium milieu, but also occurs via delicate and well-organized biologic processes, including an imbalance between osteochondrogenic signaling and anticalcific events. In addition to traditionally established osteogenic signaling, dysfunctional calcium homeostasis is prerequisite in the development of VC. Moreover, loss of defensive mechanisms, by microorganelle dysfunction, including hyper-fragmented mitochondria, mitochondrial oxidative stress, defective autophagy or mitophagy, and endoplasmic reticulum (ER) stress, may all contribute to VC. To facilitate the understanding of vascular calcification, across any number of bioscientific disciplines, we provide this review of a detailed updated molecular mechanism of VC. This encompasses a vascular smooth muscle phenotypic of osteogenic differentiation, and multiple signaling pathways of VC induction, including the roles of inflammation and cellular microorganelle genesis.

How To Build a Bone: PHOSPHO1, Biomineralization, and Beyond.

Since its characterization two decades ago, the phosphatase PHOSPHO1 has been the subject of an increasing focus of research. This work has elucidated PHOSPHO1's central role in the biomineralization of bone and other hard tissues, but has also implicated the enzyme in other biological processes in health and disease. During mineralization PHOSPHO1 liberates inorganic phosphate (Pi) to be incorporated into the mineral phase through hydrolysis of its substrates phosphocholine (PCho) and phosphoethanolamine (PEA). Localization of PHOSPHO1 within matrix vesicles allows accumulation of Pi within a protected environment where mineral crystals may nucleate and subsequently invade the organic collagenous scaffold. Here, we examine the evidence for this process, first discussing the discovery and characterization of PHOSPHO1, before considering experimental evidence for its canonical role in matrix vesicle-mediated biomineralization. We also contemplate roles for PHOSPHO1 in disorders of dysregulated mineralization such as vascular calcification, along with emerging evidence of its activity in other systems including choline synthesis and homeostasis, and energy metabolism. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Roles of Phosphate in Skeleton.

Phosphate is essential for skeletal mineralization, and its chronic deficiency leads to rickets and osteomalacia. Skeletal mineralization starts in matrix vesicles (MVs) derived from the plasma membrane of osteoblasts and chondrocytes. MVs contain high activity of tissue non-specific alkaline phosphatase (TNSALP), which hydrolyzes phosphoric esters such as pyrophosphates (PPi) to produce inorganic orthophosphates (Pi). Extracellular Pi in the skeleton is taken up by MVs through type III sodium/phosphate (Na+/Pi) cotransporters and forms hydroxyapatite. In addition to its roles in MV-mediated skeletal mineralization, accumulating evidence has revealed that extracellular Pi evokes signal transduction and regulates cellular function. Pi induces apoptosis of hypertrophic chondrocytes, which is a critical step for endochondral ossification. Extracellular Pi also regulates the expression of various genes including those related to proliferation, differentiation, and mineralization. In vitro cell studies have demonstrated that an elevation in extracellular Pi level leads to the activation of fibroblast growth factor receptor (FGFR), Raf/MEK (mitogen-activated protein kinase/ERK kinase)/ERK (extracellular signal-regulated kinase) pathway, where the type III Na+/Pi cotransporter PiT-1 may be involved. Responsiveness of skeletal cells to extracellular Pi suggests their ability to sense and adapt to an alteration in Pi availability in their environment. Involvement of FGFR in the Pi-evoked signal transduction is interesting because enhanced FGFR signaling in osteoblasts/osteocytes might be responsible for the overproduction of FGF23, a key molecule in phosphate homeostasis, in a mouse model for human X-linked hypophosphatemic rickets (XLH). Impaired Pi sensing may be a pathogenesis of XLH, which needs to be clarified in future.

RAGE/galectin-3 yields intraplaque calcification transformation via sortilin.

AIMS: Macrocalcification and microcalcification present different clinical risks, but the regulatory of their formation was unclear. Therefore, this study explored the underlying mechanisms of macrocalcification and microcalcification in diabetes mellitus. METHODS: Anterior tibial arteries of amputated diabetic feet were collected. According to the calcium content, patients were divided into less-calcification group and more-calcification group. And calcification morphology in plaques was observed. For further study, an in vivo mouse diabetic atherosclerosis model and an in vitro primary mouse aortic smooth muscle cell model were established. After the receptors for AGEs (RAGE) or galectin-3 were silenced, calcified nodule sizes and sortilin expression were determined. Scanning electron microscopy (SEM) was performed to detect the aggregation of matrix vesicles with the inhibition or promotion of sortilin. RESULTS: Both macro- and microcalcification were found in human anterior tibial artery plaques. Macrocalcification formed after the silencing of RAGE, and microcalcification formed after the silencing of galectin-3. In the process of RAGE- or galcetin-3-induced calcification, sortilin played an important role downstream. SEM showed that sortilin promoted the aggregation of MVs in the early stage of calcification and formed larger calcified nodules. CONCLUSION: RAGE downregulated sortilin and then transmitted microcalcification signals, whereas galectin-3 upregulated sortilin, which accelerated the aggregation of MVs in the early stage of calcification and mediated the formation of macrocalcifications, These data illustrate the progression of two calcification types and suggest sortilin as a potential target for early intervention of calcification and as an effective biomarker for the assessment of long-term clinical risk and prognosis.

Biomimetic mineralization using matrix vesicle nanofragments.

In vitro synthesis of bone tissue has been paid attention in recent years; however, current methods to fabricate bone tissue are still ineffective due to some remaining gaps in the understanding of real in vivo bone formation process, and application of the knowledge in bone synthesis. Therefore, the objectives of this study were first, to perform a systematic and ultrastructural investigation of the initial mineral formation during intramembranous ossification of mouse calvaria from a material scientists' viewpoint, and to develop novel mineralization methods based on the in vivo findings. First, the very initial mineral deposition was found to occur at embryonic day E14.0 in mouse calvaria. Analysis of the initial bone formation process showed that it involved the following distinct steps: collagen secretion, matrix vesicle (MV) release, MV mineralization, MV rupture, and collagen fiber mineralization. Next, we performed in vitro mineralization experiments using MVs and hydrogel scaffolds. Intact MVs embedded in collagen gel did not mineralize, whereas, interestingly, MV nanofragments obtained by ultrasonication could promote rapid mineralization. These results indicate that mechanically ruptured MV membrane can be a promising material for in vitro bone tissue synthesis. © 2019 The Authors. journal Of Biomedical Materials Research Part A Published By Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1021-1030, 2019.

Emerging Role of Vitamins D and K in Modulating Uremic Vascular Calcification: The Aspect of Passive Calcification.

Vascular calcification is a critical complication in patients with chronic kidney disease (CKD) because it is predictive of cardiovascular events and mortality. In addition to the traditional mechanisms associated with endothelial dysfunction and the osteoblastic transformation of vascular smooth muscle cells (VSMCs), the regulation of calcification inhibitors, such as calciprotein particles (CPPs) and matrix vesicles plays a vital role in uremic vascular calcification in CKD patients because of the high prevalence of vitamin K deficiency. Vitamin K governs the gamma-carboxylation of matrix Gla protein (MGP) for inhibiting vascular calcification, and the vitamin D binding protein receptor is related to vitamin K gene expression. For patients with chronic kidney disease, adequate use of vitamin D supplements may play a role in vascular calcification through modulation of the calciprotein particles and matrix vesicles (MVs).