Differential effects of the lipidic and ionic microenvironment on NPP1's phosphohydrolase and phosphodiesterase activities.

Ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) is an enzyme present in matrix vesicles (MV). NPP1 participates on the regulation of bone formation by producing pyrophosphate (PPi) from adenosine triphosphate (ATP). Here, we have used liposomes bearing dipalmitoylphosphatidylcholine (DPPC), sphingomyelin (SM), and cholesterol (Chol) harboring NPP1 to mimic the composition of MV lipid rafts to investigate ionic and lipidic influence on NPP1 activity and mineral propagation. Atomic force microscopy (AFM) revealed that DPPC-liposomes had spherical and smooth surface. The presence of SM and Chol elicited rough and smooth surface, respectively. NPP1 insertion produced protrusions in all the liposome surface. Maximum phosphodiesterase activity emerged at 0.082 M ionic strength, whereas maximum phosphomonohydrolase activity arose at low ionic strength. Phosphoserine-Calcium Phosphate Complex (PS-CPLX) and amorphous calcium-phosphate (ACP) induced mineral propagation in DPPC- and DPPC:SM-liposomes and in DPPC:Chol-liposomes, respectively. Mineral characterization revealed the presence of bands assigned to HAp in the mineral propagated by NPP1 harbored in DPPC-liposomes without nucleators or in DPPC:Chol-liposomes with ACP nucleators. These data show that studying how the ionic and lipidic environment affects NPP1 properties is important, especially for HAp obtained under controlled conditions in vitro.

Evidence that pyrophosphate acts as an extracellular signalling molecule to exert direct functional effects in primary cultures of osteoblasts and osteoclasts.

Extracellular pyrophosphate (PPi) is well known for its fundamental role as a physiochemical mineralisation inhibitor. However, information about its direct actions on bone cells remains limited. This study shows that PPi decreased osteoclast formation and resorptive activity by ≤50 %. These inhibitory actions were associated with reduced expression of genes involved in osteoclastogenesis (Tnfrsf11a, Dcstamp) and bone resorption (Ctsk, Car2, Acp5). In osteoblasts, PPi present for the entire (0-21 days) or latter stages of culture (7-21/14-21 days) decreased bone mineralisation by ≤95 %. However, PPi present for the differentiation phase only (0-7/0-14 days) increased bone formation (≤70 %). Prolonged treatment with PPi resulted in earlier matrix deposition and increased soluble collagen levels (≤2.3-fold). Expression of osteoblast (RUNX2, Bglap) and early osteocyte (E11, Dmp1) genes along with mineralisation inhibitors (Spp1, Mgp) was increased by PPi (≤3-fold). PPi levels are regulated by tissue non-specific alkaline phosphatase (TNAP) and ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1). PPi reduced NPP1 expression in both cell types whereas TNAP expression (≤2.5-fold) and activity (≤35 %) were increased in osteoblasts. Breakdown of extracellular ATP by NPP1 represents a key source of PPi. ATP release from osteoclasts and osteoblasts was decreased ≤60 % by PPi and by a selective TNAP inhibitor (CAS496014-12-2). Pertussis toxin, which prevents Gαi subunit activation, was used to investigate whether G-protein coupled receptor (GPCR) signalling mediates the effects of PPi. The actions of PPi on bone mineralisation, collagen production, ATP release, gene/protein expression and osteoclast formation were abolished or attenuated by pertussis toxin. Together these findings show that PPi, modulates differentiation, function and gene expression in osteoblasts and osteoclasts. The ability of PPi to alter ATP release and NPP1/TNAP expression and activity indicates that cells can detect PPi levels and respond accordingly. Our data also raise the possibility that some actions of PPi on bone cells could be mediated by a Gαi-linked GPCR.

Gene Therapy Using Recombinant AAV Type 8 Vector Encoding TNAP-D10 Improves the Skeletal Phenotypes in Murine Models of Osteomalacia.

Hypophosphatasia (HPP), caused by loss-of-function mutations in the ALPL gene encoding tissue-nonspecific alkaline phosphatase (TNAP), is characterized by skeletal and dental hypomineralization that can vary in severity from life-threatening to milder manifestations only in adulthood. PHOSPHO1 deficiency leads to early-onset scoliosis, osteomalacia, and fractures that mimic pseudo-HPP. Asfotase alfa, a life-saving enzyme replacement therapy approved for pediatric-onset HPP, requires subcutaneous injections 3 to 6 times per week. We recently showed that a single injection of an adeno-associated virus vector serotype 8 harboring TNAP-D10 (AAV8-TNAP-D10) effectively prevented skeletal disease and prolonged life in Alpl -/- mice phenocopying infantile HPP. Here, we aimed to determine the efficacy of AAV8-TNAP-D10 in improving the skeletal and dental phenotype in the Alpl Prx1/Prx1 and Phospho1 -/- mouse models of late-onset (adult) HPP and pseudo-HPP, respectively. A single dose of 3 × 1011 vector genomes per body (vg/b) was injected intramuscularly into 8-week-old Alpl Prx1/Prx1 and wild-type (WT) littermates, or into 3-day-old Phospho1 -/- and WT mice, and treatment efficacy was evaluated after 60 days for late-onset HPP mice and after 90 days for Phospho1 -/- mice. Biochemical analysis showed sustained serum alkaline phosphatase activity and reduced plasma PPi levels, and radiographic images, micro-computed tomography (micro-CT) analysis, and hematoxylin and eosin (H&E) staining showed improvements in the long bones in the late-onset HPP mice and corrected scoliosis in the Phospho1 -/- mice. Micro-CT analysis of the dentoalveolar complex did not reveal significant changes in the phenotype of late-onset HPP and pseudo-HPP models. Moreover, alizarin red staining analysis showed that AAV8-TNAP-D10 treatment did not promote ectopic calcification of soft organs in adult HPP mice after 60 days of treatment, even after inducing chronic kidney disease. Overall, the AAV8-TNAP-D10 treatment improved the skeletal phenotype in both the adult HPP and pseudo-HPP mouse models. This preclinical study will contribute to the advancement of gene therapy for the improvement of skeletal disease in patients with heritable forms of osteomalacia. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Inhibition of alkaline phosphatase impairs dyslipidemia and protects mice from atherosclerosis.

Calcium accumulation in atherosclerotic plaques predicts cardiovascular mortality, but the mechanisms responsible for plaque calcification and how calcification impacts plaque stability remain debated. Tissue-nonspecific alkaline phosphatase (TNAP) recently emerged as a promising therapeutic target to block cardiovascular calcification. In this study, we sought to investigate the effect of the recently developed TNAP inhibitor SBI-425 on atherosclerosis plaque calcification and progression. TNAP levels were investigated in ApoE-deficient mice fed a high-fat diet from 10 weeks of age and in plaques from the human ECLAGEN biocollection (101 calcified and 14 non-calcified carotid plaques). TNAP was inhibited in mice using SBI-425 administered from 10 to 25 weeks of age, and in human vascular smooth muscle cells (VSMCs) with MLS-0038949. Plaque calcification was imaged in vivo with 18F-NaF-PET/CT, ex vivo with osteosense, and in vitro with alizarin red. Bone architecture was determined with µCT. TNAP activation preceded and predicted calcification in human and mouse plaques, and TNAP inhibition prevented calcification in human VSMCs and in ApoE-deficient mice. More unexpectedly, TNAP inhibition reduced the blood levels of cholesterol and triglycerides, and protected mice from atherosclerosis, without impacting the skeletal architecture. Metabolomics analysis of liver extracts identified phosphocholine as a substrate of liver TNAP, who's decreased dephosphorylation upon TNAP inhibition likely reduced the release of cholesterol and triglycerides into the blood. Systemic inhibition of TNAP protects from atherosclerosis, by ameliorating dyslipidemia, and preventing plaque calcification.

NPP1 and TNAP hydrolyze ATP synergistically during biomineralization.

Matrix vesicles (MVs) are a special class of extracellular vesicles released by mineralizing cells during bone and tooth mineralization that initiate the precipitation of apatitic minerals by regulating the extracellular ratio between inorganic phosphate (Pi), a calcification promoter, and pyrophosphate (PPi), a calcification inhibitor. The Pi/PPi ratio is thought to be controlled by two ecto-phosphatases present on the outer leaflet of the MVs' membrane: ectonucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) that produces PPi as well as Pi from ATP and tissue-nonspecific alkaline phosphatase (TNAP) that hydrolyzes both ATP and PPi to generate Pi. However, if and how these enzymes act in concert in MVs are still unclear. Herein, we investigated the role of NPP1 and TNAP in ATP hydrolysis during MV-mediated biomineralization using proteoliposomes as a biomimetic model for MVs. Proteoliposomes composed by 1,2-dipalmitoylphosphatidylcholine (DPPC) and harboring NPP1 alone, TNAP alone, or both together at different molar ratios (1:1, 10:1, and 1:10) were fabricated. After 48 h of incubation with ATP, TNAP-containing proteoliposomes consumed more ATP than NPP1-containing vesicles (270 and 210 nmol, respectively). Both types of vesicles comparatively formed ADP (205 and 201 nmol, respectively), while NPP1-containing vesicles hydrolyzed AMP less efficiently than TNAP-containing proteoliposomes (10 and 25 nmol, respectively). In vitro mineralization assays showed that in the presence of ATP, TNAP-harboring proteoliposomes mineralized through a sigmoidal single-step process, while NPP1-harboring vesicles displayed a two-step mineralization process. ATR-FTIR analyses showed that the minerals produced by TNAP-harboring proteoliposomes were structurally more similar to hydroxyapatite than those produced by NPP1-harboring vesicles. Our results with proteoliposomes indicate that the pyrophosphohydrolase function of NPP1 and the phosphohydrolase activity of TNAP act synergistically to produce a Pi/PPi ratio conducive to mineralization and the synergism is maximal when the two enzymes are present at equimolar concentrations. The significance of these findings for hypophosphatasia is discussed.

Do Media Extracellular Vesicles and Extracellular Vesicles Bound to the Extracellular Matrix Represent Distinct Types of Vesicles?

Mineralization-competent cells, including hypertrophic chondrocytes, mature osteoblasts, and osteogenic-differentiated smooth muscle cells secrete media extracellular vesicles (media vesicles) and extracellular vesicles bound to the extracellular matrix (matrix vesicles). Media vesicles are purified directly from the extracellular medium. On the other hand, matrix vesicles are purified after discarding the extracellular medium and subjecting the cells embedded in the extracellular matrix or bone or cartilage tissues to an enzymatic treatment. Several pieces of experimental evidence indicated that matrix vesicles and media vesicles isolated from the same types of mineralizing cells have distinct lipid and protein composition as well as functions. These findings support the view that matrix vesicles and media vesicles released by mineralizing cells have different functions in mineralized tissues due to their location, which is anchored to the extracellular matrix versus free-floating.

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.

Perspective on Dentoalveolar Manifestations Resulting From PHOSPHO1 Loss-of-Function: A Form of Pseudohypophosphatasia?

Mineralization of the skeleton occurs by several physicochemical and biochemical processes and mechanisms that facilitate the deposition of hydroxyapatite (HA) in specific areas of the extracellular matrix (ECM). Two key phosphatases, phosphatase, orphan 1 (PHOSPHO1) and tissue-non-specific alkaline phosphatase (TNAP), play complementary roles in the mineralization process. The actions of PHOSPHO1 on phosphocholine and phosphoethanolamine in matrix vesicles (MVs) produce inorganic phosphate (Pi) for the initiation of HA mineral formation within MVs. TNAP hydrolyzes adenosine triphosphate (ATP) and the mineralization inhibitor, inorganic pyrophosphate (PPi), to generate Pi that is incorporated into MVs. Genetic mutations in the ALPL gene-encoding TNAP lead to hypophosphatasia (HPP), characterized by low circulating TNAP levels (ALP), rickets in children and/or osteomalacia in adults, and a spectrum of dentoalveolar defects, the most prevalent being lack of acellular cementum leading to premature tooth loss. Given that the skeletal manifestations of genetic ablation of the Phospho1 gene in mice resemble many of the manifestations of HPP, we propose that Phospho1 gene mutations may underlie some cases of "pseudo-HPP" where ALP may be normal to subnormal, but ALPL mutation(s) have not been identified. The goal of this perspective article is to compare and contrast the loss-of-function effects of TNAP and PHOSPHO1 on the dentoalveolar complex to predict the likely dental phenotype in humans that may result from PHOSPHO1 mutations. Potential cases of pseudo-HPP associated with PHOSPHO1 mutations may resist diagnosis, and the dental manifestations could be a key criterion for consideration.

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.

TNAP as a therapeutic target for cardiovascular calcification: a discussion of its pleiotropic functions in the body.

Cardiovascular calcification (CVC) is associated with increased morbidity and mortality. It develops in several diseases and locations, such as in the tunica intima in atherosclerosis plaques, in the tunica media in type 2 diabetes and chronic kidney disease, and in aortic valves. In spite of the wide occurrence of CVC and its detrimental effects on cardiovascular diseases (CVD), no treatment is yet available. Most of CVC involve mechanisms similar to those occurring during endochondral and/or intramembranous ossification. Logically, since tissue-nonspecific alkaline phosphatase (TNAP) is the key-enzyme responsible for skeletal/dental mineralization, it is a promising target to limit CVC. Tools have recently been developed to inhibit its activity and preclinical studies conducted in animal models of vascular calcification already provided promising results. Nevertheless, as its name indicates, TNAP is ubiquitous and recent data indicate that it dephosphorylates different substrates in vivo to participate in other important physiological functions besides mineralization. For instance, TNAP is involved in the metabolism of pyridoxal phosphate and the production of neurotransmitters. TNAP has also been described as an anti-inflammatory enzyme able to dephosphorylate adenosine nucleotides and lipopolysaccharide. A better understanding of the full spectrum of TNAP's functions is needed to better characterize the effects of TNAP inhibition in diseases associated with CVC. In this review, after a brief description of the different types of CVC, we describe the newly uncovered additional functions of TNAP and discuss the expected consequences of its systemic inhibition in vivo.

Treatment with bone maturation and average lifespan of HPP model mice by AAV8-mediated neonatal gene therapy via single muscle injection.

Hypophosphatasia (HPP) is an inherited skeletal disease characterized by defective bone and tooth mineralization due to a deficiency in tissue-nonspecific alkaline phosphatase (TNALP). Patients with the severe infantile form of HPP may appear normal at birth, but their prognosis is very poor. To develop a practical gene therapy for HPP, we endeavored to phenotypically correct TNALP knockout (Akp2 -/- ) mice through adeno-associated virus type 8 (AAV8) vector-mediated, muscle-directed, TNALP expression. Following treatment of neonatal Akp2 -/- mice with a single intramuscular injection of ARU-2801 (AAV8-TNALP-D10-vector) at 1.0 × 1012 vector genomes/body, high plasma ALP levels (19.38 ± 5.02 U/mL) were detected for up to 18 months, and computed tomography analysis showed mature bone mineralization. Histochemical staining for ALP activity in the knee joint revealed ALP activity on the surface of the endosteal bone of mice. Throughout their lives, the surviving treated Akp2 -/- mice exhibited normal physical activity and a healthy appearance, whereas untreated controls died within 3 weeks. No ectopic calcification or abnormal calcium metabolism was detected in the treated mice. These findings suggest that ARU-2801-mediated neonatal intramuscular gene therapy is both safe and effective, and that this strategy could be a practical option for treatment of the severe infantile form of HPP.

Gene Therapy Using Adeno-Associated Virus Serotype 8 Encoding TNAP-D10 Improves the Skeletal and Dentoalveolar Phenotypes in Alpl-/- Mice.

Hypophosphatasia (HPP) is caused by loss-of-function mutations in the ALPL gene that encodes tissue-nonspecific alkaline phosphatase (TNAP), whose deficiency results in the accumulation of extracellular inorganic pyrophosphate (PPi ), a potent mineralization inhibitor. Skeletal and dental hypomineralization characterizes HPP, with disease severity varying from life-threatening perinatal or infantile forms to milder forms that manifest in adulthood or only affect the dentition. Enzyme replacement therapy (ERT) using mineral-targeted recombinant TNAP (Strensiq/asfotase alfa) markedly improves the life span, skeletal phenotype, motor function, and quality of life of patients with HPP, though limitations of ERT include frequent injections due to a short elimination half-life of 2.28 days and injection site reactions. We tested the efficacy of a single intramuscular administration of adeno-associated virus 8 (AAV8) encoding TNAP-D10 to increase the life span and improve the skeletal and dentoalveolar phenotypes in TNAP knockout (Alpl-/- ) mice, a murine model for severe infantile HPP. Alpl-/- mice received 3 × 1011 vector genomes/body of AAV8-TNAP-D10 within 5 days postnatal (dpn). AAV8-TNAP-D10 elevated serum ALP activity and suppressed plasma PPi . Treatment extended life span of Alpl-/- mice, and no ectopic calcifications were observed in the kidneys, aorta, coronary arteries, or brain in the 70 dpn observational window. Treated Alpl-/- mice did not show signs of rickets, including bowing of long bones, enlargement of epiphyses, or fractures. Bone microstructure of treated Alpl-/- mice was similar to wild type, with a few persistent small cortical and trabecular defects. Histology showed no measurable osteoid accumulation but reduced bone volume fraction in treated Alpl-/- mice versus controls. Treated Alpl-/- mice featured normal molar and incisor dentoalveolar tissues, with the exceptions of slightly reduced molar enamel and alveolar bone density. Histology showed the presence of cementum and normal periodontal ligament attachment. These results support gene therapy as a promising alternative to ERT for the treatment of HPP. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Phosphate Groups in the Lipid A Moiety Determine the Effects of LPS on Hepatic Stellate Cells: A Role for LPS-Dephosphorylating Activity in Liver Fibrosis.

Alkaline phosphatase (AP) activity is highly upregulated in plasma during liver diseases. Previously, we demonstrated that AP is able to detoxify lipopolysaccharide (LPS) by dephosphorylating its lipid A moiety. Because a role of gut-derived LPS in liver fibrogenesis has become evident, we now examined the relevance of phosphate groups in the lipid A moiety in this process. The effects of mono-phosphoryl and di-phosphoryl lipid A (MPLA and DPLA, respectively) were studied in vitro and LPS-dephosphorylating activity was studied in normal and fibrotic mouse and human livers. The effects of intestinal AP were studied in mice with CCL4-induced liver fibrosis. DPLA strongly stimulated fibrogenic and inflammatory activities in primary rat hepatic stellate cells (rHSCs) and RAW264.7 macrophages with similar potency as full length LPS. However, MPLA did not affect any of the parameters. LPS-dephosphorylating activity was found in mouse and human livers and was strongly increased during fibrogenesis. Treatment of fibrotic mice with intravenous intestinal-AP significantly attenuated intrahepatic desmin+- and αSMA+ -HSC and CD68+- macrophage accumulation. In conclusion, the lack of biological activity of MPLA, contrasting with the profound activities of DPLA, shows the relevance of LPS-dephosphorylating activity. The upregulation of LPS-dephosphorylating activity in fibrotic livers and the protective effects of exogenous AP during fibrogenesis indicate an important physiological role of intestinal-derived AP during liver fibrosis.

Inhibition of vascular smooth muscle cell calcification by ATP analogues.

Arterial medial calcification (AMC) has been associated with phenotypic changes in vascular smooth muscle cells (VSMCs) that reportedly makes them more osteoblast-like. Previous work has shown that ATP/UTP can inhibit AMC directly via P2 receptors and indirectly by NPP1-mediated hydrolysis to produce the mineralisation inhibitor, pyrophosphate (PPi). This study investigated the role of P2X receptors in the inhibitory effects of extracellular nucleotides on VSMC calcification. We found that Bz-ATP, α,ß-meATP and ß,γ-meATP inhibited calcification by up to 100%. Culture in a high-phosphate medium (2 mM) was associated with increased VSMC death and apoptosis; treatment with Bz-ATP, α,ß-meATP and ß,γ-meATP reduced apoptosis to levels seen in non-calcifying cells. Calcification was also associated with alterations in the protein levels of VSMC (e.g. SM22α and SMA) and osteoblast-associated (e.g. Runx2 and osteopontin) markers; Bz-ATP, α,ß-meATP and ß,γ-meATP attenuated these changes in protein expression. Long-term culture with Bz-ATP, α,ß-meATP and ß,γ-meATP resulted in lower extracellular ATP levels and an increased rate of ATP breakdown. P2X receptor antagonists failed to prevent the inhibitory effects of these analogues suggesting that they act via P2X receptor-independent mechanisms. In agreement, the breakdown products of α,ß-meATP and ß,γ-meATP (α,ß-meADP and methylene diphosphonate, respectively) also dose-dependently inhibited VSMC calcification. Furthermore, the actions of Bz-ATP, α,ß-meATP and ß,γ-meATP were unchanged in VSMCs isolated from NPP1-knockout mice, suggesting that the functional effects of these compounds do not involve NPP1-mediated generation of PPi. Together, these results indicate that the inhibitory effects of ATP analogues on VSMC calcification and apoptosis in vitro may be mediated, at least in part, by mechanisms that are independent of purinergic signalling and PPi.

Lipid microenvironment affects the ability of proteoliposomes harboring TNAP to induce mineralization without nucleators.

Tissue-nonspecific alkaline phosphatase (TNAP), a glycosylphosphatidylinositol-anchored ectoenzyme present on the membrane of matrix vesicles (MVs), hydrolyzes the mineralization inhibitor inorganic pyrophosphate as well as ATP to generate the inorganic phosphate needed for apatite formation. Herein, we used proteoliposomes harboring TNAP as MV biomimetics with or without nucleators of mineral formation (amorphous calcium phosphate and complexes with phosphatidylserine) to assess the role of the MVs' membrane lipid composition on TNAP activity by means of turbidity assay and FTIR analysis. We found that TNAP-proteoliposomes have the ability to induce mineralization even in the absence of mineral nucleators. We also found that the addition of cholesterol or sphingomyelin to TNAP-proteoliposomes composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine reduced the ability of TNAP to induce biomineralization. Our results suggest that the lipid microenvironment is essential for the induction and propagation of minerals mediated by TNAP.

Inhibition of Tissue-Nonspecific Alkaline Phosphatase Attenuates Ectopic Mineralization in the Abcc6-/- Mouse Model of PXE but Not in the Enpp1 Mutant Mouse Models of GACI.

Pseudoxanthoma elasticum (PXE), a prototype of heritable ectopic mineralization disorders, is caused by mutations in the ABCC6 gene encoding a putative efflux transporter ABCC6. It was recently shown that the absence of ABCC6-mediated adenosine triphosphate release from the liver and, consequently, reduced inorganic pyrophosphate levels underlie the pathogenesis of PXE. Given that tissue-nonspecific alkaline phosphatase (TNAP), encoded by ALPL, is the enzyme responsible for degrading inorganic pyrophosphate, we hypothesized that reducing TNAP levels either by genetic or pharmacological means would lead to amelioration of the ectopic mineralization phenotype in the Abcc6-/- mouse model of PXE. Thus, we bred Abcc6-/- mice to heterozygous Alpl+/- mice that display approximately 50% plasma TNAP activity. The Abcc6-/-Alpl+/- double-mutant mice showed 52% reduction of mineralization in the muzzle skin compared with the Abcc6-/-Alpl+/+ mice. Subsequently, oral administration of SBI-425, a small molecule inhibitor of TNAP, resulted in 61% reduction of plasma TNAP activity and 58% reduction of mineralization in the muzzle skin of Abcc6-/- mice. By contrast, SBI-425 treatment of Enpp1 mutant mice, another model of ectopic mineralization associated with reduced inorganic pyrophosphate, failed to reduce muzzle skin mineralization. These results suggest that inhibition of TNAP might provide a promising treatment strategy for PXE, a currently intractable disease.

Enhanced phosphocholine metabolism is essential for terminal erythropoiesis.

Red cells contain a unique constellation of membrane lipids. Although much is known about regulated protein expression, the regulation of lipid metabolism during erythropoiesis is poorly studied. Here, we show that transcription of PHOSPHO1, a phosphoethanolamine and phosphocholine phosphatase that mediates the hydrolysis of phosphocholine to choline, is strongly upregulated during the terminal stages of erythropoiesis of both human and mouse erythropoiesis, concomitant with increased catabolism of phosphatidylcholine (PC) and phosphocholine as shown by global lipidomic analyses of mouse and human terminal erythropoiesis. Depletion of PHOSPHO1 impaired differentiation of fetal mouse and human erythroblasts, and, in adult mice, depletion impaired phenylhydrazine-induced stress erythropoiesis. Loss of PHOSPHO1 also impaired phosphocholine catabolism in mouse fetal liver progenitors and resulted in accumulation of several lipids; adenosine triphosphate (ATP) production was reduced as a result of decreased oxidative phosphorylation. Glycolysis replaced oxidative phosphorylation in PHOSPHO1-knockout erythroblasts and the increased glycolysis was used for the production of serine or glycine. Our study elucidates the dynamic changes in lipid metabolism during terminal erythropoiesis and reveals the key roles of PC and phosphocholine metabolism in energy balance and amino acid supply.

Matrix vesicles from chondrocytes and osteoblasts: Their biogenesis, properties, functions and biomimetic models.

BACKGROUND: Matrix vesicles (MVs) are released from hypertrophic chondrocytes and from mature osteoblasts, the cells responsible for endochondral and membranous ossification. Under pathological conditions, they can also be released from cells of non-skeletal tissues such as vascular smooth muscle cells. MVs are extracellular vesicles of approximately 100-300nm diameter harboring the biochemical machinery needed to induce mineralization. SCOPE OF THE REVIEW: The review comprehensively delineates our current knowledge of MV biology and highlights open questions aiming to stimulate further research. The review is constructed as a series of questions addressing issues of MVs ranging from their biogenesis and functions, to biomimetic models. It critically evaluates experimental data including their isolation and characterization methods, like lipidomics, proteomics, transmission electron microscopy, atomic force microscopy and proteoliposome models mimicking MVs. MAJOR CONCLUSIONS: MVs have a relatively well-defined function as initiators of mineralization. They bind to collagen and their composition reflects the composition of lipid rafts. We call attention to the as yet unclear mechanisms leading to the biogenesis of MVs, and how minerals form and when they are formed. We discuss the prospects of employing upcoming experimental models to deepen our understanding of MV-mediated mineralization and mineralization disorders such as the use of reconstituted lipid vesicles, proteoliposomes and, native sample preparations and high-resolution technologies. GENERAL SIGNIFICANCE: MVs have been extensively investigated owing to their roles in skeletal and ectopic mineralization. MVs serve as a model system for lipid raft structures, and for the mechanisms of genesis and release of extracellular vesicles.

Overexpression of tissue-nonspecific alkaline phosphatase (TNAP) in endothelial cells accelerates coronary artery disease in a mouse model of familial hypercholesterolemia.

OBJECTIVE: Overexpression of tissue-nonspecific alkaline phosphatase (TNAP) in endothelium leads to arterial calcification in mice. The purpose of this study was to examine the effect of elevated endothelial TNAP on coronary atherosclerosis. In addition, we aimed to examine endogenous TNAP activity in human myocardium. APPROACH AND RESULTS: A vascular pattern of TNAP activity was observed in human non-failing, ischemic, and idiopathic dilated hearts (5 per group); no differences were noted between groups in this study. Endothelial overexpression of TNAP was achieved in mice harboring a homozygous recessive mutation in the low density lipoprotein receptor (whc allele) utilizing a Tie2-cre recombinase (WHC-eTNAP mice). WHC-eTNAP developed significant coronary artery calcification at baseline compared WHC controls (4312 vs 0µm2 alizarin red area, p<0.001). Eight weeks after induction of atherosclerosis, lipid deposition in the coronary arteries of WHC-eTNAP was increased compared to WHC controls (121633 vs 9330µm2 oil red O area, p<0.05). Coronary lesions in WHC-eTNAP mice exhibited intimal thickening, calcifications, foam cells, and necrotic cores. This was accompanied by the reduction in body weight and left ventricular ejection fraction (19.5 vs. 23.6g, p<0.01; 35% vs. 47%, p<0.05). In a placebo-controlled experiment under atherogenic conditions, pharmacological inhibition of TNAP in WHC-eTNAP mice by a specific inhibitor SBI-425 (30mg*kg-1*d-1, for 5 weeks) reduced coronary calcium (78838 vs.144622µm2) and lipids (30754 vs. 77317µm2); improved body weight (22.4 vs.18.8g) and ejection fraction (59 vs. 47%). The effects of SBI-425 were significant in the direct comparisons with placebo but disappeared after TNAP-negative placebo-treated group was included in the models as healthy controls. CONCLUSIONS: Endogenous TNAP activity is present in human cardiac tissues. TNAP overexpression in vascular endothelium in mice leads to an unusual course of coronary atherosclerosis, in which calcification precedes lipid deposition. The prevalence and significance of this mechanism in human atherosclerosis requires further investigations.

A distinctive patchy osteomalacia characterises Phospho1-deficient mice.

The phosphatase PHOSPHO1 is involved in the initiation of biomineralisation. Bones in Phospho1 knockout (KO) mice show histological osteomalacia with frequent bowing of long bones and spontaneous fractures: they contain less mineral, with smaller mineral crystals. However, the consequences of Phospho1 ablation on the microscale structure of bone are not yet fully elucidated. Tibias and femurs obtained from wild-type and Phospho1 null (KO) mice (25-32 weeks old) were embedded in PMMA, cut and polished to produce near longitudinal sections. Block surfaces were studied using 20 kV backscattered-electron (BSE) imaging, and again after iodine staining to reveal non-mineralised matrix and cellular components. For 3D characterisation, we used X-ray micro-tomography. Bones opened with carbide milling tools to expose endosteal surfaces were macerated using an alkaline bacterial pronase enzyme detergent, 5% hydrogen peroxide and 7% sodium hypochlorite solutions to produce 3D surfaces for study with 3D BSE scanning electron microscopy (SEM). Extensive regions of both compact cortical and trabecular bone matrix in Phospho1 KO mice contained no significant mineral and/or showed arrested mineralisation fronts, characterised by a failure in the fusion of the calcospherite-like, separately mineralising, individual micro-volumes within bone. Osteoclastic resorption of the uncalcified matrix in Phospho1 KO mice was attenuated compared with surrounding normally mineralised bone. The extent and position of this aberrant biomineralisation varied considerably between animals, contralateral limbs and anatomical sites. The most frequent manifestation lay, however, in the nearly complete failure of mineralisation in the bone surrounding the numerous transverse blood vessel canals in the cortices. In conclusion, SEM disclosed defective mineralising fronts and extensive patchy osteomalacia, which has previously not been recognised. These data further confirm the role of this phosphatase in physiological skeletal mineralisation.