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.

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.

Distinct role of mitochondrial function and protein kinase C in intimal and medial calcification in vitro.

Introduction: Vascular calcification (VC) is a major risk factor for cardiovascular morbidity and mortality. Depending on the location of mineral deposition within the arterial wall, VC is classified as intimal and medial calcification. Using in vitro mineralization assays, we developed protocols triggering both types of calcification in vascular smooth muscle cells (SMCs) following diverging molecular pathways. Materials and methods and results: Human coronary artery SMCs were cultured in osteogenic medium (OM) or high calcium phosphate medium (CaP) to induce a mineralized extracellular matrix. OM induces osteoblast-like differentiation of SMCs-a key process in intimal calcification during atherosclerotic plaque remodeling. CaP mimics hyperphosphatemia, associated with chronic kidney disease-a risk factor for medial calcification. Transcriptomic analysis revealed distinct gene expression profiles of OM and CaP-calcifying SMCs. OM and CaP-treated SMCs shared 107 differentially regulated genes related to SMC contraction and metabolism. Real-time extracellular efflux analysis demonstrated decreased mitochondrial respiration and glycolysis in CaP-treated SMCs compared to increased mitochondrial respiration without altered glycolysis in OM-treated SMCs. Subsequent kinome and in silico drug repurposing analysis (Connectivity Map) suggested a distinct role of protein kinase C (PKC). In vitro validation experiments demonstrated that the PKC activators prostratin and ingenol reduced calcification triggered by OM and promoted calcification triggered by CaP. Conclusion: Our direct comparison results of two in vitro calcification models strengthen previous observations of distinct intracellular mechanisms that trigger OM and CaP-induced SMC calcification in vitro. We found a differential role of PKC in OM and CaP-calcified SMCs providing new potential cellular and molecular targets for pharmacological intervention in VC. Our data suggest that the field should limit the generalization of results found in in vitro studies using different calcification protocols.

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.

Inflammation and Microcalcification: A Never-Ending Vicious Cycle in Atherosclerosis?

Inflammatory cells and cytokines are known for long to worsen the development of atherosclerotic plaques in mice, and intense efforts are today devoted to develop anti-inflammatory therapeutic strategies to slow down plaque development. Increasing data indicate that plaque inflammation is intimately associated with microcalcifications, which exert harmful effects eventually culminating with plaque rupture. In this review article, we will first introduce microcalcification location, detection, and effects in atherosclerotic plaques. Then, we will present the numerous data suggesting that inflammatory cells and molecules are responsible for the formation of microcalcifications and the articles showing that microcalcifications stimulate macrophages and smooth muscle cells to produce more pro-inflammatory cytokines. Finally, we will discuss the possibility that microcalcifications might stimulate smooth muscle cells to produce larger and more stable calcifications to stabilize plaques, to exit the vicious cycle associating inflammation and microcalcification in atherosclerotic plaques.

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.

Simultaneous assessment of microcalcifications and morphological criteria of vulnerability in carotid artery plaque using hybrid 18F-NaF PET/MRI.

BACKGROUND: Previous studies have suggested the role of microcalcifications in plaque vulnerability. This exploratory study sought to assess the potential of hybrid positron-emission tomography (PET)/magnetic resonance imaging (MRI) using 18F-sodium fluoride (18F-NaF) to check simultaneously 18F-NaF uptake, a marker of microcalcifications, and morphological criteria of vulnerability. METHODS AND RESULTS: We included 12 patients with either recently symptomatic or asymptomatic carotid stenosis. All patients underwent 18F-NaF PET/MRI. 18F-NaF target-to-background ratio (TBR) was measured in culprit and nonculprit (including contralateral plaques of symptomatic patients) plaques as well as in other arterial walls. Morphological criteria of vulnerability were assessed on MRI. Mineral metabolism markers were also collected. 18F-NaF uptake was higher in culprit compared to nonculprit plaques (median TBR 2.6 [2.2-2.8] vs 1.7 [1.3-2.2]; P = 0.03) but was not associated with morphological criteria of vulnerability on MRI. We found a positive correlation between 18F-NaF uptake and calcium plaque volume and ratio but not with circulating tissue-nonspecific alkaline phosphatase (TNAP) activity and inorganic pyrophosphate (PPi) levels. 18F-NaF uptake in the other arterial walls did not differ between symptomatic and asymptomatic patients. CONCLUSIONS: 18F-NaF PET/MRI may be a promising tool for providing additional insights into the plaque vulnerability.

TNAP: A New Multitask Enzyme in Energy Metabolism.

Tissue-nonspecific alkaline phosphatase (TNAP) is mainly known for its necessary role in skeletal and dental mineralization, which relies on the hydrolysis of the mineralization inhibitor inorganic pyrophosphate (PPi). Mutations in the gene encoding TNAP leading to severe hypophosphatasia result in strongly reduced mineralization and perinatal death. Fortunately, the relatively recent development of a recombinant TNAP with a bone anchor has allowed to correct the bone defects and prolong the life of affected babies and children. Researches on TNAP must however not be slowed down, because accumulating evidence indicates that TNAP activation in individuals with metabolic syndrome (MetS) is associated with enhanced cardiovascular mortality, presumably in relation with cardiovascular calcification. On the other hand, TNAP appears to be necessary to prevent the development of steatohepatitis in mice, suggesting that TNAP plays protective roles. The aim of the present review is to highlight the known or suspected functions of TNAP in energy metabolism that may be associated with the development of MetS. The location of TNAP in liver and its function in bile excretion, lipopolysaccharide (LPS) detoxification and fatty acid transport will be presented. The expression and function of TNAP in adipocyte differentiation and thermogenesis will also be discussed. Given that TNAP is a tissue- and substrate-nonspecific phosphatase, we believe that it exerts several crucial pathophysiological functions that are just beginning to be discovered.

Hydrolysis of Extracellular ATP by Vascular Smooth Muscle Cells Transdifferentiated into Chondrocytes Generates Pi but Not PPi.

(1) Background: Tissue non-specific alkaline phosphatase (TNAP) is suspected to induce atherosclerosis plaque calcification. TNAP, during physiological mineralization, hydrolyzes the mineralization inhibitor inorganic pyrophosphate (PPi). Since atherosclerosis plaques are characterized by the presence of necrotic cells that probably release supraphysiological concentrations of ATP, we explored whether this extracellular adenosine triphosphate (ATP) is hydrolyzed into the mineralization inhibitor PPi or the mineralization stimulator inorganic phosphate (Pi), and whether TNAP is involved. (2) Methods: Murine aortic smooth muscle cell line (MOVAS cells) were transdifferentiated into chondrocyte-like cells in calcifying medium, containing ascorbic acid and ß-glycerophosphate. ATP hydrolysis rates were determined in extracellular medium extracted from MOVAS cultures during their transdifferentiation, using 31P-NMR and IR spectroscopy. (3) Results: ATP and PPi hydrolysis by MOVAS cells increased during transdifferentiation. ATP hydrolysis was sequential, yielding adenosine diphosphate (ADP), adenosine monophosphate (AMP), and adenosine without any detectable PPi. The addition of levamisole partially inhibited ATP hydrolysis, indicating that TNAP and other types of ectonucleoside triphoshatediphosphohydrolases contributed to ATP hydrolysis. (4) Conclusions: Our findings suggest that high ATP levels released by cells in proximity to vascular smooth muscle cells (VSMCs) in atherosclerosis plaques generate Pi and not PPi, which may exacerbate plaque calcification.

The Elusive Origin of Atherosclerotic Plaque Calcification.

It has been known for decades or even centuries that arteries calcify as they age. Vascular calcification probably affects all adults, since virtually all have atherosclerotic plaques: an accumulation of lipids, inflammatory cells, necrotic debris, and calcium phosphate crystals. A high vascular calcium score is associated with a high cardiovascular mortality risk, and relatively recent data suggest that even microcalcifications that form in early plaques may destabilize plaques and trigger a cardiovascular event. If the cellular and molecular mechanisms of plaque calcification have been relatively well characterized in mice, human plaques appear to calcify through different mechanisms that remain obscure. In this context, we will first review articles reporting the location and features of early calcifications in human plaques and then review the articles that explored the mechanisms though which human and mouse plaques calcify.

Enhanced BMP-2/BMP-4 ratio in patients with peripheral spondyloarthritis and in cytokine- and stretch-stimulated mouse chondrocytes.

BACKGROUND: Excessive bone formation in the entheses is one of the features of peripheral spondyloarthritis (SpA). Complex pathological mechanisms connecting inflammation, mechanical stress, and ossification are probably involved. We focused on bone morphogenetic protein (BMP)-2, -4, and -7 as possible mediators of this process. METHODS: BMP-2, -4, and -7 concentration was measured by ELISA in synovial fluids (SFs) of SpA (n = 56) and osteoarthritic (n = 21) patients. Mouse organotypic ankle cultures were challenged by a pro-inflammatory cocktail. Mouse primary chondrocytes, osteoblasts, or tenocytes were treated with TNF-α, interleukin (IL)-17, or IL-22 and/or subjected to cyclic stretch, or with recombinant BMP-2 or -4. RESULTS: In SpA SFs, if BMP-7 was barely detectable, BMP-2 concentration was higher and BMP-4 was lower than in osteoarthritic samples, so that BMP-2/BMP-4 ratio augmented 6.5 folds (p < 0.001). In SpA patients, TNF-α, IL-6, and IL-17 levels correlated this ratio (n = 21). Bmp-2/Bmp-4 ratio was similarly enhanced by cytokine treatment in explant and cell cultures, at mRNA level. In particular, simultaneous application of TNF-α and cyclical stretch induced a 30-fold increase of the Bmp-2/Bmp-4 ratio in chondrocytes (p = 0.027). Blockade of prostaglandin E2 and IL-6 production had almost no effect on the stretch-induced regulation of Bmp-2 or -4. Osteoinductive effects of BMP-4, and to a lesser extend BMP-2, were identified on cultured chondrocytes and tenocytes. CONCLUSIONS: Our results first settle that BMP factors are locally deregulated in the SpA joint. An unexpected decrease in BMP-4 could be associated to an increase in BMP-2, possibly in response to mechanical and/or cytokine stimulations.

Prostate cancer-derived exosomes promote osteoblast differentiation and activity through phospholipase D2.

Prostate cancer (PCa) is the most frequent cancer in men aged 65 and over. PCa mainly metastasizes in the bone, forming osteosclerotic lesions, inducing pain, fractures, and nerve compression. Cancer cell-derived exosomes participate in the metastatic spread, ranging from oncogenic reprogramming to the formation of pre-metastatic niches. Moreover, exosomes were recently involved in the dialog between PCa cells and the bone metastasis microenvironment. Phospholipase D (PLD) isoforms PLD1/2 catalyze the hydrolysis of phosphatidylcholine to yield phosphatidic acid (PA), regulating tumor progression and metastasis. PLD is suspected to play a role in exosomes biogenesis. We aimed to determine whether PCa-derived exosomes, through PLD, interact with the bone microenvironment, especially osteoblasts, during the metastatic process. Here we demonstrate for the first time that PLD2 is present in exosomes of C4-2B and PC-3 cells. C4-2B-derived exosomes activate proliferation and differentiation of osteoblasts models, by stimulating ERK 1/2 phosphorylation, by increasing the tissue-nonspecific alkaline phosphatase activity and the expression of osteogenic differentiation markers. Contrariwise, when C4-2B exosomes are generated in the presence of halopemide, a PLD pan-inhibitor, they lose their ability to stimulate osteoblasts. Furthermore, the number of released exosomes diminishes significantly (-40%). When the PLD product PA is combined with halopemide, exosome secretion is fully restored. Taken together, our results indicate that PLD2 stimulates exosome secretion in PCa cell models as well as their ability to increase osteoblast activity. Thus, PLD2 could be considered as a potent player in the establishment of PCa bone metastasis acting through tumor cell derived-exosomes.

Increased phospholipase D activity contributes to tumorigenesis in prostate cancer cell models.

Prostate cancer (PCa) is the most frequent cancer among men and the first cause of death over 65. Approximately 90% of patients with advanced disease will develop bone metastasis, which dramatically reduces long-term survival. Therefore, effective therapies need to be developed, especially when disease is still well-localized. Phospholipase D (PLD), an enzyme that hydrolyzes phosphatidylcholine to yield phosphatidic acid, regulates several cellular functions as proliferation, survival, migration or vesicular trafficking. PLD is implicated in numerous diseases such as neurodegenerative, cardiovascular, autoimmune disorders or cancer. Indeed, PLD controls different aspects of oncogenesis including tumor progression and resistance to targeted therapies such as radiotherapy. PLD1 and PLD2 are the only isoforms with catalytic activity involved in cancer. Surprisingly, studies deciphering the role of PLD in the pathophysiology of PCa are scarce. Here we describe the correlation between PLD activity and PLD1 and PLD2 expression in PCa bone metastasis-derived cell lines C4-2B and PC-3. Next, by using PLD pharmacological inhibitors and RNA interference strategy, we validate the implication of PLD1 and PLD2 in cell viability, clonogenicity and proliferation of C4-2B and PC-3 cells and in migration capacity of PC-3 cells. Last, we show an increase in PLD activity as well as PLD2 protein expression during controlled starvation of PC-3 cells, concomitant with an augmentation of its migration capacity. Specifically, upregulation of PLD activity appears to be PKC-independent. Taken together, our results indicate that PLD, and in particular PLD2, could be considered as a potential therapeutic target for the treatment of PCa-derived bone metastasis.

Localization of Annexin A6 in Matrix Vesicles During Physiological Mineralization.

Annexin A6 (AnxA6) is the largest member of the annexin family of proteins present in matrix vesicles (MVs). MVs are a special class of extracellular vesicles that serve as a nucleation site during cartilage, bone, and mantle dentin mineralization. In this study, we assessed the localization of AnxA6 in the MV membrane bilayer using native MVs and MV biomimetics. Biochemical analyses revealed that AnxA6 in MVs can be divided into three distinct groups. The first group corresponds to Ca2+-bound AnxA6 interacting with the inner leaflet of the MV membrane. The second group corresponds to AnxA6 localized on the surface of the outer leaflet. The third group corresponds to AnxA6 inserted in the membrane's hydrophobic bilayer and co-localized with cholesterol (Chol). Using monolayers and proteoliposomes composed of either dipalmitoylphosphatidylcholine (DPPC) to mimic the outer leaflet of the MV membrane bilayer or a 9:1 DPPC:dipalmitoylphosphatidylserine (DPPS) mixture to mimic the inner leaflet, with and without Ca2+, we confirmed that, in agreement with the biochemical data, AnxA6 interacted differently with the MV membrane. Thermodynamic analyses based on the measurement of surface pressure exclusion (πexc), enthalpy (ΔH), and phase transition cooperativity (Δt1/2) showed that AnxA6 interacted with DPPC and 9:1 DPPC:DPPS systems and that this interaction increased in the presence of Chol. The selective recruitment of AnxA6 by Chol was observed in MVs as probed by the addition of methyl-ß-cyclodextrin (MßCD). AnxA6-lipid interaction was also Ca2+-dependent, as evidenced by the increase in πexc in negatively charged 9:1 DPPC:DPPS monolayers and the decrease in ΔH in 9:1 DPPC:DPPS proteoliposomes caused by the addition of AnxA6 in the presence of Ca2+ compared to DPPC zwitterionic bilayers. The interaction of AnxA6 with DPPC and 9:1 DPPC:DPPS systems was distinct even in the absence of Ca2+ as observed by the larger change in Δt1/2 in 9:1 DPPC:DPPS vesicles as compared to DPPC vesicles. Protrusions on the surface of DPPC proteoliposomes observed by atomic force microscopy suggested that oligomeric AnxA6 interacted with the vesicle membrane. Further work is needed to delineate possible functions of AnxA6 at its different localizations and ways of interaction with lipids.

The role of sphingosine 1-phosphate metabolism in bone and joint pathologies and ectopic calcification.

Sphingolipids display important functions in various pathologies such as cancer, obesity, diabetes, cardiovascular or neurodegenerative diseases. Sphingosine, sphingosine 1-phosphate (S1P), and ceramide are the central molecules of sphingolipid metabolism. Sphingosine kinases 1 and 2 (SK1 and SK2) catalyze the conversion of the sphingolipid metabolite sphingosine into S1P. The balance between the levels of S1P and its metabolic precursors ceramide and sphingosine has been considered as a switch that could determine whether a cell proliferates or dies. This balance, also called « sphingolipid rheostat ¼, is mainly under the control of SKs. Several studies have recently pointed out the contribution of SK/S1P metabolic pathway in skeletal development, mineralization and bone homeostasis. Indeed, SK/S1P metabolism participates in different diseases including rheumatoid arthritis, spondyloarthritis, osteoarthritis, osteoporosis, cancer-derived bone metastasis or calcification disorders as vascular calcification. In this review, we will summarize the most important data regarding the implication of SK/S1P axis in bone and joint diseases and ectopic calcification, and discuss the therapeutic potential of targeting SK/S1P metabolism for the treatment of these pathologies.

Cytokine-Induced and Stretch-Induced Sphingosine 1-Phosphate Production by Enthesis Cells Could Favor Abnormal Ossification in Spondyloarthritis.

Spondyloarthritis (SpA) is a common rheumatic disease characterized by enthesis inflammation (enthesitis) and ectopic ossification (enthesophytes). The current pathogenesis model suggests that inflammation and mechanical stress are both strongly involved in SpA pathophysiology. We have previously observed that the levels of sphingosine 1-phosphate (S1P), a bone anabolic molecule, were particularly high in SpA patients' serum compared to healthy donors. Therefore, we wondered how this deregulation was related to SpA molecular mechanisms. Mouse primary osteoblasts, chondrocytes, and tenocytes were used as cell culture models. The sphingosine kinase 1 (Sphk1) gene expression and S1P secretion were significantly enhanced by cyclic stretch in osteoblasts and chondrocytes. Further, TNF-α and IL-17, cytokines implicated in enthesitis, increased Sphk1 mRNA in chondrocytes in an additive manner when combined to stretch. The immunochemistry on mouse ankles showed that sphingosine kinase 1 (SK1) was localized in some chondrocytes; the addition of a pro-inflammatory cocktail augmented Sphk1 expression in cultured ankles. Subsequently, fingolimod was used to block S1P metabolism in cell cultures. It inhibited S1P receptors (S1PRs) signaling and SK1 and SK2 activity in both osteoblasts and chondrocytes. Fingolimod also reduced S1PR-induced activation by SpA patients' synovial fluid (SF), demonstrating that the stimulation of chondrocytes by SFs from SpA patients involves S1P. In addition, when the osteogenic culture medium was supplemented with fingolimod, alkaline phosphatase activity, matrix mineralization, and bone formation markers were significantly reduced in osteoblasts and hypertrophic chondrocytes. Osteogenic differentiation was accompanied by an increase in S1prs mRNA, especially S1P1/3 , but their contribution to S1P-impact on mineralization seemed limited. Our results suggest that S1P might be overproduced in SpA enthesis in response to cytokines and mechanical stress, most likely by chondrocytes. Moreover, S1P could locally favor the abnormal ossification of the enthesis; therefore, blocking the S1P metabolic pathway could be a potential therapeutic approach for the treatment of SpA. © 2019 American Society for Bone and Mineral Research.

Quantitative atomic force microscopy provides new insight into matrix vesicle mineralization.

Matrix vesicles (MVs) are a class of extracellular vesicles that initiate mineralization in cartilage, bone, and other vertebrate tissues by accumulating calcium ions (Ca2+) and inorganic phosphate (Pi) within their lumen and forming a nucleation core (NC). After further sequestration of Ca2+ and Pi, the NC transforms into crystalline complexes. Direct evidence of the existence of the NC and its maturation have been provided solely by analyses of dried samples. We isolated MVs from chicken embryo cartilage and used atomic force microscopy peak force quantitative nanomechanical property mapping (AFM-PFQNM) to measure the nanomechanical and morphological properties of individual MVs under both mineralizing (+Ca2+) and non-mineralizing (-Ca2+) fluid conditions. The elastic modulus of MVs significantly increased by 4-fold after incubation in mineralization buffer. From AFM mapping data, we inferred the morphological changes of MVs as mineralization progresses: prior to mineralization, a punctate feature, the NC, is present within MVs and this feature grows and stiffens during mineralization until it occupies most of the MV lumen. Dynamic light scattering showed a significant increase in hydrodynamic diameter and no change in the zeta potential of hydrated MVs after incubation with Ca2+. This validates that crystalline complexes, which are strongly negative relative to MVs, were forming within the lumen of MVs. These data were substantiated by transmission electron microscopy energy dispersive X-ray and Fourier transform infrared spectroscopic analyses of dried MVs, which provide evidence that the complexes increased in size, crystallinity, and Ca/P ratio within MVs during the mineralization process.

Design, synthesis and biological evaluation of inhibitors of cathepsin K on dedifferentiated chondrocytes.

Selective proteinase inhibitors have demonstrated utility in the investigation of cartilage degeneration mechanisms and may have clinical use in the management of osteoarthritis. The cysteine protease cathepsin K (CatK) is an attractive target for arthritis therapy. Here we report the synthesis of two cathepsin K inhibitors (CKIs): racemic azanitrile derivatives CKI-E and CKI-F, which have better inhibition properties on CatK than the commercial inhibitor odanacatib (ODN). Their IC50 values and inhibition constants (Ki) have been determined in vitro. Inhibitors demonstrate differential selectivity for CatK over cathepsin B, L and S in vitro, with Ki amounting to 1.14 and 7.21 nM respectively. We analyzed the effect of these racemic inhibitors on viability in different cell types. The human osteoblast-like cell line MG63, MOVAS cells (a murine vascular smooth muscle cell line) or murine primary chondrocytes, were treated either with CKI-E or with CKI-F, which were not toxic at doses of up to 5 µM. Primary chondrocytes subjected to several passages were used as a model of phenotypic loss of articular chondrocytes, occurring in osteoarthritic cartilage. The efficiency of CKIs regarding CatK inhibition and their specificity over other proteases were validated in primary chondrocytes subjected to several passages. Racemic CKI-E and CKI-F at 0.1 and 1 µM significantly inhibited CatK activity in dedifferentiated chondrocytes, even better than the commercial CatK inhibitor ODN. The enzymatic activity of other proteases such as matrix metalloproteinases or aggrecanases were not affected. Taken together, these findings support the possibility to design CatK inhibitors for preventing cartilage degradation in different pathologies.

Effects of phospholipase D during cultured osteoblast mineralization and bone formation.

Mammalian phospholipase D (PLD) mostly hydrolyzes phosphatidylcholine producing phosphatidic acid. PLD activity was previously detected in different osteoblastic cell models, and was increased by several growth factors involved in bone homeostasis. To confirm possible actions of PLD isoforms during mineralization process, we analyzed their effects in osteoblastic cell models and during bone formation. PLD1 expression, along with PLD activity, increased during differentiation of primary osteoblasts and Saos-2 cells, and peaked at the onset of mineralization. Subsequently, both PLD1 expression and PLD activity decreased, suggesting that PLD1 function is regulated during osteoblast maturation. In contrast, PLD2 expression was not significantly affected during differentiation of osteoblasts. Overexpression of PLD1 in Saos-2 cells improved their mineralization potential. PLD inhibitor Halopemide or PLD1-selective inhibitor, led to a decrease in mineralization in both cell types. On the contrary, the selective inhibitor of PLD2, did not affect the mineralization process. Moreover, primary osteoblasts isolated from PLD1 knockout (KO) mice were significantly less efficient in mineralization as compared with those isolated from wild type (WT) or PLD2 KO mice. In contrast, bone formation, as monitored by high-resolution microcomputed tomography analysis, was not impaired in PLD1 KO nor in PLD2 KO mice, indicating that the lack of PLD1 or that of PLD2 did not affect the bone structure in adult mice. Taken together, our findings indicate that PLD activity, especially which of PLD1 isoform, may enhance the mineralization process in osteoblastic cells. Nonetheless, the lack of PLD1 or PLD2 do not seem to significantly affect bone formation in adult mice.

Long-chain polyphosphate in osteoblast matrix vesicles: Enrichment and inhibition of mineralization.

BACKGROUND: Inorganic polyphosphate (polyP) is a fundamental and ubiquitous molecule in prokaryotes and eukaryotes. PolyP has been found in mammalian tissues with particularly high levels of long-chain polyP in bone and cartilage where critical questions remain as to its localization and function. Here, we investigated polyP presence and function in osteoblast-like SaOS-2 cells and cell-derived matrix vesicles (MVs), the initial sites of bone mineral formation. METHODS: PolyP was quantified by 4',6-diamidino-2-phenylindole (DAPI) fluorescence and characterized by enzymatic methods coupled to urea polyacrylamide gel electrophoresis. Transmission electron microscopy and confocal microscopy were used to investigate polyP localization. A chicken embryo cartilage model was used to investigate the effect of polyP on mineralization. RESULTS: PolyP increased in concentration as SaOS-2 cells matured and mineralized. Particularly high levels of polyP were observed in MVs. The average length of MV polyP was determined to be longer than 196 Pi residues by gel chromatography. Electron micrographs of MVs, stained by two polyP-specific staining approaches, revealed polyP localization in the vicinity of the MV membrane. Additional extracellular polyP binds to MVs and inhibits MV-induced hydroxyapatite formation. CONCLUSION: PolyP is highly enriched in matrix vesicles and can inhibit apatite formation. PolyP may be hydrolysed to phosphate for further mineralization in the extracellular matrix. GENERAL SIGNIFICANCE: PolyP is a unique yet underappreciated macromolecule which plays a critical role in extracellular mineralization in matrix vesicles.