Differing calcification processes in cultured vascular smooth muscle cells and osteoblasts

© 2019 Published by Elsevier Inc.Arterial medial calcification (AMC) is the deposition of calcium phosphate mineral, often as hydroxyapatite, inthe medial layer of the arteries. AMC shares some similarities to skeletal mineralisation and has been associatedwith the transdifferentiation of vascular smooth muscle cells (VSMCs) towards an osteoblast-like phenotype. Thisstudy used primary mouse VSMCs and calvarial osteoblasts to directly compare the established and widely usedin vitromodels of AMC and bone formation. Significant differences were identified between osteoblasts andcalcifying VSMCs. First, osteoblasts formed large mineralised bone nodules that were associated with widespreaddeposition of an extracellular collagenous matrix. In contrast, VSMCs formed small discrete regions of calcifi-cation that were not associated with collagen deposition and did not resemble bone. Second, calcifying VSMCsdisplayed a progressive reduction in cell viability over time (≤7-fold), with a 50% increase in apoptosis,whereas osteoblast and control VSMCs viability remained unchanged. Third, osteoblasts expressed high levels ofalkaline phosphatase (TNAP) activity and TNAP inhibition reduced bone formation by to 90%. TNAP activity incalcifying VSMCs was∼100-fold lower than that of bone-forming osteoblasts and cultures treated withβ-gly-cerophosphate, a TNAP substrate, did not calcify. Furthermore, TNAP inhibition had no effect on VSMC calci-fication. Although, VSMC calcification was associated with increased mRNA expression of osteoblast-relatedgenes (e.g. Runx2, osterix, osteocalcin, osteopontin), the relative expression of these genes was up to 40-foldlower in calcifying VSMCs versus bone-forming osteoblasts. In summary, calcifying VSMCsin vitrodisplay somelimited osteoblast-like characteristics but also differ in several key respects: 1) their inability to form collagen-containing bone; 2) their lack of reliance on TNAP to promote mineral deposition; and, 3) the deleterious effectof calcification on their viability.Peer reviewedFinal Published versio

The P2X7 Receptor is an Important Regulator of Extracellular ATP Levels

Controlled ATP release has been demonstrated from many neuronal and non-neuronal cell types. Once released, extracellular ATP acts on cells in a paracrine manner via purinergic receptors. Considerable evidence now suggests that extracellular nucleotides, signaling via P2 receptors, play important roles in bone homeostasis modulating both osteoblast and osteoclast function. In this study, we demonstrate that mouse osteoclasts and their precursors constitutively release ATP into their extracellular environment. Levels were highest at day 2 (precursor cells), possibly reflecting the high number of red blood cells and accessory cells present. Mature osteoclasts constitutively released ATP in the range 0.05–0.5 pmol/ml/cell. Both osteoclasts and osteoblasts express mRNA and protein for the P2X7 receptor. We found that in osteoclasts, expression levels are fourfold higher in mature cells relative to precursors, whilst in osteoblasts expression remains relatively constant during differentiation. Selective antagonists (0.1–100 μM AZ10606120, A438079, and KN-62) were used to determine whether this release was mediated via P2X7 receptors. AZ10606120, A438079, and KN-62, at 0.1–10 μM, decreased ATP release by mature osteoclasts by up to 70, 60, and 80%, respectively. No differences in cell viability were observed. ATP release also occurs via vesicular exocytosis; inhibitors of this process (1–100 μM NEM or brefeldin A) had no effect on ATP release from osteoclasts. P2X7 receptor antagonists (0.1–10 μM) also decreased ATP release from primary rat osteoblasts by up to 80%. These data show that ATP release via the P2X7 receptor contributes to extracellular ATP levels in osteoclast and osteoblast cultures, suggesting an important additional role for this receptor in autocrine/paracrine purinergic signaling in bone

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,β-meATP and β,γ-meATP, inhibited calcification by up to 100%. Culture in a high phosphate medium (2mM) was associated with increased VSMC death and apoptosis; treatment with Bz-ATP, α,β-meATP and β,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, SMA) and osteoblast-associated (e.g. Runx2, osteopontin) markers; Bz-ATP, α,β-meATP and β,γ-meATP attenuated these changes in protein expression. Long-term culture with Bz-ATP, α,β-meATP and β,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,β-meATP and β,γ-meATP (α,β-meADP and methylene diphosphonate, respectively) also dose dependently inhibited VSMC calcification. Furthermore, the actions of Bz-ATP, α,β-meATP and β,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

Hypoxia mimetics restore bone biomineralisation in hyperglycaemic environments

Diabetic patients have an increased risk of fracture and an increased occurrence of impaired fracture healing. Diabetic and hyperglycaemic conditions have been shown to impair the cellular response to hypoxia, via an inhibited hypoxia inducible factor (HIF)-1α pathway. We investigated, using an in vitro hyperglycaemia bone tissue engineering model (and a multidisciplinary bone characterisation approach), the differing effects of glucose levels, hypoxia and chemicals known to stabilise HIF-1α (CoCl2 and DMOG) on bone formation. Hypoxia (1% O2) inhibited bone nodule formation and resulted in discrete biomineralisation as opposed to the mineralised extracellular collagen fibres found in normoxia (20% O2). Unlike hypoxia, the use of hypoxia mimetics did not prevent nodule formation in normal glucose level. Hyperglycaemic conditions (25 mM and 50 mM glucose) inhibited biomineralisation. Interestingly, both hypoxia mimetics (CoCl2 and DMOG) partly restored hyperglycaemia inhibited bone nodule formation. These results highlight the difference in osteoblast responses between hypoxia mimetics and actual hypoxia and suggests a role of HIF-1α stabilisation in bone biomineralisation that extends that of promoting neovascularisation, or other system effects associated with hypoxia and bone regeneration in vivo. This study demonstrates that targeting the HIF pathway may represent a promising strategy for bone regeneration in diabetic patients

Peculiar Type II Supernovae from Blue Supergiants

The vast majority of Type II supernovae (SNe) are produced by red supergiants (RSGs), but SN 1987A revealed that blue supergiants (BSGs) can produce members of this class as well, albeit with some peculiar properties. This best studied event revolutionized our understanding of SNe, and linking it to the bulk of Type II events is essential. We present here optical photometry and spectroscopy gathered for SN 2000cb, which is clearly not a standard Type II SN and yet is not a SN 1987A analog. The light curve of SN 2000cb is reminiscent of that of SN 1987A in shape, with a slow rise to a late optical peak, but on substantially different time scales. Spectroscopically, SN 2000cb resembles a normal SN II but with ejecta velocities that far exceed those measured for SN 1987A or normal SNe II, above 18000 km/s for H-alpha at early times. The red colours, high velocities, late photometric peak, and our modeling of this object all point toward a scenario involving the high-energy explosion of a small-radius star, most likely a BSG, producing 0.1 solar masses of Ni-56. Adding a similar object to the sample, SN 2005ci, we derive a rate of about 2% of the core-collapse rate for this loosely defined class of BSG explosions.Comment: Accepted to MNRAS on March 14, 201

Extracellular ATP released by osteoblasts is a key local inhibitor of bone mineralisation

Previous studies have shown that exogenous ATP (>1µM) prevents bone formation in vitro by blocking mineralisation of the collagenous matrix. This effect is thought to be mediated via both P2 receptor-dependent pathways and a receptor-independent mechanism (hydrolysis of ATP to produce the mineralisation inhibitor pyrophosphate, PPi). Osteoblasts are also known to release ATP constitutively. To determine whether this endogenous ATP might exert significant biological effects, bone-forming primary rat osteoblasts were cultured with 0.5-2.5U/ml apyrase (which sequentially hydrolyses ATP to ADP to AMP + 2Pi). Addition of 0.5U/ml apyrase to osteoblast culture medium degraded extracellular ATP to <1% of control levels within 2 minutes; continuous exposure to apyrase maintained this inhibition for up to 14 days. Apyrase treatment for the first 72 hours of culture caused small decreases (≤25%) in osteoblast number, suggesting a role for endogenous ATP in stimulating cell proliferation. Continuous apyrase treatment for 14 days (≥0.5U/ml) increased mineralisation of bone nodules by up to 3-fold. Increases in bone mineralisation were also seen when osteoblasts were cultured with the ATP release inhibitors, NEM and brefeldin A, as well as with P2X1 and P2X7 receptor antagonists. Apyrase decreased alkaline phosphatase (TNAP) activity by up to 60%, whilst increasing the activity of the PPi-generating ecto-nucleotide pyrophosphatase/phosphodiesterases (NPPs) up to 2.7-fold. Both collagen production and adipocyte formation were unaffected. These data suggest that nucleotides released by osteoblasts in bone could act locally, via multiple mechanisms, to limit mineralisation

Mineralisation of collagen rich soft tissues and osteocyte lacunae in Enpp1(-/-) mice

Ecto-nucleotide pyrophosphatase/phosphodiesterases (NPPs) hydrolyse nucleotide triphosphates to the corresponding nucleotide monophosphates and the mineralisation inhibitor, pyrophosphate (PPi). This study examined the role of NPP1 in osteocytes, osteoclasts and cortical bone, using a mouse model lacking NPP1 (Enpp1−/−). We used microcomputed tomography (μCT) to investigate how NPP1 deletion affects cortical bone structure; excised humerus bones from 8, 15 and 22-week old mice were scanned at 0.9 μm. Although no changes were evident in the cortical bone of 8-week old Enpp1−/− mice, significant differences were observed in older animals. Cortical bone volume was decreased 28% in 22-week Enpp1−/− mice, whilst cortical porosity was reduced 30% and 60% at 15 and 22-weeks, respectively. This was accompanied by up to a 15% decrease in closed pore diameter and a 55% reduction in the number of pores. Cortical thickness was reduced up to 35% in 15 and 22-week Enpp1−/− animals and the endosteal diameter was increased up to 23%. Thus, the cortical bone from Enpp1−/− mice was thinner and less porous, with a larger marrow space. Scanning electron microscopy (SEM) revealed a decrease in the size and number of blood vessel channels in the cortical bone as well as a 40% reduction in the mean plan area of osteocyte lacunae. We noted that the number of viable osteocytes isolated from the long bones of Enpp1−/− mice was decreased ≤ 50%. In contrast, osteoclast formation and resorptive activity were unaffected by NPP1 deletion. μCT and histological analysis of Enpp1−/− mice also revealed calcification of the joints and vertebrae as well as soft tissues including the whisker follicles, ear pinna and trachea. This calcification worsened as the animals aged. Together, these data highlight the key role of NPP1 in regulating calcification of both soft and skeletal tissues

Allopurinol and oxypurinol promote osteoblast differentiation and increase bone formation

AbstractAllopurinol and its active metabolite, oxypurinol are widely used in the treatment of gout and hyperuricemia. They inhibit xanthine oxidase (XO) an enzyme in the purine degradation pathway that converts xanthine to uric acid. This investigation examined the effect of allopurinol and oxypurinol on bone formation, cell number and viability, gene expression and enzyme activity in differentiating and mature, bone-forming osteoblasts. Although mRNA expression remained relatively constant, XO activity decreased over time with mature osteoblasts displaying reduced levels of uric acid (20% decrease). Treatment with allopurinol and oxypurinol (0.1–1µM) reduced XO activity by up to 30%. At these concentrations, allopurinol and oxypurinol increased bone formation by osteoblasts ~4-fold and ~3-fold, respectively. Cell number and viability were unaffected. Both drugs increased tissue non-specific alkaline phosphatase (TNAP) activity up to 65%. Osteocalcin and TNAP mRNA expression was increased, 5-fold and 2-fold, respectively. Expression of NPP1, the enzyme responsible for generating the mineralisation inhibitor, pyrophosphate, was decreased 5-fold. Col1α1 mRNA expression and soluble collagen levels were unchanged. Osteoclast formation and resorptive activity were not affected by treatment with allopurinol or oxypurinol. Our data suggest that inhibition of XO activity promotes osteoblast differentiation, leading to increased bone formation in vitro

Optimisation of the differing conditions required for bone formation in vitro by primary osteoblasts from mice and rats

The in vitro culture of calvarial osteoblasts from neonatal rodents remains an important method for studying the regulation of bone formation. The widespread use of transgenic mice has created a particular need for a reliable, simple method that allows the differentiation and bone‑forming activity of murine osteoblasts to be studied. In the present study, we established such a method and identified key differences in optimal culture conditions between mouse and rat osteoblasts. Cells isolated from neonatal rodent calvariae by collagenase digestion were cultured for 14‑28 days before staining for tissue non-specific alkaline phosphatase (TNAP) and bone mineralisation (alizarin red). The reliable differentiation of mouse osteoblasts, resulting in abundant TNAP expression and the formation of mineralised ‘trabecular‑shaped’ bone nodules, occurred only following culture in α minimum essential medium (αMEM) and took 21‑28 days. Dexamethasone (10 nM) inhibited bone mineralisation in the mouse osteoblasts. By contrast, TNAP expression and bone formation by rat osteoblasts were observed following culture in both αMEM and Dulbecco's modified Eagle's medium (DMEM) after approximately 14 days (although ~3‑fold more effectively in αMEM) and was strongly dependent on dexamethasone. Both the mouse and rat osteoblasts required ascorbate (50 µg/ml) for osteogenic differentiation and β‑glycerophosphate (2 mM) for mineralisation. The rat and mouse osteoblasts showed similar sensitivity to the well‑established inhibitors of mineralisation, inorganic pyrophosphate (PPi) and adenosine triphosphate (ATP; 1‑100 µM). The high efficiency of osteogenic differentiation observed following culture in αMEM, compared with culture in DMEM possibly reflects the richer formulation of the former. These findings offer a reliable technique for inducing mouse osteoblasts to form bone in vitro and a more effective method for culturing bone‑forming rat osteoblasts

Epigenetic patterns in blood associated with lipid traits predict incident coronary heart disease events and are enriched for results from genome-wide association studies

Background- Genome-wide association studies have identified loci influencing circulating lipid concentrations in humans; further information on novel contributing genes, pathways, and biology may be gained through studies of epigenetic modifications. Methods and Results- To identify epigenetic changes associated with lipid concentrations, we assayed genome-wide DNA methylation at cytosine-guanine dinucleotides (CpGs) in whole blood from 2306 individuals from 2 population-based cohorts, with replication of findings in 2025 additional individuals. We identified 193 CpGs associated with lipid levels in the discovery stage (P &lt; 1.08E-07) and replicated 33 (at Bonferroni-corrected P &lt; 0.05), including 25 novel CpGs not previously associated with lipids. Genes at lipid-associated CpGs were enriched in lipid and amino acid metabolism processes. A differentially methylated locus associated with triglyceridesand high-density lipoprotein cholesterol (HDL- C; cg27243685; P= 8.1E-26 and 9.3E-19) was associated with cis-expression of a reverse cholesterol transporter (ABCG1; P= 7.2E-28) and incident cardiovascular disease events (hazard ratio per SD increment, 1.38; 95% confidence interval, 1.15-1.66; P= 0.0007). We found significant cis-methylation quantitative trait loci at 64% of the 193 CpGs with an enrichment of signals from genome-wide association studies of lipid levels (P-TC = 0.004, PHDL-C = 0.008 and P-triglycerides = 0.00003) and coronary heart disease ( P= 0.0007). For example, genome-wide significant variants associated with low-density lipoprotein cholesterol and coronary heart disease at APOB were cis-methylation quantitative trait loci for a low-density lipoprotein cholesterol-related differentially methylated locus. Conclusions-We report novel associations of DNA methylation with lipid levels, describe epigenetic mechanisms related to previous genome-wide association studies discoveries, and provide evidence implicating epigenetic regulation of reverse cholesterol transport in blood in relation to occurrence of cardiovascular disease events