Jinxiu Lu

Hyperphosphatemia-induced nanocrystals upregulate the expression of bone morphogenetic protein-2 and osteopontin genes in mouse smooth muscle cells in vitro

Vascular calcification, which contributes to cardiovascular disease in patients with uremic hyperphosphatemia, is associated with vascular cell expression of osteogenic genes, including bone morphogenetic protein (BMP)-2 and osteopontin (OPN). High inorganic phosphate levels in vitro stimulate the osteogenic conversion of smooth muscle cells; however, the mechanism governing this is not clear. We found that high-phosphate medium increased the expression of BMP-2 and OPN in mouse smooth muscle cells in culture. However, this effect was lost in the presence of the mineralization inhibitor, pyrophosphate, suggesting a contribution of calcium phosphate crystals. Addition of 1-2 mmol/l phosphate alone to growth medium was sufficient to induce nanosized crystals after 1 day at 37 °C. Isolated crystals were about 160 nm in diameter and had a calcium to phosphate ratio of 1.35, consistent with the hydroxyapatite precursor octacalcium phosphate. Nanocrystal formation increased fourfold in the absence of serum, was blocked by fetuin-A, and was dependent on time and on the concentrations of phosphate and calcium. Purified synthetic hydroxyapatite nanocrystals and isolated high-phosphate-induced nanocrystals, but not nanocrystal-free high-phosphate medium, also induced BMP-2 and OPN. Thus, our results suggest that BMP-2 and OPN are induced by calcium phosphate nanocrystals, rather than soluble phosphate. This mechanism may contribute, in part, to hyperphosphatemia-related vascular cell differentiation and calcification.

Adverse Effects of Hyperlipidemia on Bone Regeneration and Strength

Hyperlipidemia increases the risk for generation of lipid oxidation products, which accumulate in the subendothelial spaces of vasculature and bone. Atherogenic high‐fat diets increase serum levels of oxidized lipids, which are known to attenuate osteogenesis in culture and to promote bone loss in mice. In this study, we investigated whether oxidized lipids affect bone regeneration and mechanical strength. Wild‐type (WT) and hyperlipidemic (Ldlr−/−) mice were placed on a high‐fat (HF) diet for 13 weeks. Bilateral cranial defects were introduced on each side of the sagittal suture, and 5 weeks postsurgery on the respective diets, the repair/regeneration of cranial bones and mechanical properties of femoral bones were assessed. MicroCT and histological analyses demonstrated that bone regeneration was significantly impaired by the HF diet in WT and Ldlr−/− mice. In femoral bone, cortical bone volume fraction (bone volume [BV]/tissue volume [TV]) was significantly reduced, whereas cortical porosity was increased by the HF diet in Ldlr−/− but not in WT mice. Femoral bone strength and stiffness, measured by three‐point bending analysis, were significantly reduced by the HF diet in Ldlr−/−, but not in WT mice. Serum analysis showed that the HF diet significantly increased levels of parathyroid hormone, tumor necrosis factor (TNF)‐α, calcium, and phosphorus, whereas it reduced procollagen type I N‐terminal propeptide, a serum marker of bone formation, in Ldlr−/−, but not in WT mice. The serum level of carboxyl‐terminal collagen crosslinks, a marker for bone resorption, was also 1.7‐fold greater in Ldlr−/− mice. These findings suggest that hyperlipidemia induces secondary hyperparathyroidism and impairs bone regeneration and mechanical strength.

Hyperlipidemia induces resistance to PTH bone anabolism in mice via oxidized lipids

In hyperlipidemia, oxidized lipids accumulate in vascular tissues and trigger atherosclerosis. Such lipids also deposit in bone tissues, where they may promote osteoporosis. We found previously that oxidized lipids attenuate osteogenesis and that parathyroid hormone (PTH) bone anabolism is blunted in hyperlipidemic mice, suggesting that osteoporotic patients with hyperlipidemia may develop resistance to PTH therapy. To determine if oxidized lipids account for this PTH resistance, we blocked lipid oxidation products in hyperlipidemic mice with an ApoA‐I mimetic peptide, D‐4F, and the bone anabolic response to PTH treatment was assessed. Skeletally immature Ldlr−/− mice were placed on a high‐fat diet and treated with D‐4F peptide and/or with intermittent PTH(1–34) injections. As expected, D‐4F attenuated serum lipid oxidation products and tissue lipid deposition induced by the diet. Importantly, D‐4F treatment attenuated the adverse effects of dietary hyperlipidemia on PTH anabolism by restoring micro–computed tomographic parameters of bone quality—cortical mineral content, area, and thickness. D‐4F significantly reduced serum markers of bone resorption but not bone formation. PTH and D‐4F, together but not separately, also promoted bone anabolism in an alternative model of hyperlipidemia, Apoe−/− mice. In normolipemic mice, D‐4F cotreatment did not further enhance the anabolic effects of PTH, indicating that the mechanism is through its effects on lipids. These findings suggest that oxidized lipids mediate hyperlipidemia‐induced PTH resistance in bone through modulation of bone resorption.

Phosphate and pyrophosphate mediate PKA-induced vascular cell calcification

Vascular calcification is associated with increased cardiovascular risk and occurs by osteochondrogenic differentiation of vascular cells. Many of the same regulatory factors that control skeletal mineralization, including the complex metabolic pathway controlling levels of the activator, inorganic phosphate, and the potent inhibitor, pyrophosphate, also govern vascular calcification. We previously found that the cAMP/PKA signaling pathway mediates in vitro vascular cell calcification induced by inflammatory factors including tumor necrosis factor-alpha 1 and oxidized phospholipids. In this report, we tested whether this signaling pathway modulates phosphate and pyrophosphate metabolism. Treatment of primary murine aortic cells with the PKA activator, forskolin, significantly induced osteoblastic differentiation markers, including alkaline phosphatase (ALP), osteopontin, and osteocalcin as well as the pyrophosphate generator, ectonucleotide-pyrophosphatase/phosphodiesterase-1 (Enpp1) and the pyrophosphate transporter, ankylosis protein, but not the sodium/phosphate cotransporter, Pit-1. In the presence of a substrate for ALP, beta-glycerophosphate, which generates inorganic phosphate, forskolin also enhanced matrix mineralization. Inhibitors of ALP or Pit-1 abrogated forskolin-induced osteopontin expression and mineralization but not forskolin-induced osteocalcin or ALP. These results suggest that phosphate is necessary for PKA-induced calcification of vascular cells and that the extent of PKA-induced calcification is controlled by feedback induction of the inhibitor, pyrophosphate.

Atherogenic Phospholipids Attenuate Osteogenic Signaling by BMP-2 and Parathyroid Hormone in Osteoblasts

Cardiovascular disease, such as atherosclerosis, has been associated with reduced bone mineral density and fracture risk. A major etiologic factor in atherogenesis is believed to be oxidized phospholipids. We previously found that these phospholipids inhibit spontaneous osteogenic differentiation of marrow stromal cells, suggesting that they may account for the clinical link between atherosclerosis and osteoporosis. Currently, anabolic agents that promote bone formation are increasingly used as a new treatment for osteoporosis. It is not known, however, whether atherogenic phospholipids alter the effects of bone anabolic agents, such as bone morphogenetic protein (BMP)-2 and parathyroid hormone (PTH). Therefore we investigated the effects of oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (ox-PAPC) on osteogenic signaling induced by BMP-2 and PTH in MC3T3-E1 cells. Results showed that ox-PAPC attenuated BMP-2 induction of osteogenic markers alkaline phosphatase and osteocalcin. Ox-PAPC also inhibited both spontaneous and BMP-induced expression of PTH receptor. Consistently, pretreatment of cells with ox-PAPC inhibited PTH-induced cAMP production and expression of immediate early genes Nurr1 and IL-6. Results from immunofluorescence and Western blot analyses showed that inhibitory effects of ox-PAPC on BMP-2 signaling were associated with inhibition of SMAD 1/5/8 but not p38-MAPK activation. These effects appear to be due to ox-PAPC activation of the ERK pathway, as the ERK inhibitor PD98059 reversed ox-PAPC inhibitory effects on BMP-2-induced alkaline phosphatase activity, osteocalcin expression, and SMAD activation. These results suggest that atherogenic lipids inhibit osteogenic signaling induced by BMP-2 and PTH, raising the possibility that hyperlipidemia and atherogenic phospholipids may interfere with anabolic therapy.

Hyperlipidemia Impairs Osteoanabolic Effects of PTH

Epidemiological and in vitro studies have suggested that hyperlipidemia/oxidized phospholipids adversely affect bone. We recently found that oxidized phospholipids attenuate PTH‐induced cAMP and immediate‐early gene (IEG) expression in MC3T3‐E1 cells, raising concerns that clinical hyperlipidemia may attenuate osteoanabolic effects of PTH in vivo. Thus, we studied whether intermittent PTH treatment has differential osteoanabolic effects in wildtype (C57BL/6) and hyperlipidemic (LDLR−/−) mice. Consistent with our previous in vitro studies, induction of IEGs in calvarial tissue, 45 min after a single dose of recombinant hPTH(1‐34), was attenuated in LDLR−/− mice compared with C57BL/6 mice. Daily hPTH(1‐34) injections for 5 wk significantly increased total and cortical BMD and BMC, assessed by pQCT, in C57BL/6 mice. However, this induction was completely abrogated in LDLR−/− mice. Similarly, PTH(1‐34) failed to increase BMD in another hyperlipidemic mouse model, ApoE−/− mice. Histomorphometric analysis showed that trabecular bone of both mice responded similarly to PTH(1‐34). Structural parameters improved significantly in response to PTH(1‐34) in both mouse strains, although to a lesser degree in LDLR−/− mice. With PTH(1‐34) treatment, osteoblast surface trended toward an increase in C57BL/6 mice and increased significantly in LDLR−/− mice. PTH(1‐34) did not alter resorption parameters significantly, except for the eroded surface (ES/BS), which was reduced in the C57BL/6 but not in the LDLR−/− mice. These results show that PTH(1‐34) has adverse effects on cortical bones of the hyperlipidemic mice, suggesting that the therapeutic effects of PTH may be compromised in the presence of hyperlipidemia.

PKA-induced Receptor Activator of NF-κB Ligand (RANKL) Expression in Vascular Cells Mediates Osteoclastogenesis but Not Matrix Calcification

Vascular calcification is a predictor of cardiovascular mortality and is prevalent in patients with atherosclerosis and chronic renal disease. It resembles skeletal osteogenesis, and many bone cells as well as bone-related factors involved in both formation and resorption have been localized in calcified arteries. Previously, we showed that aortic medial cells undergo osteoblastic differentiation and matrix calcification both spontaneously and in response to PKA agonists. The PKA signaling pathway is also involved in regulating bone resorption in skeletal tissue by stimulating osteoblast-production of osteoclast regulating cytokines, including receptor-activator of nuclear κB ligand (RANKL) and interleukins. Therefore, we investigated whether PKA activators regulate osteoclastogenesis in aortic smooth muscle cells (SMC). Treatment of murine SMC with the PKA agonist forskolin stimulated RANKL expression at both mRNA and protein levels. Forskolin also stimulated expression of interleukin-6 but not osteoprotegerin (OPG), an inhibitor of RANKL. Consistent with these results, osteoclastic differentiation was induced when monocytic preosteoclasts (RAW264.7) were cocultured with forskolin-treated aortic SMC. Oxidized phospholipids also slightly induced RANKL expression in T lymphocytes, another potential source of RANKL in the vasculature. Because previous studies have shown that RANKL treatment alone induces matrix calcification of valvular and vascular cells, we next examined whether RANKL mediates forskolin-induced matrix calcification by aortic SMC. RANKL inhibition with OPG had little or no effect on osteoblastic differentiation and matrix calcification of aortic SMC. These findings suggest that, as in skeletal tissues, PKA activation induces bone resorptive factors in the vasculature and that aortic SMC calcification specifically induced by PKA, is not mediated by RANKL.

Role of Cellular Cholesterol Metabolism in Vascular Cell Calcification

Vascular calcification impairs vessel compliance and increases the risk of cardiovascular events. We found previously that liver X receptor agonists, which regulate intracellular cholesterol homeostasis, augment PKA agonist- or high phosphate-induced osteogenic differentiation of vascular smooth muscle cells. Because cholesterol is an integral component of the matrix vesicles that nucleate calcium mineral, we examined the role of cellular cholesterol metabolism in vascular cell mineralization. The results showed that vascular smooth muscle cells isolated from LDL receptor null (Ldlr−/−) mice, which have impaired cholesterol uptake, had lower levels of intracellular cholesterol and less osteogenic differentiation, as indicated by alkaline phosphatase activity and matrix mineralization, compared with WT cells. PKA activation with forskolin acutely induced genes that promote cholesterol uptake (LDL receptor) and biosynthesis (HMG-CoA reductase). In WT cells, inhibition of cholesterol uptake by lipoprotein-deficient serum attenuated forskolin-induced matrix mineralization, which was partially reversed by the addition of cell-permeable cholesterol. Prolonged activation of both uptake and biosynthesis pathways by cotreatment with a liver X receptor agonist further augmented forskolin-induced matrix mineralization. Inhibition of either cholesterol uptake, using Ldlr−/− cells, or of cholesterol biosynthesis, using mevastatin-treated WT cells, failed to inhibit matrix mineralization due to up-regulation of the respective compensatory pathway. Inhibition of both pathways simultaneously using mevastatin-treated Ldlr−/− cells did inhibit forskolin-induced matrix mineralization. Altogether, the results suggest that up-regulation of cholesterol metabolism is essential for matrix mineralization by vascular cells.

Protective Role of Smad6 in Inflammation‐Induced Valvular Cell Calcification

Calcific aortic vascular and valvular disease (CAVD) is associated with hyperlipidemia, the effects of which occur through chronic inflammation. Evidence suggests that inhibitory small mothers against decapentaplegic (I‐Smads; Smad6 and 7) regulate valve embryogenesis and may serve as a mitigating factor in CAVD. However, whether I‐Smads regulate inflammation‐induced calcific vasculopathy is not clear. Therefore, we investigated the role of I‐Smads in atherosclerotic calcification. Results showed that expression of Smad6, but not Smad7, was reduced in aortic and valve tissues of hyperlipidemic compared with normolipemic mice, while expression of tumor necrosis factor alpha (TNF‐α) was upregulated. To test whether the effects are in response to inflammatory cytokines, we isolated murine aortic valve leaflets and cultured valvular interstitial cells (mVIC) from the normolipemic mice. By immunochemistry, mVICs were strongly positive for vimentin, weakly positive for smooth muscle α actin, and negative for an endothelial cell marker. TNF‐α upregulated alkaline phosphatase (ALP) activity and matrix mineralization in mVICs. By gene expression analysis, TNF‐α significantly upregulated bone morphogenetic protein 2 (BMP‐2) expression while downregulating Smad6 expression. Smad7 expression was not significantly affected. To further test the role of Smad6 on TNF‐α‐induced valvular cell calcification, we knocked down Smad6 expression using lentiviral transfection. In cells transfected with Smad6 shRNA, TNF‐α further augmented ALP activity, expression of BMP‐2, Wnt‐ and redox‐regulated genes, and matrix mineralization compared with the control cells. These findings suggest that TNF‐α induces valvular and vascular cell calcification, in part, by specifically reducing the expression of a BMP‐2 signaling inhibitor, Smad6. J. Cell. Biochem. 116: 2354–2364, 2015.

Regulation of interleukin-6 expression in osteoblasts by oxidized phospholipids

Epidemiological evidence suggests that cardiovascular disease is associated with osteoporosis, independent of age. Bone resorptive surface is increased in mice on a high-fat diet, and osteoclastic differentiation of bone marrow preosteoclasts is promoted by oxidized phospholipids. Because osteoclastic differentiation requires cytokines produced by osteoblasts, we hypothesized that the stimulatory mechanism of oxidized phospholipids is via induction of osteoclast-regulating cytokines in osteoblasts. To investigate the effects of oxidized phospholipids on expression of such cytokines, murine calvarial preosteoblasts, MC3T3-E1, were treated with oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine (ox-PAPC), an active component of oxidized lipoproteins. Results showed that ox-PAPC increased expression of interleukin-6 (IL-6) and tumor necrosis factor-α. IL-6 expression was also elevated in calvarial tissues from hyperlipidemic but not in wild-type mice. Ox-PAPC also induced IL-6 protein levels in both MC3T3-E1 and primary calvarial cells. Promoter-reporter assay analysis showed that ox-PAPC, but not PAPC, induced murine IL-6 promoter activity. Effects of ox-PAPC on IL-6 expression and the promoter activity were attenuated by H89, a PKA inhibitor. Analysis of deletion and mutant IL-6 promoter constructs suggested that CAAT/enhancer binding protein (C/EBP) partly mediates the ox-PAPC effects. Taken together, the data suggest that oxidized phospholipids induce IL-6 expression in osteoblasts in part via C/EBP.