Inactivation of a non-canonical gp130 signaling arm attenuates chronic systemic inflammation and multimorbidity induced by a high-fat diet.

Interleukin-6 (IL-6) is a major pro-inflammatory cytokine for which the levels in plasma demonstrate a robust correlation with age and body mass index (BMI) as part of the senescence-associated secretory phenotype. IL-6 cytokines also play a crucial role in metabolic homeostasis and regenerative processes, primarily via the canonical STAT3 pathway. Thus, selective modulation of IL-6 signaling may offer a unique opportunity for therapeutic interventions. Recently, we discovered that a non-canonical signaling pathway downstream of tyrosine (Y) 814 within the intracellular domain of gp130, the IL-6 co-receptor, is responsible for the recruitment and activation of SRC family of kinases (SFK). Mice with constitutive genetic inactivation of gp130 Y814 (F814 mice) show accelerated resolution of inflammatory response and superior regenerative outcomes in skin wound healing and posttraumatic models of osteoarthritis. The current study was designed to explore if selective genetic or pharmacological inhibition of the non-canonical gp130-Y814/SFK signaling reduces systemic chronic inflammation and multimorbidity in a high-fat diet (HFD)-induced model of accelerated aging. F814 mice showed significantly reduced inflammatory response to HFD in adipose and liver tissue, with significantly reduced levels of systemic inflammation compared to wild type mice. F814 mice were also protected from HFD-induced bone loss and cartilage degeneration. Pharmacological inhibition of gp130-Y814/SFK in mice on HFD mirrored the effects observed in F814 mice on HFD; furthermore, this pharmacological treatment also demonstrated a marked increase in physical activity levels and protective effects against inflammation-associated suppression of neurogenesis in the brain tissue compared to the control group. These findings suggest that selective inhibition of SFK signaling downstream of gp130 receptor represents a promising strategy to alleviate systemic chronic inflammation. Increased degenerative changes and tissue senescence are inevitable in obese and aged organisms, but we demonstrated that the systemic response and inflammation-associated multi-morbidity can be therapeutically mitigated.

High-frequency ultrasound imaging to assess fetal pancreas: a promising application.

Objectives: The purpose of this study is to establish a comprehensive reference range of quantitative characteristics of the fetal pancreas using a high-frequency transducer, and assess the growth and development of the fetal pancreas.Methods: Pregnant women referred to a tertiary center were recruited to undergo a detailed fetal scan from 16 to 37 weeks. We evaluated the visualization rate of the fetal pancreas with high-frequency and low-frequency transducers and measured the head, neck, body, tail, circumference, area, and abdominal circumference(AC) of the fetal pancreas at different gestational ages(GA) with the high-frequency transducer. Regression analysis was used to analyze the relationship between biological parameters and GA and AC.Results: During the time periods of 16+1∼21+6 weeks and 22+1∼27+6 weeks, the visualization rate of high-frequency transducers was higher compared to low-frequency transducers (83.33% vs 45% and 95.65% vs 70%, respectively). However, in the third trimester of pregnancy, the performance of the two transducers was similar (70.37% vs 74.07% for 28+1∼33+6 weeks and 41.67% vs 53.85% for 34+1∼37+6 weeks). The head, neck, body, and tail as well as the circumference and area of the pancreas were significantly positively correlated with GA (R2＝0.87, 0.94, 0.92, 0.92,0.96, and 0.92) and AC (R2＝0.87, 0.93, 0.91, 0.93,0.96, and 0.92).Conclusions: The high-frequency transducer was utilized to establish the normal reference, which can be used to evaluate normal pancreatic development and may help in the accurate diagnosis of fetal pancreatic abnormalities.

STAT3 promotes a youthful epigenetic state in articular chondrocytes.

Epigenetic mechanisms guiding articular cartilage regeneration and age-related disease such as osteoarthritis (OA) are poorly understood. STAT3 is a critical age-patterned transcription factor highly active in fetal and OA chondrocytes, but the context-specific role of STAT3 in regulating the epigenome of cartilage cells remain elusive. In this study, DNA methylation profiling was performed across human chondrocyte ontogeny to build an epigenetic clock and establish an association between CpG methylation and human chondrocyte age. Exposure of adult chondrocytes to a small molecule STAT3 agonist decreased DNA methylation, while genetic ablation of STAT3 in fetal chondrocytes induced global hypermethylation. CUT&RUN assay and subsequent transcriptional validation revealed DNA methyltransferase 3 beta (DNMT3B) as one of the putative STAT3 targets in chondrocyte development and OA. Functional assessment of human OA chondrocytes showed the acquisition of progenitor-like immature phenotype by a significant subset of cells. Finally, conditional deletion of Stat3 in cartilage cells increased DNMT3B expression in articular chondrocytes in the knee joint in vivo and resulted in a more prominent OA progression in a post-traumatic OA (PTOA) mouse model induced by destabilization of the medial meniscus (DMM). Taken together these data reveal a novel role for STAT3 in regulating DNA methylation in cartilage development and disease. Our findings also suggest that elevated levels of active STAT3 in OA chondrocytes may indicate an intrinsic attempt of the tissue to regenerate by promoting a progenitor-like phenotype. However, it is likely that chronic activation of this pathway, induced by IL-6 cytokines, is detrimental and leads to tissue degeneration.

Identification and validation of quantitative trait loci for chlorophyll content of flag leaf in wheat under different phosphorus treatments.

In wheat, the leaf chlorophyll content in flag leaves is closely related to the degree of phosphorus stress. Identifying major genes/loci associated with chlorophyll content in flag leaves under different phosphorus conditions is critical for breeding wheat varieties resistant to low phosphorus (P). Under normal, medium, and low phosphorus conditions, the chlorophyll content of flag leaves was investigated by a double haploid (DH) population derived from a cross between two popular wheat varieties Jinmai 47 and Jinmai 84, at different grain filling stages. Chlorophyll content of the DH population and parents decreased gradually during the S1 to the S3 stages and rapidly at the S4 stage. At the S4 stage, the chlorophyll content of the DH population under low phosphorus conditions was significantly lower than under normal phosphate conditions. Using a wheat 15K single-nucleotide polymorphism (SNP) panel, a total of 157 QTLs were found to be associated with chlorophyll content in flag leaf and were identified under three phosphorus conditions. The phenotypic variation explained (PVE) ranged from 3.07 to 31.66%. Under three different phosphorus conditions, 36, 30, and 48 QTLs for chlorophyll content were identified, respectively. Six major QTLs Qchl.saw-2B.1, Qchl.saw-3B.1, Qchl.saw-4D.1, Qchl.saw-4D.2, Qchl.saw-5A.9 and Qchl.saw-6A.4 could be detected under multiple phosphorus conditions in which Qchl.saw-4D.1, Qchl.saw-4D.2, and Qchl.saw-6A.4 were revealed to be novel major QTLs. Moreover, the closely linked SNP markers of Qchl.saw-4D.1 and Qchl.saw-4D.2 were validated as KASP markers in a DH population sharing the common parent Jinmai 84, showed extreme significance (P <0.01) in more than three environments under different phosphorus conditions, which has the potential to be utilized in molecular marker-assisted breeding for low phosphorus tolerance in wheat.

gp130/STAT3 signaling is required for homeostatic proliferation and anabolism in postnatal growth plate and articular chondrocytes.

Growth of long bones and vertebrae is maintained postnatally by a long-lasting pool of progenitor cells. Little is known about the molecular mechanisms that regulate the output and maintenance of the cells that give rise to mature cartilage. Here we demonstrate that postnatal chondrocyte-specific deletion of a transcription factor Stat3 results in severely reduced proliferation coupled with increased hypertrophy, growth plate fusion, stunting and signs of progressive dysfunction of the articular cartilage. This effect is dimorphic, with females more strongly affected than males. Chondrocyte-specific deletion of the IL-6 family cytokine receptor gp130, which activates Stat3, phenocopied Stat3-deletion; deletion of Lifr, one of many co-receptors that signals through gp130, resulted in a milder phenotype. These data define a molecular circuit that regulates chondrogenic cell maintenance and output and reveals a pivotal positive function of IL-6 family cytokines in the skeletal system with direct implications for skeletal development and regeneration.

A Single-Cell Culture System for Dissecting Microenvironmental Signaling in Development and Disease of Cartilage Tissue.

Cartilage tissue is comprised of extracellular matrix and chondrocytes, a cell type with very low cellular turnover in adults, providing limited capacity for regeneration. However, in development a significant number of chondrocytes actively proliferate and remodel the surrounding matrix. Uncoupling the microenvironmental influences that determine the balance between clonogenic potential and terminal differentiation of these cells is essential for the development of novel approaches for cartilage regeneration. Unfortunately, most of the existing methods are not applicable for the analysis of functional properties of chondrocytes at a single cell resolution. Here we demonstrate that a novel 3D culture method provides a long-term and permissive in vitro niche that selects for highly clonogenic, colony-forming chondrocytes which maintain cartilage-specific matrix production, thus recapitulating the in vivo niche. As a proof of concept, clonogenicity of Sox9 IRES-EGFP mouse chondrocytes is almost exclusively found in the highest GFP+ fraction known to be enriched for chondrocyte progenitor cells. Although clonogenic chondrocytes are very rare in adult cartilage, we have optimized this system to support large, single cell-derived chondrogenic organoids with complex zonal architecture and robust chondrogenic phenotype from adult pig and human articular chondrocytes. Moreover, we have demonstrated that growth trajectory and matrix biosynthesis in these organoids respond to a pro-inflammatory environment. This culture method offers a robust, defined and controllable system that can be further used to interrogate the effects of various microenvironmental signals on chondrocytes, providing a high throughput platform to assess genetic and environmental factors in development and disease.

Effects of teriparatide on morphology of aortic calcification in aged hyperlipidemic mice.

Calcific aortic vasculopathy correlates with bone loss in osteoporosis in an age-independent manner. Prior work suggests that teriparatide, the bone anabolic treatment for postmenopausal osteoporosis, may inhibit the onset of aortic calcification. Whether teriparatide affects the progression of preexisting aortic calcification, widespread among this patient population, is unknown. Female apolipoprotein E-deficient mice were aged for over 1 yr to induce aortic calcification, treated for 4.5 wk with daily injections of control vehicle (PBS), 40 µg/kg teriparatide (PTH40), or 400 µg/kg teriparatide (PTH400), and assayed for aortic calcification by microcomputed tomography (microCT) before and after treatment. In a followup cohort, aged female apolipoprotein E-deficient mice were treated with PBS or PTH400 and assayed for aortic calcification by serial microCT and micropositron emission tomography. In both cohorts, aortic calcification detected by microCT progressed similarly in all groups. Mean aortic 18F-NaF incorporation, detected by serial micropositron emission tomography, increased in the PBS-treated group (+14 ± 5%). In contrast, 18F-NaF incorporation decreased in the PTH400-treated group (-33 ± 20%, P = 0.03). Quantitative histochemical analysis by Alizarin red staining revealed a lower mineral surface area index in the PTH400-treated group compared with the PBS-treated group ( P = 0.04). Furthermore, Masson trichrome staining showed a significant increase in collagen deposition in the left ventricular myocardium of mice that received PTH400 [2.1 ± 0.6% vs. control mice (0.5 ± 0.1%), P = 0.02]. In summary, although teriparatide may not affect the calcium mineral content of aortic calcification, it reduces 18F-NaF uptake in calcified lesions, suggesting the possibility that it may reduce mineral surface area with potential impact on plaque stability. NEW & NOTEWORTHY Parathyroid hormone regulates bone mineralization and may also affect vascular calcification, which is an important issue, given that its active fragment, teriparatide, is widely used for the treatment of osteoporosis. To determine whether teriparatide alters vascular calcification, we imaged aortic calcification in mice treated with teriparatide and control mice. Although teriparatide did not affect the calcium content of cardiovascular deposits, it reduced their fluoride tracer uptake.

Highly expressed long non-coding RNA FOXD2-AS1 promotes non-small cell lung cancer progression via Wnt/ß-catenin signaling.

Non-small cell lung cancer (NSCLC) is one of the most common and aggressive tumors around the world. Long-noncoding RNAs (lncRNAs) have been recently shown to play important roles in regulating numerous biological processes including tumor progression. However, the role of lncRNA FOXD2-AS1 in NSCLC remains unclear. In this study, we found that lncRNA FOXD2-AS1 is significantly up-regulated in NSCLC tissues. Loss- and gain-function assays revealed that FOXD2-AS1 promotes NSCLC cell growth and NSCLC tumor progression. Furthermore, we also revealed that FOXD2-AS1 modulates Wnt/ß-catenin signaling in NSCLC cells. Taken together, we conclude that lncRNA FOXD2-AS1 promotes NSCLC progression though Wnt/ß-catenin signaling. These results suggest that lncRNA FOXD2-AS1 might act as a novel therapeutic target for NSCLC treatment.

Inflammation Drives Retraction, Stiffening, and Nodule Formation via Cytoskeletal Machinery in a Three-Dimensional Culture Model of Aortic Stenosis.

In calcific aortic valve disease, the valve cusps undergo retraction, stiffening, and nodular calcification. The inflammatory cytokine, tumor necrosis factor (TNF)-α, contributes to valve disease progression; however, the mechanisms of its actions on cusp retraction and stiffening are unclear. We investigated effects of TNF-α on murine aortic valvular interstitial cells (VICs) within three-dimensional, free-floating, compliant, collagen hydrogels, simulating their natural substrate and biomechanics. TNF-α increased retraction (percentage of diameter), stiffness, and formation of macroscopic, nodular structures with calcification in the VIC-laden hydrogels. The effects of TNF-α were attenuated by blebbistatin inhibition of myosin II-mediated cytoskeletal contraction. Inhibition of actin polymerization with cytochalasin-D, but not inhibition of Rho kinase with Y27632, blocked TNF-α-induced retraction in three-dimensional VIC hydrogels, suggesting that actin stress fibers mediate TNF-α-induced effects. In the hydrogels, inhibitors of NF-κB blocked TNF-α-induced retraction, whereas simultaneous inhibition of c-Jun N-terminal kinase was required to block TNF-α-induced stiffness. TNF-α also significantly increased collagen deposition, as visualized by Masson's trichrome staining, and up-regulated mRNA expression of discoidin domain receptor tyrosine kinase 2, fibronectin, and α-smooth muscle actin. In human aortic valves, calcified cusps were stiffer and had more collagen deposition than noncalcified cusps. These findings suggest that inflammation, through stimulation of cytoskeletal contractile activity, may be responsible for valvular cusp retraction, stiffening, and formation of calcified nodules.

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.

Roles of parathyroid hormone (PTH) receptor and reactive oxygen species in hyperlipidemia-induced PTH resistance in preosteoblasts.

Bioactive lipids initiate inflammatory reactions leading to pathogenesis of atherosclerosis. Evidence shows that they also contribute to bone loss by inhibiting parathyroid hormone receptor (PTH1R) expression and differentiation of osteoblasts. We previously demonstrated that bone anabolic effects of PTH(1-34) are blunted in hyperlipidemic mice and that these PTH effects are restored by antioxidants. However, it is not clear which osteoblastic cell developmental stage is targeted by bioactive lipids. To investigate the effects of hyperlipidemia at the cellular level, hyperlipidemic Ldlr(-/-) mice were bred with Col3.6GFPtpz mice, in which preosteoblasts/osteoblasts carry a topaz fluorescent label, and with Col2.3GFPcyan mice, in which more mature osteoblasts/osteocytes carry a cyan fluorescent label. Histological analyses of trabecular bone surfaces in femoral as well as calvarial bones showed that intermittent PTH(1-34) increased fluorescence intensity in WT-Tpz mice, but not in Tpz-Ldlr(-/-) mice. In contrast, PTH(1-34) did not alter fluorescence intensity in femoral cortical envelopes of either WT-Cyan or Ldlr(-/-)-Cyan mice. To test the mechanism of PTH1R downregulation, preosteoblastic MC3T3-E1 cells were treated with bioactive lipids and the antioxidant Trolox. Results showed that inhibitory effects of PTH1R levels by bioactive lipids were rescued by pretreatment with Trolox. The inhibitory effects on expression of PTH1R as well as on PTH-induced osteoblastic genes were mimicked by xanthine/xanthine oxidase, a known generator of reactive oxygen species. These findings suggest an important role of the preosteoblastic development stage as the target and downregulation of PTH receptor expression mediated by intracellular oxidant stress as a mechanism in hyperlipidemia-induced PTH resistance.

Role of paraoxonase-1 in bone anabolic effects of parathyroid hormone in hyperlipidemic mice.

Hyperlipidemia blunts anabolic effects of intermittent parathyroid hormone (PTH) on cortical bone, and the responsiveness to PTH are restored in part by oral administration of the antioxidant ApoA-I mimetic peptide, D-4F. To evaluate the mechanism of this rescue, hyperlipidemic mice overexpressing the high-density lipoprotein-associated antioxidant enzyme, paraoxonase 1 (Ldlr(-/-)PON1(tg)) were generated, and daily PTH injections were administered to Ldlr(-/-)PON1(tg) and to littermate Ldlr(-/-) mice. Expression of bone regulatory genes was determined by realtime RT-qPCR, and cortical bone parameters of the femoral bones by micro-computed tomographic analyses. PTH-treated Ldlr(-/-)PON1(tg) mice had significantly greater expression of PTH receptor (PTH1R), activating transcription factor-4 (ATF4), and osteoprotegerin (OPG) in femoral cortical bone, as well as significantly greater cortical bone mineral content, thickness, and area in femoral diaphyses compared with untreated Ldlr(-/-)PON1(tg) mice. In contrast, in control mice (Ldlr(-/-)) without PON1 overexpression, PTH treatment did not induce these markers. Calvarial bone of PTH-treated Ldlr(-/-)PON1(tg) mice also had significantly greater expression of osteoblastic differentiation marker genes as well as BMP-2-target and Wnt-target genes. Untreated Ldlr(-/-)PON1(tg) mice had significantly greater expression of PTHR1 than untreated Ldlr(-/-) mice, whereas sclerostin expression was reduced. In femoral cortical bones, expression levels of transcription factors, FoxO1 and ATF4, were also elevated in the untreated, control Ldlr(-/-)PON1(tg) mice, suggesting enhancement of cellular protection against oxidants. These findings suggest that PON1 restores responsiveness to PTH through effects on oxidant stress, PTH receptor expression, and/or Wnt signaling.

Focal high cell density generates a gradient of patterns in self-organizing vascular mesenchymal cells.

In embryogenesis, structural patterns, such as vascular branching, may form via a reaction-diffusion mechanism in which activator and inhibitor morphogens guide cells into periodic aggregates. We previously found that vascular mesenchymal cells (VMCs) spontaneously aggregate into nodular structures and that morphogen pairs regulate the aggregation into patterns of spots and stripes. To test the effect of a focal change in activator morphogen on VMC pattern formation, we created a focal zone of high cell density by plating a second VMC layer within a cloning ring over a confluent monolayer. After 24 h, the ring was removed and pattern formation monitored by phase-contrast microscopy. At days 2-8, the patterns progressed from uniform distributions to swirl, labyrinthine and spot patterns. Within the focal high-density zone (HDZ) and a narrow halo zone, cells aggregated into spot patterns, whilst in the outermost zone of the plate, cells formed a labyrinthine pattern. The area occupied by aggregates was significantly greater in the outermost zone than in the HDZ or halo. The rate of pattern progression within the HDZ increased as a function of its plating density. Thus, focal differences in cell density may drive pattern formation gradients in tissue architecture, such as vascular branching.

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.

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.

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.

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.

PKA-induced receptor activator of NF-kappaB 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.

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-alpha. 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.