Overexpression of secreted frizzled-related protein 1 inhibits bone formation and attenuates parathyroid hormone bone anabolic effects

Authors

Yao, W. and Cheng, Z. and Shahnazari, M. and Dai, W. and Johnson, M.L. and Lane, N.E.

Abstract

Secreted frizzled-related protein 1 (sFRP1) is an antagonist of Wnt signaling, an important pathway in maintaining bone homeostasis. In this study we evaluated the skeletal phenotype of mice overexpressing sFRP1 (sFRP1 Tg) and the interaction of parathyroid hormone (PTH) treatment and sFRP1 (over)expression. Bone mass and microarchitecture were measured by micro-computed tomography (µCT). Osteoblastic and osteoclastic cell maturation and function were assessed in primary bone marrow cell cultures. Bone turnover was assessed by biochemical markers and dynamic bone histomorphometry. Real-time PCR was used to monitor the expression of several genes that regulate osteoblast maturation and function in whole bone. We found that trabecular bone mass measurements in distal femurs and lumbar vertebral bodies were 22% and 51% lower in female and 9% and 33% lower in male sFRP1 Tg mice, respectively, compared with wild-type (WT) controls at 3 months of age. Genes associated with osteoblast maturation and function, serum bone formation markers, and surface based bone formation were significantly decreased in sFRP1 Tg mice of both sexes. Bone resorption was similar between sFRP1 Tg and WT females and was higher in sFRP1 Tg male mice. Treatment with hPTH(1-34) (40 µg/kg/d) for 2 weeks increased trabecular bone volume in WT mice (females: +30% to 50%; males: +35% to 150%) compared with sFRP1 Tg mice (females: +5%; males: +18% to 54%). Percentage increases in bone formation also were lower in PTH-treated sFRP1 Tg mice compared with PTH-treated WT mice. In conclusion, overexpression of sFRP1 inhibited bone formation as well as attenuated PTH anabolic action on bone. The gender differences in the bone phenotype of the sFRP1 Tg animal warrants further investigation.

Link to Article

http://www3.interscience.wiley.com/journal/123210016/abstract

Ex Vivo Transfer of the Hoxc-8-interacting Domain of Smad1 by a Tropism-modified Adenoviral Vector Results in Efficient Bone Formation in a Rabbit Model of Spinal Fusion

Authors

Douglas, J.T. and Rivera, A.A. and Lyons, G.R. and Lott, P.F. and Wang, D. and Zayzafoon, M. and Siegal, G.P. and Cao, X. and Theiss, S.M.

Abstract

Study Design: Ex vivo gene transfer for spinal fusion.

Objective: This study aimed to evaluate ex vivo transfer of the nuclear-localized Hoxc-8-interacting domain of Smad1 (termed Smad1C) to rabbit bone marrow stromal cells (BMSCs) by a tropism-modified human adenovirus serotype 5 (Ad5) vector as a novel therapeutic approach for spinal fusion.

Summary of Background Data: Novel approaches are needed to improve the success of bone union after spinal fusion. One such approach is the ex vivo transfer of a gene encoding an osteoinductive factor to BMSCs which are subsequently reimplanted into the host. We have previously shown that heterologous expression of the Hoxc-8-interacting domain of Smad1 in the nuclei of osteoblast precursor cells is able to stimulate the expression of genes related to osteoblast differentiation and induce osteogenesis in vivo. Gene delivery vehicles based on human Ad5 are well suited for gene transfer for spinal fusion because they can mediate high-level, short-term gene expression. However, Ad5-based vectors with native tropism poorly transduce BMSCs, necessitating the use of vectors with modified tropism to achieve efficient gene transfer.

Methods: The gene encoding Smad1C was transferred to rabbit BMSCs by an Ad5 vector with native tropism or a vector retargeted to αv integrins, which are abundantly expressed on rabbit BMSCs. Transduced BMSCs were maintained in osteoblastic differentiation medium for 30 days. Alkaline phosphatase activity was determined and cells stained for calcium deposition. As positive controls for osteogenesis, we used Ad5 vectors expressing bone morphogenetic protein 2. As negative controls, BMSCs were mock-transduced or transduced with an Ad5 vector expressing β-galactosidase. In an immunocompetent rabbit model of spinal fusion, transduced BMSCs were coated onto absorbable gelatin sponge and implanted between decorticated transverse processes L6 and L7 of 8-week-old female New Zealand white rabbits. Animals were killed 4 weeks after implantation of the sponges, the fusion masses harvested and the area of new bone quantified using image analysis software.

Results: The Smad1C-expressing tropism-modified Ad5 vector mediated a significantly higher level of alkaline phosphatase activity and calcium deposition in transduced rabbit BMSCs than all other vectors. The rabbit BMSCs transduced ex vivo with the Smad1C-expressing tropism-modified Ad5 vector mediated a greater amount of new bone formation than BMSCs transduced with any other vector.

Conclusions: Delivery of the Smad1C gene construct to BMSCs by an αv integrin-targeted Ad5 vector shows promise for spinal fusion and other applications requiring the formation of new bone in vivo.

Link to Article

http://journals.lww.com/jspinaldisorders/Abstract/2010/02000/Ex_Vivo_Transfer_of_the_Hoxc_8_interacting_Domain.13.aspx

Multiple roles for CCR2 during fracture healing

Authors

Xing, Z. and Lu, C. and Hu, D. and Yu, Y. and Wang, X. and Colnot, C. and Nakamura, M. and Wu, Y. and Miclau, T. and Marcucio, R.S.

Abstract

Bone injury induces an inflammatory response that involves neutrophils, macrophages and other inflammatory cells. The recruitment of inflammatory Ccr2 transcripts and the filtration of macrophages into fracture calluses were most robust during the early phases of fracture healing. We then determined that the number of macrophages at the fracture site was significantly lower in Ccr2–/– mice compared with wild-type controls at 3 days injury. As a result, impaired vascularization, decreased formation of callus, and delayed maturation of cartilage were observed at 7 days after in mutant mice. At day 14, Ccr2–/– mice had less bone in their calluses. At day 21, Ccr2–/– mice had larger calluses and more bone compared with wild-type mice, suggesting a delayed remodeling. In addition, we examined the effect of Ccr2 mutation on osteoclasts. We found that a lack of Ccr2 did not affect the number of osteoclasts within fracture calluses at 21 days after injury. However, Ccr2–/– osteoclasts exhibited a decreased ability to resorb bone compared with wild-type cells, which could contribute to the delayed remodeling of fracture calluses observed in Ccr2–/– mice. Collectively, these results indicate that a deficiency of Ccr2 reduces the infiltration of macrophages and impairs the function of osteoclasts, leading to delayed fracture healing.

Link to Article

http://dmm.biologists.org/content/early/2010/03/25/dmm.003186.full.pdf

Influence of Different-Frequency GlucocorticoidInduction on Morphological Structures of Humeri,Soft Tissues and Immune System in Rats

Authors

Li Jian-min, Li Heng

Abstract

Objective: To explore the influence of different-frequency glucocorticoid (GC) induction on morphological structures of humeri and soft tissues as well as immune system in rats. 

Methods: A total of 32 specific pathogen-free (SPF) SD rats at the age of 3 months were selected and randomly divided into 4 groups, 8 cases in each group. The rats in control group were not given any treatment, while those in low-, moderate- and high-frequency groups were treated with intramuscular injection of dexamethasone 1 mg/kg per time for twice, 4 times and 6 times per week, respectively. All the rats were sacrificed on d30 to measure their body mass and qualities of soft tissues and immune organs, and bone histomorphometry was applied to analyze humeral bone mass and bone structural changes.

Results: Compared with control group, there was no change in cancellous bone mass and bone structures of upper humeri in low-frequency group, but serious loss of bone mass, significantly degenerated bone structure, markedly reduced trabecular thickness and number as well as notably increased trabecular separation was all observed in moderate- and high-frequency groups. The size of cortical bones, total size of bone structure, thickness of cortical bones and size percentage of cortical bones in middle humeri reduced apparently, while the size percentage of medullary cavity increased dramatically in high-frequency group. Growth plate thickness of upper humeri decreased in low-, moderate- and high-frequency groups, and the diameters of mastocytes diminished in moderate- and high-frequency groups. Compared with control group, body mass decreased obviously, qualities and indexes of spleen and thymus showed decreasing tendency along with the increase of drug administration frequency in low-, moderate- and high-frequency groups.

Conclusion: Low-frequency GC cannot change humeral morphology. The higher the frequency of drug administration is, the more the loss of cancellous bone mass is. When the frequency reaches to 6 times per week, the loss of cortical and cancellous bones is much severer. However, with the increase of drug administration frequency, thymic degeneration, splenic atrophy and immunosuppression can be induced. Therefore, the influence of different-frequency drug administration on bones and soft tissues in different locations as well as immune function should be fully considered and reasonable drug administration protocols should be designed for the establishment of SD rat models with osteoporosis.

Link to Article

http://scholar.google.com/scholar_url?url=http://www.jitm.hk/CN/article/downloadArticleFile.do%3FattachType%3DPDF%26id%3D196&hl=en&sa=X&scisig=AAGBfm28q6Urn7yc_UONqiUYfhESjzbqFA&nossl=1&oi=scholaralrt

Lifelong challenge of calcium homeostasis in male mice lacking TRPV5 leads to changes in bone and calcium metabolism

Authors

Bram C. J. van der Eerden, W. Nadia H. Koek, Paul Roschger, M. Carola Zillikens, Jan H. Waarsing, Annemiete van der Kemp, Marijke Schreuders-Koedam, Nadja Fratzl-Zelman, Pieter J. M. Leenen, Joost G. J. Hoenderop,
Klaus Klaushofer, René J. M. Bindels and Johannes P.T.M. van Leeuwen

Abstract

Trpv5 plays an important role in calcium (Ca2+) homeostasis, among others by mediating renal calcium reabsorption. Accordingly, Trpv5 deficiency strongly stresses Ca2+ homeostasis in order to maintain stable serum Ca2+. We addressed the impact of lifelong challenge of calcium homeostasis on the bone phenotype of these mice. Aging significantly increased serum 1,25(OH)2D3 and PTH levels in both genotypes but they were more elevated in Trpv5-/- mice, whereas serum Ca2+ was not affected by age or genotype. Age-related changes in trabecular and cortical bone mass were accelerated in Trpv5-/- mice, including reduced trabecular and cortical bone thickness as well as reduced bone mineralization. No effect of Trpv5 deficiency on bone strength was observed. In 78-week-old mice no differences were observed between the genotypes regarding urinary deoxypyridinoline, osteoclast number, differentiation and activity as well as osteoclast precursor numbers, as assessed by flow cytometry. In conclusion, life-long challenge of Ca2+ homeostasis present in Trpv5-/- mice causes accelerated bone aging and a low cortical and trabecular bone mass phenotype. The phenotype of the Trpv5-/- mice suggests that maintenance of adequate circulatory Ca2+ levels in patients with disturbances in Ca2+ homeostasis should be a priority in order to prevent bone loss at older age.

Link to Article

http://www.physiomics.eu/media/210426/j_biol_chem_xx_xx-xx__2016.1.pdf