Inhibition of CaMKK2 reverses age-associated decline in bone mass

Authors

Zachary J. Pritchard, Rachel L. Cary, Chang Yang, Deborah V. Novack, Michael J. Voor, Uma Sankar

Abstract

Decline in bone formation is a major contributing factor to the loss of bone mass associated with aging. We previously showed that the genetic ablation of the tissue-restricted and multifunctional Ca2+/calmodulin (CaM)-dependent protein kinase kinase 2 (CaMKK2) stimulates trabecular bone mass accrual, mainly by promoting anabolic pathways and inhibiting catabolic pathways of bone remodeling. In this study, we investigated whether inhibition of this kinase using its selective cell-permeable inhibitor STO-609 will stimulate bone formation in 32 week old male WT mice and reverse age-associated of decline in bone volume and strength. Tri-weekly intraperitoneal injections of saline or STO-609 (10 μM) were performed for six weeks followed by metabolic labeling with calcein and alizarin red. New bone formation was assessed by dynamic histomorphometry whereas micro-computed tomography was employed to measure trabecular bone volume, microarchitecture and femoral mid-shaft geometry. Cortical and trabecular bone biomechanical properties were assessed using three-point bending and punch compression methods respectively. Our results reveal that as they progress from 12 to 32 weeks of age, WT mice sustain a significant decline in trabecular bone volume, microarchitecture and strength as well as cortical bone strength. However, treatment of the 32 week old WT mice with STO-609 stimulated apposition of new bone and completely reversed the age-associated decrease in bone volume, quality, as well as trabecular and cortical bone strength. We also observed that regardless of age, male Camkk2−/− mice possessed significantly elevated trabecular bone volume, microarchitecture and compressive strength as well as cortical bone strength compared to age-matched WT mice, implying that the chronic loss of this kinase attenuates age-associated decline in bone mass. Further, whereas STO-609 treatment and/or the absence of CaMKK2 significantly enhanced the femoral mid-shaft geometry, the mid-shaft cortical wall thickness and material bending stress remained similar among the cohorts, implying that regardless of treatment, the material properties of the bone remain similar. Thus, our cumulative results provide evidence for the pharmacological inhibition of CaMKK2 as a bone anabolic strategy in combating age-associated osteoporosis.

Link To Article

http://dx.doi.org/10.1016/j.bone.2015.01.021

Extra- oral defect augmentation using autologous, bovine and equine bone blocks: a preclinical histomorphometrical comparative study

Authors

Tobias Moest, DDS, Falk Wehrhan, DDS, MD, PhD, Rainer Lutz, DDS, MD, Christian Martin Schmitt, DDS, Friedrich Wilhelm Neukam, DDS, MD, PhD, Karl Andreas Schlegel, DDS, MD, PhD

Abstract

Objectives This study aimed to compare autologous bone (AB), bovine bone (BB), and equine bone (EB) blocks with regard to de novo bone formation, connective tissue, and residual bone substitute material portions in a standardized defect animal model.

Material and Methods In the frontal skull of 20 pigs, 106 standardized cylindrical “critical size defects” were prepared. Defects were randomly filled with AB, BB, and EB blocks. After a healing period of 30 and 60 days, de novo bone formation, residual bone substitute material, and connective tissue portion was assessed by means of histomorphometry (Toluidine blue O staining). Mann-Whitney U-tests were used to evaluate differences between the groups.

Results The de novo bone formation was significantly higher in the AB group in comparison to the xenogeneic groups (p < 0.05). After 30 days, EB showed significantly (p < 0.05) more newly formed bone compared to the BB group. The soft tissue formation was significantly higher in the BB and EB group. Defects augmented with BB showed significantly (p < 0.05) higher portions of bone substitute materials compared to sides augmented with EB after 30 days.

Conclusion In the extra-oral model, AB blocks were superior concerning de novo bone formation. No clinical advantages of EB blocks could be observed.

Link To The Article

http://dx.doi.org/10.1016/j.jcms.2015.02.012

Development of a Mouse Model of Ischemic Osteonecrosis

Authors

Nobuhiro Kamiya MD, PhD, Ryosuke Yamaguchi MD, PhD, Olumide Aruwajoye MS, Naga Suresh Adapala PhD, Harry K. W. Kim MD, MS

Abstract

Availability of a reliable mouse model of ischemic osteonecrosis could accelerate the development of novel therapeutic strategies to stimulate bone healing after ischemic osteonecrosis; however, no mouse model of ischemic osteonecrosis is currently available.

Questions/purposes To develop a surgical mouse model of ischemic osteonecrosis, we asked, (1) if the blood vessels that contribute to the blood supply of the distal femoral epiphysis are cauterized, can we generate an osteonecrosis mouse model; (2) what are the histologic changes observed in this mouse model, and (3) what are the morphologic changes in the model.

Methods We performed microangiography to identify blood vessels supplying the distal femoral epiphysis in mice, and four vessels were cauterized using microsurgical techniques to induce ischemic osteonecrosis. Histologic assessment of cell death in the trabecular bone was performed using terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling (TUNEL) and counting the empty lacunae in three serial sections. Quantitation of osteoclast and osteoblast numbers was performed using image analysis software. Morphologic assessments of the distal femoral epiphysis for deformity and for trabecular bone parameters were performed using micro-CT.

Results We identified four blood vessels about the knee that had to be cauterized to induce total ischemic osteonecrosis of the distal femoral epiphysis. Qualitative assessment of histologic sections of the epiphysis showed a loss of nuclear staining of marrow cells, disorganized marrow structure, and necrotic blood vessels at 1 week. By 2 weeks, vascular tissue invasion of the necrotic marrow space was observed with a progressive increase in infiltration of the necrotic marrow space with the vascular tissue at 4 and 6 weeks. TUNEL staining showed extensive cell death in the marrow and trabecular bone 24 hours after the induction of ischemia. The mean percent of TUNEL-positive osteocytes in the trabecular bone increased from 2% ± 1% in the control group to a peak of 98% ± 3% in the ischemic group 1 week after induction of ischemia (mean difference, 96%; 95% CI, 81%–111%; p < 0.0001). The mean percent of empty lacunae increased from 1% ± 1% in the control group to a peak of 78% ± 15% in the ischemic group at 4 weeks (mean difference, 77%; 95% CI, 56%–97%; p < 0.0001). Quantitative analysis showed that the mean number of osteoclasts per bone surface was decreased in the ischemic group at 1, 2, and 4 weeks (p < 0.0001, < 0.0001, and p = 0.02, respectively) compared with the control group. The mean number of osteoclasts increased to a level similar to that of the control group at 6 weeks (p = 0.23). The numbers of osteoblasts per bone surface were decreased in the ischemic group at 1, 2 and 4 weeks (p < 0.0001 for each) compared with the numbers in the control group. The mean number of osteoblasts also increased to a level similar to that of the control group at 6 weeks (p = 0.91). Mean bone volume percent assessed by micro-CT was lower in the ischemic group compared with the control group from 2 to 6 weeks. The mean differences in the percent bone volume between the control and ischemic groups at 2, 4, and 6 weeks were 5.5% (95% CI, 0.9%–10.2%; p = 0.01), 5.3% (95% CI, 0.6%–9.9%; p = 0.02), and 6.0% (95% CI, 1.1%–10.9%; p = 0.008), respectively. A deformity of the distal femoral epiphysis was observed at 6 weeks with the mean epiphyseal height to width ratio of 0.74 ± 0.03 in the control group compared with 0.66 ± 0.06 in the ischemic group (mean difference, 0.08; 95% CI, 0.00–0.16; p = 0.03).

Conclusion We developed a novel mouse model of ischemic osteonecrosis that produced extensive cell death in the distal femoral epiphysis which developed a deformity with time.

Clinical Relevance The new mouse model may be a useful tool to test potential therapeutic strategies to improve bone healing after ischemic osteonecrosis.

Link To Article

http://dx.doi.org/10.1007/s11999-015-4172-6

β-PDGF Receptor Expressed by Hepatic Stellate Cells Regulates Fibrosis in Murine Liver Injury, but Not Carcinogenesis

Authors

Peri Kocabayoglu, Abigale Lade, Youngmin A. Lee, Ana-Cristina Dragomir, Xiaochen Sun, M. Isabel Fiel, Swan Thung, Costica Aloman, Philippe Soriano, Yujin Hoshida, Scott L. Friedman

Abstract

Background & Aims Rapid induction of β-PDGF receptor (β-PDGFR) is a core feature of hepatic stellate cell activation, but its cellular impact in vivo is not well characterized. We explored the contribution of β-PDGFR-mediated pathway activation to hepatic stellate cell responses in liver injury, fibrogenesis, and carcinogenesis in vivo using genetic models with divergent β-PDGFR activity, and assessed its prognostic implications in human cirrhosis.

Methods The impact of either loss or constitutive activation of β-PDGFR in stellate cells on fibrosis was assessed following carbon tetrachloride (CCl4) or bile duct ligation. Hepatocarcinogenesis in fibrotic liver was tracked after a single dose of diethylnitrosamine (DEN) followed by repeated injections of CCl4. Genome-wide expression profiling was performed from isolated stellate cells that expressed or lacked β-PDGFR to determine deregulated pathways and evaluate their association with prognostic gene signatures in human cirrhosis.

Results Depletion of β-PDGFR in hepatic stellate cells decreased injury and fibrosis in vivo, while its auto-activation accelerated fibrosis. However, there was no difference in development of DEN-induced pre-neoplastic foci. Genomic profiling revealed ERK, AKT, and NF-kB pathways and a subset of a previously identified 186-gene prognostic signature in hepatitis C virus (HCV)-related cirrhosis as downstream of β-PDGFR in stellate cells. In the human cohort, the β-PDGFR signature was not associated with HCC development, but was significantly associated with a poorer outcome in HCV cirrhosis.

Conclusions β-PDGFR is a key mediator of hepatic injury and fibrogenesis in vivo and contributes to the poor prognosis of human cirrhosis, but not by increasing HCC development.

Link To Article

http://dx.doi.org/10.1016/j.jhep.2015.01.036

Development of an in vivo rabbit ulnar loading model

Authors

Andrew P. Baumann, Mohammad W. Aref, Travis L. Turnbull, Alex G. Robling, Glen L. Niebur, Matthew R. Allen, Ryan K. Roeder

Abstract

Ulnar and tibial cyclic compression in rats and mice have become the preferred animal models for investigating the effects of mechanical loading on bone modeling/remodeling. Unlike rodents, rabbits provide a larger bone volume and normally exhibit intracortical Haversian remodeling, which may be advantageous for investigating mechanobiology and pharmaceutical interventions in cortical bone. Therefore, the objective of this study was to develop and validate an in vivo rabbit ulnar loading model. Ulnar tissue strains during loading of intact forelimbs were characterized and calibrated to applied loads using strain gauge measurements and specimen-specific finite element models. Periosteal bone formation in response to varying strain levels was measured by dynamic histomorphometry at the location of maximum strain in the ulnar diaphysis. Ulnae loaded at 3000 microstrain did not exhibit periosteal bone formation greater than the contralateral controls. Ulnae loaded at 3500, 4000, and 4500 microstrain exhibited a dose-dependent increase in periosteal mineralizing surface (MS/BS) compared with contralateral controls during the second week of loading. Ulnae loaded at 4500 microstrain exhibited the most robust response with significantly increased MS/BS at multiple time points extending at least 2 weeks after loading was ceased. Ulnae loaded at 5250 microstrain exhibited significant woven bone formation. Rabbits required greater strain levels to produce lamellar and woven bone on periosteal surfaces compared with rats and mice, perhaps due to lower basal levels of MS/BS. In summary, bone adaptation during rabbit ulnar loading was tightly controlled and may provide a translatable model for human bone biology in preclinical investigations of metabolic bone disease and pharmacological treatments.

Link To Article

http://dx.doi.org/10.1016/j.bone.2015.01.022

Decitabine represses osteoclastogenesis through inhibition of RANK and NF-κB

Authors

Hanfeng Guan, Baoguo Mi, Yong Li, Wei Wu, Peng Tan, Zhong Fang, Jing Li, Yong Zhang, Feng Li

Abstract

DNA methylation is essential for maintenance of stable repression of gene transcription during differentiation and tumorigenesis. Demethylating reagents including decitabine could release the repression, leading to perturbed transcription program. Recently others and we showed that, in B cell lymphomas, decitabine repressed B cell specific gene transcription and activated NF-κB signaling, causing decreased expression of translocated oncogenes including MYC and attenuated tumor cell proliferation. During osteoclastogenesis, changes in DNA methylation occurred in numerous genes, implicating important roles for DNA methylation in osteoclastogenesis. In the present study, we found that decitabine inhibited osteoclastogenesis. The inhibitory effect could be at least partially attributed to reduced expression of multiple osteoclast specific genes including RANK by decitabine. Moreover, decitabine inhibited activity of NF-κB, AP-1 and extracellular signal-regulated kinase (ERK), but not PI3K/Akt pathway. In vivo, using ovariectomized mouse as a model, we observed that decitabine reduced the osteoclast activity and bone loss. In conclusion, our findings demonstrated that decitabine was an inhibitor of osteoclastogenesis by repression of osteoclast specific transcription program including the RANK, NF-κB and AP-1 pathways. DNA methylation might be indispensable for osteoclastogenesis. The use of decitabine could represent a novel strategy in treatment of diseases associated with increased osteoclast activity.

Link To Article

http://dx.doi.org/10.1016/j.cellsig.2015.02.006