Abaloparatide has the same catabolic effects on bones of mice when infused as PTH (1-34)

AUTHORS

Carole Le Henaff Ph.D., Brandon Finnie B.S., Maria Pacheco M.S., Zhiming He M.S., Joshua Johnson M.S., Nicola C. Partridge Ph.D.

ABSTRACT

Abaloparatide is a peptide analog of parathyroid hormone-related protein (PTHrP 1-34) and was approved in 2017 as the second osteoanabolic peptide for treating osteoporosis. We have previously shown that intermittent abaloparatide is equally as effective as PTH (1-34). This study was designed to compare the catabolic effects of PTH (1-34) and abaloparatide on bone in young female wild-type mice. Two-month-old C57Bl/6J female mice were continuously infused with human PTH (1-34) or abaloparatide at 80  μg/kg BW/day or vehicle for 2 weeks. At euthanasia, DEXA-PIXImus was performed to assess bone mineral density (BMD) in the whole body, femurs, tibiae and vertebrae. Bone turnover marker levels were measured in sera, femurs were harvested for μCT analyses and histomorphometry, and tibiae were separated into cortical and trabecular fractions for gene expression analyses. Our results demonstrated that infusion of abaloparatide resulted in a similar decrease in BMD as infused PTH (1-34) at all sites. MicroCT and histomorphometry analyses showed similar decreases in cortical bone thickness and BMD associated with an increase in bone turnover from the increased bone formation rate found by in vivo double labeling and serum P1NP, and the increased bone resorption as shown by osteoclast numbers and serum CTX. Trabecular bone did not show major changes with either treatment. Osteoblastic gene expression analyses of trabecular and cortical bone revealed that infusion of PTH (1-34) or abaloparatide led to similar and different actions in genes of osteoblast differentiation and activity. As with intermittent and in vitro treatment, both infused PTH (1-34) and abaloparatide similarly regulated downstream genes of the PTHR1/SIK/HDAC4 pathway such as Sost and Mmp13, but differed for those of the PTHR1/SIK/CRTC pathway. Taken together, at the same dose, infused abaloparatide causes the same high bone turnover as infused PTH (1-34) with a net resorption in female wild-type mice.

A Novel RANKL-Targeted Furoquinoline Alkaloid Ameliorates Bone Loss in Ovariectomized Osteoporosis through Inhibiting the NF-κB Signal Pathway and Reducing Reactive Oxygen Species

AUTHORS

Puiian Wong, Zheng Lv, Jinglan Li, Qiushi Wei, LiangLiang Xu, Bin Fang, Yiwen Luo, and Mincong He

ABSTRACT

Dysregulation of osteoclast-osteoblast balance, resulting in abnormal bone remodeling, is responsible for postmenopausal osteoporosis (PMOP) or other secondary forms of osteoporosis. We demonstrated that dictamnine (DIC), a novel RANKL-targeted furoquinoline alkaloid, inhibits osteoclastogenesis by facilitating the activities of reactive oxygen species (ROS), NF-κB, and NFATc1 in vitro and prevents the development of OVX-induced osteoporosis mouse models in vivo. Methods. The docking mechanism of DIC and RANKL was initially identified by protein–ligand molecular docking. RNA sequencing was performed and analyzed to reveal the potential mechanism and signaling pathway of the antiosteoporosis effects of DIC. To verify the sequencing results, we examined the impact of DIC on RANKL-induced osteoclast differentiation, bone resorption, F-actin ring production, ROS generation, and NF-κB activation in osteoclasts in vitro. Moreover, a luciferase assay was performed to determine the binding and transcriptional activity of Nrf2 and NF-κB. The in vivo efficacy of DIC was assessed with an ovariectomy- (OVX-) induced osteoporosis model, which was analyzed using micro-CT and bone histomorphometry. Results. The molecular docking results indicated that DIC could bind particularly to RANKL. RNA-seq confirmed that DIC could regulate the osteoclast-related pathway. DIC suppressed osteoclastogenesis, bone resorption, F-actin belt formation, osteoclast-specific gene expression, and ROS activity by preventing NFATc1 expression and affecting NF-κB signaling pathways in vitro. The luciferase assay showed that DIC not only suppressed the activity of Nrf2 but also contributed to the combination of Nrf2 and NF-κB. Our in vivo study indicated that DIC protects against OVX-induced osteoporosis and preserves bone volume by inhibiting osteoclast activity and function. Conclusions. DIC can ameliorate osteoclast formation and OVX-induced osteoporosis and therefore is a potential therapeutic treatment for osteoporosis.

Osteocytes directly regulate osteolysis via MYD88 signaling in bacterial bone infection

AUTHORS

Tetsuya Yoshimoto, Mizuho Kittaka, Andrew Anh Phuong Doan, Rina Urata, Matthew Prideaux, Roxana E. Rojas, Clifford V. Harding, W. Henry Boom, Lynda F. Bonewald, Edward M. Greenfield & Yasuyoshi Ueki

ABSTRACT

The impact of bone cell activation on bacterially-induced osteolysis remains elusive. Here, we show that matrix-embedded osteocytes stimulated with bacterial pathogen-associated molecular patterns (PAMPs) directly drive bone resorption through an MYD88-regulated signaling pathway. Mice lacking MYD88, primarily in osteocytes, protect against osteolysis caused by calvarial injections of bacterial PAMPs and resist alveolar bone resorption induced by oral Porphyromonas gingivalis (Pg) infection. In contrast, mice with targeted MYD88 restoration in osteocytes exhibit osteolysis with inflammatory cell infiltration. In vitro, bacterial PAMPs induce significantly higher expression of the cytokine RANKL in osteocytes than osteoblasts. Mechanistically, activation of the osteocyte MYD88 pathway up-regulates RANKL by increasing binding of the transcription factors CREB and STAT3 to Rankl enhancers and by suppressing K48-ubiquitination of CREB/CREB binding protein and STAT3. Systemic administration of an MYD88 inhibitor prevents jawbone loss in Pg-driven periodontitis. These findings reveal that osteocytes directly regulate inflammatory osteolysis in bone infection, suggesting that MYD88 and downstream RANKL regulators in osteocytes are therapeutic targets for osteolysis in periodontitis and osteomyelitis.

Aberrant adenosine signaling in patients with focal cortical dysplasia

AUTHORS

Mengyi Guo, Jing Zhang, Jing Wang, Xiongfei Wang, Qing Gao, Chongyang Tang, Jiahui Deng, Zhonghua Xiong, Xiangru Kong, Yuguang Guan, Jian Zhou, Detlev Boison, Guoming Luan, Tianfu Li

ABSTRACT

Focal cortical dysplasia (FCD), a common malformation of cortical development, is frequently associated with pharmacoresistant epilepsy in both children and adults. Adenosine is an inhibitory modulator of brain activity and a prospective anti-seizure agent with potential for clinical translation. Our previous results demonstrated that the major adenosine-metabolizing enzyme adenosine kinase (ADK) was upregulated in balloon cells (BCs) within FCD type IIB lesions, suggesting that dysfunction of the adenosine system is implicated in the pathophysiology of FCD. In our current study, we therefore performed a comprehensive analysis of adenosine metabolism and signaling in surgically resected cortical specimens from patients with FCD type I and type II via immunohistochemistry and immunoblot analysis. Adenosine metabolism was assessed by quantifying the levels of the key enzymes of adenosine metabolism, i.e., ADK, adenosine deaminase (ADA), and 5’-ectonucleotidase (CD73). Adenosine signaling was assessed by quantifying the levels of adenosine A2A receptor (A2AR) and putative downstream mediators of adenosine, namely, glutamate transporter-1 (GLT-1) and mammalian target of rapamycin (mTOR). Within lesions in FCD specimens, we found that the adenosine-metabolizing enzymes ADK and ADA, as well as the adenosine-producing enzyme CD73, were upregulated. We also observed an increase in A2AR expression, as well as a decrease in GLT-1 levels and an increase in mTOR levels, in FCD specimens compared with control tissue. These results suggest that dysregulation of the adenosine system is a common pathologic feature of both FCD type I and type II. The adenosine system might therefore be a therapeutic target for the treatment of epilepsy associated with FCD.

3D-printed Strontium-Titanium Scaffolds Incorporated with Highly Bioactive Serum Exosomes Promotes Critical Bone Defect Repair by Enhancing Osteogenesis and Angiogenesis

AUTHORS

Hao Liu, Ranli Gu, Wei Li, Lijun Zeng, Yuan Zhu, Siyi Wang, Xuenan Liu, Boon Chin Heng, Yunsong Liu, Yongsheng Zhou

ABSTRACT

Background

Large bone defect healing faces significant challenges because of inadequate bone regeneration and revascularization. Serum exosomes (sEXO) during bone defect repair are rich in osteogenic factors. Titanium (Ti) scaffolds and low dose strontium (Sr) can promote bone regeneration. Here, a “cell-free scaffold engineering” strategy that incorporates strontium and highly bioactive sEXO within a 3D-printed Ti scaffold is developed.

Methods

Sr-Ti-sEXO composite was prepared by ion implantation and ultra-high-speed centrifugation. Alkaline phosphatase (ALP), Alizarin red (ARS), immunofluorescence (IF) staining, and polymerase chain reaction (PCR) were used to detect the osteogenic effect of Sr-Ti-sExo on bone marrow mesenchymal stem cells (BMSCs). Tartrate-resistant acid phosphatase (TRAP) staining, and PCR were used to detect the osteoclast effect of Sr-Ti-sEXO on RAW264.7. The vascularization effect of Sr-Ti-sEXO on human umbilical vein endothelial cells (HUVECs) was investigated by scratch and migration experiments. Micro-CT and histological staining were used to study the osteogenic and vasculogenic effects of Sr-Ti-sEXO implanted in rabbit large radius defect at 6 and 12 weeks in vivo. RNA-seq was used to explore the potential mechanism.

Results

Sr-Ti-sEXO composite promoted early osteogenesis and inhibited osteoclast formation through the combined release of Sr ions and sEXO, and sustained release of Sr ions enhanced bone conduction, bone induction and inhibited fibroblasts. sEXO can promote the vascular reconstruction of CBD in fracture stage, which has the dual effect of promoting bone and promoting angiogenesis in critical bone defect repair. These effects are regulated by multiple miRNAs that shuttle in sEXO.

Conclusions

Sr-Ti-sEXO has favourable sustained release performance, osteogenic and vasogenic effects, which is a biocompatible and clinically feasible critical bone defect repair strategy. This study also broadens the biomedical potential of exosomes with specific functions such as sEXO in fracture stage. Based on the relative abundance of sEXO, a sEXO library for clinical treatment can be established.

Bone-targeting delivery of platelet lysate exosomes ameliorates glucocorticoid-induced osteoporosis by enhancing bone-vessel coupling

AUTHORS

Gang Zheng, Hai-Wei Ma, Guang-Heng Xiang, Gao-Lu He, Han-Chen Cai, Zi-Han Dai, Yan-Lin Chen, Yan Lin, Hua-Zi Xu, Wen-Fei Ni, Cong Xu, Hai-Xiao Liu & Xiang-Yang Wang

ABSTRACT

Background

Glucocorticoids (GCs) overuse is associated with decreased bone mass and osseous vasculature destruction, leading to severe osteoporosis. Platelet lysates (PL) as a pool of growth factors (GFs) were widely used in local bone repair by its potent pro-regeneration and pro-angiogenesis. However, it is still seldom applied for treating systemic osteopathia due to the lack of a suitable delivery strategy. The non-targeted distribution of GFs might cause tumorigenesis in other organs.

Results

In this study, PL-derived exosomes (PL-exo) were isolated to enrich the platelet-derived GFs, followed by conjugating with alendronate (ALN) grafted PEGylated phospholipid (DSPE-PEG-ALN) to establish a bone-targeting PL-exo (PL-exo-ALN). The in vitro hydroxyapatite binding affinity and in vivo bone targeting aggregation of PL-exo were significantly enhanced after ALN modification. Besides directly modulating the osteogenic and angiogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and endothelial progenitor cells (EPCs), respectively, PL-exo-ALN also facilitate their coupling under GCs’ stimulation. Additionally, intravenous injection of PL-exo-ALN could successfully rescue GCs induced osteoporosis (GIOP) in vivo.

Conclusions

PL-exo-ALN may be utilized as a novel nanoplatform for precise infusion of GFs to bone sites and exerts promising therapeutic potential for GIOP.