Buds of new bone formation within the femoral head of hip fracture patients coincide with zones of low osteocyte sclerostin

AUTHORS

Hiroshige Sano, Tristan Whitmarsh, Linda Skingle, Taketoshi Shimakura, Noriaki Yamamoto, Juliet E. Compston, Hideaki E. Takahashi, Kenneth E. S. Poole

ABSTRACT

Romosozumab treatment reduces the rate of hip fractures and increases hip bone density, increasing bone formation by inhibiting sclerostin protein. We studied the normal pattern of bone formation and osteocyte expression in the human proximal femur because it is relevant to both anti-sclerostin treatment effects and fracture. Having visualized and quantified buds of new bone formation in trabeculae, we hypothesized that they would coincide with areas of a) higher mechanical stress and b) low sclerostin expression by osteocytes. In patients with hip fracture, we visualised each bud of active modeling-based formation, (Forming Minimodeling Structure, FMiS) in trabecular cores taken from different parts of the femoral head. Trabecular bone structure was also measured with high resolution imaging.

More buds of new bone formation (by volume) were present in the higher stress supero-medial zone (FMiS density, N.FMiS/T.Ar) than lower stress supero-lateral (p < 0.05), and inferomedial (p < 0.001) regions. There were fewer sclerostin expressing osteocytes close to, or within FMiS. FMiS density correlated with greater amount, thickness, number and connectivity of trabeculae (bone volume BV/TV, r = 0.65, p < 0.0001; bone surface BS/TV, r = 0.47, p < 0.01; trabecular thickness Tb.Th, r = 0.55, p < 0.001; trabecular number Tb.N, r = 0.47, p < 0.01; and connectivity density Conn.D, r = 0.40, p < 0.05) and lower trabecular separation (Tb.Sp, r = −0.56, p < 0.001).

These results demonstrate modeling-based bone formation in femoral trabeculae from patients with hip fracture as a potential therapeutic target to enhance bone structure.

Reactivation of Bone Lining Cells are Attenuated Over Repeated Anti-sclerostin Antibody Administration

AUTHORS

A Ram Hong, Jae-Yeon Yang, Ji Yeon Lee, Joonho Suh, Yun-Sil Lee, Jung-Eun Kim & Sang Wan Kim

ABSTRACT

Reactivation of bone lining cells (BLCs) is a crucial mechanism governing the anabolic action of anti-sclerostin antibody (Scl-Ab) via modeling-based bone formation; however, it remains unclear whether this reactivation can be attenuated after persistent administration of Scl-Ab. Here, we aimed to investigate the reproducibility of persistent Scl-Ab administration for the reactivation of BLCs, and to elucidate the relationship between the activity of BLCs and serum levels of N-terminal procollagen type I (P1NP) during chronic Scl-Ab administration. We conducted an osteoblast lineage tracing study. Briefly, Dmp1-CreERt2(+):Rosa26R mice were injected with 1 mg of 4-hydroxy-tamoxifen weekly from postnatal weeks four to eight. Mice were treated twice with either vehicle or Scl-Ab (25 mg/kg) at weeks 12, 16, and 20, and were euthanized at weeks 8, 12, 13, 16, 17, 20, and 21 (4–6 mice in each group). After euthanization, the number and thickness of X-gal (+) cells on the periosteum of the femoral bones and the serum levels of P1NP were quantified at each time point. Scl-Ab induced a significant increase in the thickness of X-gal (+) cells on periosteal bone surfaces at postnatal weeks 13 (after 1st dose), 17 (after 2nd dose), and 21 (after 3rd dose) compared to that in vehicle-treated mice (all P < 0.001). In the Scl-Ab group, significant increases in the thickness of labeled cells were observed between weeks 16 and 17 and weeks 20 and 21 (both P < 0.001). The percentage increase in X-gal (+) cell thickness was 108.9% from week 12 to week 13, 54.6% from week 16 to week 17, and 49.2% from week 20 to week 21 in the Scl-Ab group. Although Scl-Ab treatment increased the serum levels of P1NP at postnatal weeks 13 and 17 compared with those at week 12 (P = 0.017 and P = 0.038, respectively), the same was not observed at week 21 (P = 0.296). A significant increase in P1NP levels was observed between weeks 16 and 17 and weeks 20 and 21 in the Scl-Ab group (P = 0.005 and P = 0.007, respectively). The percentage increase in P1NP levels was 141.7% from weeks 12 to 13, 114.8% from weeks 16 to 17, and 99.4% from weeks 20 to 21. Serum P1NP levels were positively correlated with X-gal (+) cell thickness (R2 = 0.732, P < 0.001). Reactivation of BLCs is modestly attenuated, but reproducible, during persistent Scl-Ab administration. Serum P1NP levels appear to be an indicator of the impact of Scl-Ab on the conversion of BLCs into mature osteoblasts on periosteal bone surfaces, thus contributing to modeling-based bone formation.

Targeting loop3 of sclerostin preserves its cardiovascular protective action and promotes bone formation

AUTHORS

Yuanyuan Yu, Luyao Wang, Shuaijian Ni, Dijie Li, Jin Liu, Hang Yin Chu, Ning Zhang, Meiheng Sun, Nanxi Li, Qing Ren, Zhenjian Zhuo, Chuanxin Zhong, Duoli Xie, Yongshu Li, Zong-Kang Zhang, Huarui Zhang, Mei Li, Zhenlin Zhang, Lin Chen, Xiaohua Pan, Weibo Xia, Shu Zhang, Aiping Lu, Bao-Ting Zhang & Ge Zhang

ABSTRACT

Sclerostin negatively regulates bone formation by antagonizing Wnt signalling. An antibody targeting sclerostin for the treatment of postmenopausal osteoporosis was approved by the U.S. Food and Drug Administration, with a boxed warning for cardiovascular risk. Here we demonstrate that sclerostin participates in protecting cardiovascular system and inhibiting bone formation via different loops. Loop3 deficiency by genetic truncation could maintain sclerostin’s protective effect on the cardiovascular system while attenuating its inhibitory effect on bone formation. We identify an aptamer, named aptscl56, which specifically targets sclerostin loop3 and use a modified aptscl56 version, called Apc001PE, as specific in vivo pharmacologic tool to validate the above effect of loop3. Apc001PE has no effect on aortic aneurysm and atherosclerotic development in ApoE−/− mice and hSOSTki.ApoE−/− mice with angiotensin II infusion. Apc001PE can promote bone formation in hSOSTki mice and ovariectomy-induced osteoporotic rats. In summary, sclerostin loop3 cannot participate in protecting the cardiovascular system, but participates in inhibiting bone formation.

HDAC inhibitor quisinostat prevents estrogen deficiency-induced bone loss by suppressing bone resorption and promoting bone formation in mice

AUTHORS

Shengxuan Sun, Chunmei Xiu, Langhui Chai, Xinyu Chen, Lei Zhang, Qingbai Liu, Jianquan Chen, Haibin Zhou

ABSTRACT

Postmenopausal osteoporosis (PMOP) is a metabolic skeletal disorder characterized by reduced bone mass and impaired bone microarchitecture resulting in increased bone fragility and fracture risk. PMOP is primarily caused by excessive osteoclastogenesis induced by estrogen deficiency. Quisinostat (Qst) is a potent hydroxamate-based second-generation inhibitor of histone deacetylases (HDACs) that can inhibit osteoclast differentiation in vitro, and protect mice from titanium particle-induced osteolysis in vivo. However, whether Qst has therapeutic potential against PMOP remains unclear. In the present study, we evaluated the therapeutic efficacy of Qst on PMOP, using a murine model of ovariectomy (OVX)-induced osteoporosis. We examined the body weight, femur length, and histology of major organs, and showed that Qst did not cause obvious toxicity in mice. Micro-computed tomography and histological analyses revealed that Qst treatment prevented OVX-induced trabecular bone loss both in femurs and vertebrae. Moreover, ELISA showed that Qst decreased the serum levels of the osteoclastic bone resorption marker CTX-1, whereas increased the levels of the osteoblastic bone formation marker Osteocalcin in OVX mice. Consistent with the CTX-1 results, TRAP staining showed that Qst suppressed OVX-induced osteoclastogenesis. Mechanistically, we showed that Qst suppressed RANKL-induced osteoclast differentiation in part by inhibiting p65 nuclear translocation. Collectively, our results demonstrated that Qst can ameliorate estrogen deficiency-induced osteoporosis by inhibiting bone resorption and promoting bone formation in vivo. In summary, our study provided the first preclinical evidence to support Qst as a potential therapeutic agent for PMOP prevention and treatment.

Reduced bone mass in collagen prolyl 4-hydroxylase P4ha1+/-;P4ha2-/- compound mutant mice

AUTHORS

Jussi-Pekka Tolonen, Antti M. Salo, Mikko Finnilä, Ellinoora Aro, Emma Karjalainen, Veli-Pekka Ronkainen, Kati Drushinin, Christophe Merceron, Valerio Izzi, Ernestina Schipani, Johanna Myllyharju

ABSTRACT

Proper deposition of the extracellular matrix and its major components, the collagens, is essential for endochondral ossification and bone mass accrual. Collagen prolyl 4-hydroxylases (C-P4Hs) hydroxylate proline residues in the -X-Pro-Gly- repeats of all known collagen types. Their product, 4-hydroxyproline, is essential for correct folding and thermal stability of the triple-helical collagen molecules in physiological body temperatures. We have previously shown that inactivation of the mouse P4ha1 gene, which codes for the catalytic α subunit of the major C-P4H isoform, is embryonic lethal, while inactivation of the P4ha2 gene produced only a minor phenotype. Instead, mice with a haploinsufficiency of the P4ha1 gene combined with a homozygous deletion of the P4ha2 gene present with a moderate chondrodysplasia due to transient cell death of the growth plate chondrocytes. Here, to further characterize the bone phenotype of the P4ha1+/-;P4ha2-/- mice, we have carried out gene expression analyses at whole tissue and single cell levels, biochemical analyses, microcomputed tomography and histomorphometric analyses and second harmonic generation microscopy to show that C-P4H α subunit expression peaks early and that the C-P4H deficiency leads to reduced collagen amount, a reduced rate of bone formation and a loss of trabecular and cortical bone volume in the long bones. The total osteoblast number in the proximal P4ha1+/-;P4ha2-/- tibia and the C-P4H activity in primary P4ha1+/-;P4ha2-/- osteoblasts were reduced, while the population of osteoprogenitor colony forming-unit fibroblasts was increased in the P4ha1+/-;P4ha2-/- marrow. Thus, the P4ha1+/-;P4ha2-/- mouse model recapitulates key aspects of a recently recognized congenital connective tissue disorder with short stature and bone dysplasia caused by bi-allelic variants of the human P4HA1 gene. Altogether, the data demonstrate the allele-dose dependent importance of the C-P4Hs to the developing organism and a threshold effect of C-P4H activity in the proper production of bone matrix.

Conditional loss of IKKα in Osterix + cells has no effect on bone but leads to age-related loss of peripheral fat

AUTHORS

Jennifer L. Davis, Nitin Kumar Pokhrel, Linda Cox, Nidhi Rohatgi, Roberta Faccio & Deborah J. Veis

ABSTRACT

NF-κB has been reported to both promote and inhibit bone formation. To explore its role in osteolineage cells, we conditionally deleted IKKα, an upstream kinase required for non-canonical NF-κB activation, using Osterix (Osx)-Cre. Surprisingly, we found no effect on either cancellous or cortical bone, even following mechanical loading. However, we noted that IKKα conditional knockout (cKO) mice began to lose body weight after 6 months of age with severe reductions in fat mass and lower adipocyte size in geriatric animals. qPCR analysis of adipogenic markers in fat pads of cKO mice indicated no difference in early differentiation, but instead markedly lower leptin with age. We challenged young mice with a high fat diet finding that cKO mice gained less weight and showed improved glucose metabolism. Low levels of recombination at the IKKα locus were detected in fat pads isolated from old cKO mice. To determine whether recombination occurs in adipocytes, we examined fat pads in Osx-Cre;TdT reporter mice; these showed increasing Osx-Cre-mediated expression in peripheral adipocytes from 6 weeks to 18 months. Since Osx-Cre drives recombination in peripheral adipocytes with age, we conclude that fat loss in cKO mice is most likely caused by progressive deficits of IKKα in adipocytes