IGF‐1 Regulates Vertebral Bone Aging Through Sex‐Specific and Time‐Dependent Mechanisms

Advanced aging is associated with increased risk of bone fracture, especially within the vertebrae, which exhibit significant reductions in trabecular bone structure. Aging is also associated with a reduction in circulating levels of insulin‐like growth factor (IGF‐1). Studies have suggested that the reduction in IGF‐1 compromises healthspan, whereas others report that loss of IGF‐1 is beneficial because it increases healthspan and lifespan. To date, the effect of decreases in circulating IGF‐1 on vertebral bone aging has not been thoroughly investigated. Here, we delineate the consequences of a loss of circulating IGF‐1 on vertebral bone aging in male and female Igf^f/f mice. IGF‐1 was reduced at multiple specific time points during the mouse lifespan: early in postnatal development (crossing albumin–cyclic recombinase [Cre] mice with Igf^f/f mice); and in early adulthood and in late adulthood using hepatic‐specific viral vectors (AAV8‐TBG‐Cre). Vertebrae bone structure was analyzed at 27 months of age using micro–computed tomography (μCT) and quantitative bone histomorphometry. Consistent with previous studies, both male and female mice exhibited age‐related reductions in vertebral bone structure. In male mice, reduction of circulating IGF‐1 induced at any age did not diminish vertebral bone loss. Interestingly, early‐life loss of IGF‐1 in females resulted in a 67% increase in vertebral bone volume fraction, as well as increased connectivity density and increased trabecular number. The maintenance of bone structure in the early‐life IGF‐1–deficient females was associated with increased osteoblast surface and an increased ratio of osteoprotegerin/receptor‐activator of NF‐κB‐ligand (RANKL) levels in circulation. Within 3 months of a loss of IGF‐1, there was a 2.2‐fold increase in insulin receptor expression within the vertebral bones of our female mice, suggesting that local signaling may compensate for the loss of circulating IGF‐1. Together, these data suggest the age‐related loss of vertebral bone density in females can be reduced by modifying circulating IGF‐1 levels early in life. © 2015 American Society for Bone and Mineral Research.     

The RhoGAP Myo9b Promotes Bone Growth by Mediating Osteoblastic Responsiveness to IGF‐1

The Ras homolog A (RhoA) subfamily of Rho guanosine triphosphatases (GTPases) regulates actin‐based cellular functions in bone such as differentiation, migration, and mechanotransduction. Polymorphisms or genetic ablation of RHOA and some of its regulatory guanine exchange factors (GEFs) have been linked to poor bone health in humans and mice, but the effects of RhoA‐specific GTPase‐activating proteins (GAPs) on bone quality have not yet been identified. Therefore, we examined the consequences of RhoGAP Myo9b gene knockout on bone growth, phenotype, and cellular activity. Male and female mice lacking both alleles demonstrated growth retardation and decreased bone formation rates during early puberty. These mice had smaller, weaker bones by 4 weeks of age, but only female KOs had altered cellular numbers, with fewer osteoblasts and more osteoclasts. By 12 weeks of age, bone quality in KOs worsened. In contrast, 4‐week‐old heterozygotes demonstrated bone defects that resolved by 12 weeks of age. Throughout, Myo9b ablation affected females more than males. Osteoclast activity appeared unaffected. In primary osteogenic cells, Myo9b was distributed in stress fibers and focal adhesions, and its absence resulted in poor spreading and eventual detachment from culture dishes. Similarly, MC3T3‐E1 preosteoblasts with transiently suppressed Myo9b levels spread poorly and contained decreased numbers of focal adhesions. These cells also demonstrated reduced ability to undergo IGF‐1–induced spreading or chemotaxis toward IGF‐1, though responses to PDGF and BMP‐2 were unaffected. IGF‐1 receptor (IGF1R) activation was normal in cells with diminished Myo9b levels, but the activated receptor was redistributed from stress fibers and focal adhesions into nuclei, potentially affecting receptor accessibility and gene expression. These results demonstrate that Myo9b regulates a subset of RhoA‐activated processes necessary for IGF‐1 responsiveness in osteogenic cells, and is critical for normal bone formation in growing mice. © 2017 American Society for Bone and Mineral Research     

Effect of Low‐Magnitude Mechanical Stimuli on Bone Density and Structure in Pediatric Crohn's Disease: A Randomized Placebo‐Controlled Trial

Pediatric Crohn's Disease (CD) is associated with low trabecular bone mineral density (BMD), cortical area, and muscle mass. Low‐magnitude mechanical stimulation (LMMS) may be anabolic. We conducted a 12‐month randomized double‐blind placebo‐controlled trial of 10 minutes daily exposure to LMMS (30 Hz frequency, 0.3 g peak‐to‐peak acceleration). The primary outcomes were tibia trabecular BMD and cortical area by peripheral quantitative CT (pQCT) and vertebral trabecular BMD by QCT; additional outcomes included dual‐energy X‐ray absorptiometry (DXA) whole body, hip and spine BMD, and leg lean mass. Results were expressed as sex‐specific Z‐scores relative to age. CD participants, ages 8 to 21 years with tibia trabecular BMD <25th percentile for age, were eligible and received daily cholecalciferol (800 IU) and calcium (1000 mg). In total, 138 enrolled (48% male), and 121 (61 active, 60 placebo) completed the 12‐month trial. Median adherence measured with an electronic monitor was 79% and did not differ between arms. By intention‐to‐treat analysis, LMMS had no significant effect on pQCT or DXA outcomes. The mean change in spine QCT trabecular BMD Z‐score was +0.22 in the active arm and –0.02 in the placebo arm (difference in change 0.24 [95% CI 0.04, 0.44]; p = 0.02). Among those with >50% adherence, the effect was 0.38 (95% CI 0.17, 0.58, p < 0.0005). Within the active arm, each 10% greater adherence was associated with a 0.06 (95% CI 0.01, 1.17, p = 0.03) greater increase in spine QCT BMD Z‐score. Treatment response did not vary according to baseline body mass index (BMI) Z‐score, pubertal status, CD severity, or concurrent glucocorticoid or biologic medications. In all participants combined, height, pQCT trabecular BMD, and cortical area and DXA outcomes improved significantly. In conclusion, LMMS was associated with increases in vertebral trabecular BMD by QCT; however, no effects were observed at DXA or pQCT sites. © 2016 American Society for Bone and Mineral Research.     

Neuropeptide Y Attenuates Stress‐Induced Bone Loss Through Suppression of Noradrenaline Circuits

Chronic stress and depression have adverse consequences on many organ systems, including the skeleton, but the mechanisms underlying stress‐induced bone loss remain unclear. Here we demonstrate that neuropeptide Y (NPY), centrally and peripherally, plays a critical role in protecting against stress‐induced bone loss. Mice lacking the anxiolytic factor NPY exhibit more anxious behavior and elevated corticosterone levels. Additionally, following a 6‐week restraint, or cold‐stress protocol, Npy‐null mice exhibit three‐fold greater bone loss compared to wild‐type mice, owing to suppression of osteoblast activity. This stress‐protective NPY pathway acts specifically through Y2 receptors. Centrally, Y2 receptors suppress corticotropin‐releasing factor expression and inhibit activation of noradrenergic neurons in the paraventricular nucleus. In the periphery, they act to control noradrenaline release from sympathetic neurons. Specific deletion of arcuate Y2 receptors recapitulates the Npy‐null stress response, coincident with elevated serum noradrenaline. Importantly, specific reintroduction of NPY solely in noradrenergic neurons of otherwise Npy‐null mice blocks the increase in circulating noradrenaline and the stress‐induced bone loss. Thus, NPY protects against excessive stress‐induced bone loss, through Y2 receptor‐mediated modulation of central and peripheral noradrenergic neurons. © 2014 American Society for Bone and Mineral Research.     

Cross‐sectional Versus Longitudinal Change in a Prospective HR‐pQCT Study

Longitudinal studies assessing age‐related changes using high‐resolution peripheral quantitative computed tomography (HR‐pQCT) provide novel insight compared with cross‐sectional analyses. The purpose of this cohort study was 1) to determine individuals’ change in HR‐pQCT parameters over 5 years relative to least significant change (LSC), and 2) to evaluate if predicted rate of change from cross‐sectional data is comparable to actual change from longitudinal investigation. A cohort of 466 (162 male, 304 female) participants completed two HR‐pQCT scans with 5 years between assessments. After image registration, standard and cortical morphological analyses were conducted. Rate of bone microarchitectural change was compared between cross‐sectional models and actual change calculated from longitudinal analyses. At the young end of the life span, we observed gains in total bone density of +0.2% to +2.9% per year, whereas the older participants (aged >50 years) lost total bone density at a rate of –0.3% to –1.3% per year. Declines in total bone density begin at age 40 years in females and 60 years in males, and significant adaptation was found at both ends of the age spectrum with respect to the LSC. Models predicting rate of change from cross‐sectional data were similar to the actual change reported in this longitudinal study for total density and cortical thickness at the radius and cortical density at the tibia, but we found that changes in comparison to our 5‐year longitudinal results were often overestimated from cross‐sectional data. Studies aimed at observing age‐related changes in a normative cohort, especially in a follow‐up period of less than 5 years, are better to focus on the tibia rather than the radius because of the increased sensitivity to change at the tibia. © 2017 American Society for Bone and Mineral Research.     

Bisphosphonate Withdrawal: Effects on Bone Formation and Bone Resorption in Maturing Male Mice

Bisphosphonates are being increasingly used to treat pediatric patients with skeletal disorders. However, the effects of long‐term bisphosphonate therapy and cessation of therapy during growth are unclear. Thus, studies were undertaken to determine the effects of alendronate discontinuation after treatment of C57Bl/6 mice during the period of rapid skeletal growth. Compared with vehicle‐treated mice, 16 weeks of alendronate treatment starting at age 18 days resulted in a 3.7‐fold increase in trabecular bone in the setting of suppressed bone formation. Alendronate therapy for 8 weeks followed by 8 weeks of vehicle treatment resulted in a more pronounced increase in trabecular bone compared with mice treated with alendronate for 16 weeks (1.7‐fold) and to vehicle‐treated controls (6.5‐fold). Mice that received alendronate for 8 weeks followed by 8 weeks of vehicle exhibited increased osteoblast surface (2.5‐fold), mineralizing surface (5.7‐fold), and bone formation rate (5.1‐fold) compared with mice treated continuously with alendronate. However, these parameters were not restored to the levels observed in the vehicle‐treated mice. Thus, partial resumption of bone formation upon cessation of bisphosphonate therapy leads to a greater increase in trabecular bone than that found when bisphosphonates are administered continuously to growing mice. These data suggest that intermittent administration of bisphosphonates may optimize their beneficial effects on the growing skeleton. © 2017 American Society for Bone and Mineral Research.     

Bone Mineral Density and Microarchitecture in Patients With Autosomal Dominant Osteopetrosis: A Report of Two Cases

The aim of this case study is to describe changes in areal bone mineral density (aBMD) by dual‐energy X‐ray absorptiometry (DXA) scan, as well as volumetric bone density and microarchitecture by high‐resolution peripheral quantitative computed tomography (HR‐pQCT) in two patients with autosomal dominant osteopetrosis (ADO) and compare with 20 healthy subjects. We describe a 44‐year‐old male patient with six low‐impact fractures since he was age 16 years, and a 32‐year‐old female patient with four low‐impact fractures on her past history. Radiographic changes were typical of ADO. Consistent with the much higher aBMD, total volumetric BMD (average bone density of the whole bone, including trabecular and cortical compartments) at distal radius and tibia (HR‐pQCT) was more than twice the mean values found in healthy subjects in both patients. Trabecular number and thickness were higher, leading to an evident increase in trabecular bone volume to tissue volume. Also, an enormous increase in cortical thickness was found. Most important, a great heterogeneity in bone microstructure of the affected patients was evident on HR‐pQCT images: islets of very dense bone were interposed with areas with apparent normal density. The increase in aBMD, volumetric BMD, and most indices of trabecular and cortical bone, associated with the great heterogeneity on bone tridimensional microarchitecture, reflect the accumulation of old and fragile bone randomly distributed along the skeleton. These alterations in bone microstructure probably compromise bone quality, which might justify the high prevalence of low‐impact fractures in patients with ADO, despite abnormally elevated BMD. © 2015 American Society for Bone and Mineral Research.     

BMP‐9‐induced muscle heterotopic ossification requires changes to the skeletal muscle microenvironment

Heterotopic ossification (HO) is defined as the formation of bone inside soft tissue. Symptoms include joint stiffness, swelling, and pain. Apart from the inherited form, the common traumatic form generally occurs at sites of injury in damaged muscles and is often associated with brain injury. We investigated bone morphogenetic protein 9 (BMP‐9), which possesses a strong osteoinductive capacity, for its involvement in muscle HO physiopathology. We found that BMP‐9 had an osteoinductive influence on mouse muscle resident stromal cells by increasing their alkaline phosphatase activity and bone‐specific marker expression. Interestingly, BMP‐9 induced HO only in damaged muscle, whereas BMP‐2 promoted HO in skeletal muscle regardless of its state. The addition of the soluble form of the ALK1 protein (the BMP‐9 receptor) significantly inhibited the osteoinductive potential of BMP‐9 in cells and HO in damaged muscles. BMP‐9 thus should be considered a candidate for involvement in HO physiopathology, with its activity depending on the skeletal muscle microenvironment. © 2011 American Society for Bone and Mineral Research.     

The Lrp4R1170Q Homozygous Knock‐In Mouse Recapitulates the Bone Phenotype of Sclerosteosis in Humans

Sclerosteosis is a rare autosomal recessive bone disorder marked by hyperostosis of the skull and tubular bones. Initially, we and others reported that sclerosteosis was caused by loss‐of‐function mutations in SOST, encoding sclerostin. More recently, we identified disease‐causing mutations in LRP4, a binding partner of sclerostin, in three sclerosteosis patients. Upon binding to sclerostin, LRP4 can inhibit the canonical WNT signaling that is known to be an important pathway in the regulation of bone formation. To further investigate the role of LRP4 in the bone formation process, we generated an Lrp4 mutated sclerosteosis mouse model by introducing the p.Arg1170Gln mutation in the mouse genome. Extensive analysis of the bone phenotype of the Lrp4R1170Q/R1170Q knock‐in (KI) mouse showed the presence of increased trabecular and cortical bone mass as a consequence of increased bone formation by the osteoblasts. In addition, three‐point bending analysis also showed that the increased bone mass results in increased bone strength. In contrast to the human sclerosteosis phenotype, we could not observe syndactyly in the forelimbs or hindlimbs of the Lrp4 KI animals. Finally, we could not detect any significant changes in the bone formation and resorption markers in the serum of the mutant mice. However, the serum sclerostin levels were strongly increased and the level of sclerostin in the tibia was decreased in Lrp4R1170Q/R1170Q mice, confirming the role of LRP4 as an anchor for sclerostin in bone. In conclusion, the Lrp4R1170Q/R1170Q mouse is a good model for the human sclerosteosis phenotype caused by mutations in LRP4 and can be used in the future for further investigation of the mechanism whereby LRP4 regulates bone formation. © 2017 American Society for Bone and Mineral Research.     

Myelosuppressive Therapies Significantly Increase Pro‐Inflammatory Cytokines and Directly Cause Bone Loss

Skeletal‐related events resulting from accelerated bone loss are common complications in patients treated for a range of cancers. However, the mechanisms and rate of bone loss after myelosuppression are unclear. We, therefore, investigated this in mice and humans. We treated mice with different myelosuppressive therapies (chemotherapy or irradiation with or without transplantation) and studied their effects on bone structure. Myelosuppression of mice rapidly caused an increase in bone resorption that was not matched by bone formation. The resultant significant and persistent bone loss early after therapy was associated with increased inflammatory cytokines, in particular, monocyte chemoattractant protein 1 (MCP1). Therapy‐induced bone loss was prevented with a single dose of the bisphosphonate zoledronic acid (ZA), administered before myelosuppression. Importantly, ZA treatment of mice did not impair hematopoiesis, including hematopoietic stem cell function. Furthermore, examination of serum from patients before and after autologous or allogeneic stem cell transplantion (SCT) revealed altered levels of bone turnover markers and elevated inflammatory cytokines. MCP1 levels in serum obtained between days 7 and 14 post‐SCT positively correlated with bone loss observed at 100 days after allogeneic SCT. Similar to that observed in our studies in mice, the bone loss was long term, persisting at 12 months post‐SCT. Furthermore, patients who received chemotherapy less than 100 days before SCT had significantly more bone loss at the hip. In these patients, serum levels of MCP1, but not routine biomarkers of bone turnover, including C‐terminal cross‐linking telopeptide of type‐1 collagen (β‐CTx), positively correlated with their bone loss. Hence, myelosuppressive therapies increase inflammation and directly contribute to bone loss. Administration of an osteoclast inhibitor before the initiation of cancer therapy is likely to have the best outcome in preventing bone loss in patients with cancer. © 2014 American Society for Bone and Mineral Research.     

Age‐ and Sex‐Dependent Changes of Intra‐articular Cortical and Trabecular Bone Structure and the Effects of Rheumatoid Arthritis

The objective of this cross‐sectional study was to define normal sex‐ and age‐dependent values of intra‐articular bone mass and microstructures in the metacarpal heads of healthy individuals by high‐resolution peripheral quantitative computed tomography (HR‐pQCT) and test the effect of rheumatoid arthritis (RA) on these parameters. Human cadaveric metacarpal heads were used to exactly define intra‐articular bone. Healthy individuals of different sex and age categories and RA patients with similar age and sex distribution received HR‐pQCT scans of the second metacarpal head and the radius. Total, cortical, and trabecular bone densities as well as microstructural parameters were compared between 1) the different ages and sexes in healthy individuals; 2) between metacarpal heads and the radius; and 3) between healthy individuals and RA patients. The cadaveric study allowed exact definition of the intra‐articular (intracapsular) bone margins. These data were applied in measuring intra‐articular and radial bone parameters in 214 women and men (108 healthy individuals, 106 RA patients). Correlations between intra‐articular and radial bone parameters were good (r = 0.51 to 0.62, p < 0.001). In contrast to radial bone, intra‐articular bone remained stable until age 60 years (between 297 and 312 mg HA/cm³) but decreased significantly (p < 0.001) in women thereafter (237.5 ± 44.3) with loss of both cortical and trabecular bone. Similarly, RA patients showed significant (p < 0.001) loss of intra‐articular total (263.0 ± 44.8), trabecular (171.2 ± 35.6), and cortical bone (610.2 ± 62.0) compared with sex‐ and age‐adjusted controls. Standard sex‐ and age‐dependent values for physiological intra‐articular bone were defined. Postmenopausal state and RA led to significant decrease of intra‐articular bone. © 2016 American Society for Bone and Mineral Research.     

Cortical and Trabecular Bone Microstructure Did Not Recover at Weight‐Bearing Skeletal Sites and Progressively Deteriorated at Non‐Weight‐Bearing Sites During the Year Following International Space Station Missions

Risk for premature osteoporosis is a major health concern in astronauts and cosmonauts; the reversibility of the bone lost at the weight‐bearing bone sites is not established, although it is suspected to take longer than the mission length. The bone three‐dimensional structure and strength that could be uniquely affected by weightlessness is currently unknown. Our objective is to evaluate bone mass, microarchitecture, and strength of weight‐bearing and non‐weight‐bearing bone in 13 cosmonauts before and for 12 months after a 4‐month to 6‐month sojourn in the International Space Station (ISS). Standard and advanced evaluations of trabecular and cortical parameters were performed using high‐resolution peripheral quantitative computed tomography. In particular, cortical analyses involved determination of the largest common volume of each successive individual scan to improve the precision of cortical porosity and density measurements. Bone resorption and formation serum markers, and markers reflecting osteocyte activity or periosteal metabolism (sclerostin, periostin) were evaluated. At the tibia, in addition to decreased bone mineral densities at cortical and trabecular compartments, a 4% decrease in cortical thickness and a 15% increase in cortical porosity were observed at landing. Cortical size and density subsequently recovered and serum periostin changes were associated with cortical recovery during the year after landing. However, tibial cortical porosity or trabecular bone failed to recover, resulting in compromised strength. The radius, preserved at landing, unexpectedly developed postflight fragility, from 3 months post‐landing onward, particularly in its cortical structure. Remodeling markers, uncoupled in favor of bone resorption at landing, returned to preflight values within 6 months, then declined farther to lower than preflight values. Our findings highlight the need for specific protective measures not only during, but also after spaceflight, because of continuing uncertainties regarding skeletal recovery long after landing. © 2017 American Society for Bone and Mineral Research.     

Compromised Exercise Capacity and Mitochondrial Dysfunction in the Osteogenesis Imperfecta Murine (oim) Mouse Model

Osteogenesis imperfecta (OI) is a heritable connective tissue disorder that most often arises from type I collagen—COL1A1 and COL1A2—gene defects leading to skeletal fragility, short stature, blue-gray sclera, and muscle weakness. Relative to the skeletal fragility, muscle weakness is much less understood. Recent investigations into OI muscle weakness in both patients and mouse models have revealed the presence of an inherent muscle pathology. Understanding the mechanisms responsible for OI muscle weakness is critical, particularly in light of the extensive cross-talk between muscle and bone via mechanotransduction and biochemical signaling. In the following study we initially subjected WT and oim/oim mice, modeling severe human OI type III, to either weight-bearing (voluntary wheel-running) or non-weight-bearing (swimming) exercise regimens as a modality to improve muscle strength and ultimately bone strength. The oim/oim mice ran only 35% to 42% of the distance run by age- and sex-matched WT mice and exhibited little improvement with either exercise regimen. Upon further investigation, we determined that oim/oim gastrocnemius muscle exhibited severe mitochondrial dysfunction as characterized by a 52% to 65% decrease in mitochondrial respiration rates, alterations in markers of mitochondrial biogenesis, mitophagy, and the electron transport chain components, as well as decreased mitochondrial citrate synthase activity, relative to age- and sex-matched WT gastrocnemius muscle. Thus, mitochondrial dysfunction in the oim/oim mouse likely contributes to compromised muscle function and reduced physical activity levels. © 2019 American Society for Bone and Mineral Research.