Growth hormone and testosterone delay vertebral fractures in boys with muscular dystrophy on chronic glucocorticoids.

Glucocorticoid use in Duchenne and Becker muscular dystrophy prolongs ambulation but cause significant skeletal toxicity. Our analysis has immediate clinical implications, suggesting that growth hormone and testosterone have a stronger effect prior to first and subsequent vertebral fracture, respectively, relative to bisphosphonates alone in children with dystrophinopathies on chronic glucocorticoids. PURPOSE: Glucocorticoids prolong ambulation in boys with Duchenne muscular dystrophy; however, they have significant endocrine side effects. We assessed the impact of growth hormone (GH), testosterone, and/or zoledronic acid (ZA) on vertebral fracture (VF) incidence in patients with dystrophinopathies on chronic glucocorticoids. METHODS: We conducted a longitudinal retrospective review of 27 males with muscular dystrophy. Accelerated failure time (AFT) models were used to estimate the relative time to VF while on GH, testosterone, and/or ZA compared to ZA alone. Results are reported as failure time ratio, where >1 indicates prolonged time versus <1 indicates shorter time to next VF. RESULTS: The prevalence of growth impairment was 96% (52% utilized GH), pubertal delay was 86% (72% utilized testosterone), and low trauma fractures were 87% (72% utilized ZA). Multivariable analysis of the AFT models showed that participants on either GH or testosterone treatment relative to ZA alone experienced prolonged time to next VF (1.253, P<0.001), with GH being the significant contributor when analyzed independently from testosterone (1.229, P<0.001). Use of ZA with GH or testosterone relative to ZA alone resulted in prolonged time to next VF (1.171, P<0.001), with testosterone being a significant contributor (1.130, P=0.033). CONCLUSION: GH and testosterone each decreased VF risk in patients independent of or in combination with ZA, respectively.

Management of RANKL-mediated disorders with denosumab in children and adolescents: a global expert guidance document.

CONTEXT: Denosumab is an effective treatment for many receptor activator of nuclear factor kappa-B ligand (RANKL)-mediated disorders but there are potential safety considerations and limited data to guide its use in children and adolescents. OBJECTIVE: This document seeks to summarise the evidence and provide expert opinion on safe and appropriate use of denosumab in paediatric RANKL-mediated disorders. PARTICIPANTS: Ten experts in paediatric bone and mineral medicine from six countries with experience in the use of denosumab participated in the creation of this document. EVIDENCE: Data was sourced from the published literature, primarily consisting of case reports/series and review articles due to the lack of higher-level evidence. Expert opinion of the authors was used substantially, where no published data was available. CONCLUSIONS: Denosumab is an effective treatment for RANKL-mediated disorders in children and adolescents but is often not curative and in some cases is best used in conjunction with surgical or other medical treatments. Careful multidisciplinary planning is required to define the goals of treatment and expert oversight needed to manage the risk of mineral abnormalities. Substantive, collaborative research efforts are needed to determine optimal treatment regimens and minimise risks.

Asfotase alfa improved skeletal mineralization and fracture healing in a child with MCAHS.

Tissue non-specific alkaline phosphatase (TNSALP) is an enzyme that is tethered to the cell membrane by glycosylphosphatidylinositol (GPI) and converts inorganic pyrophosphate to inorganic phosphate. Inorganic phosphate combines with calcium to form hydroxyapatite, the main mineral in the skeleton. When TNSALP is defective, conversion of inorganic pyrophosphate to inorganic phosphate is impaired and the skeleton is at risk of under-mineralization. Phosphatidylinositol glycan anchor biosynthesis class N (PIGN) is one of more than 20 genes in the GPI-biosynthesis family. Pathogenic variants in PIGN have been identified in multiple congenital anomalies-hypotonia-seizures syndrome (OMIM 614080), although a metabolic bone disease or skeletal fragility phenotype has not been reported. We describe a female child with multiple congenital anomalies-hypotonia-seizures syndrome due to a compound heterozygous pathogenic variant in PIGN who sustained a low-trauma distal femur fracture at age 7.4 years. We hypothesized that the GPI synthesis defect may result in metabolic bone disease from inadequate anchoring of TNSALP in bone and initiated asfotase alfa, a human bone-targeted recombinant TNSALP-Fc-deca-aspartate peptide, as it could bypass the PIGN genetic defect that possibly caused her skeletal fragility. Asfotase alfa was begun at 8.5 years. Baseline X-rays revealed mild rachitic findings of wrists and knees, which resolved by 5 months of treatment. Bone mineral density (BMD) assessed by dual-energy X-ray absorptiometry (DXA) showed mild improvement in spine, hip and total body less head after 16 months of treatment, while radius declined. She sustained additional low trauma fractures at right tibia and left humeral neck at 11 and 15 months into treatment, which healed quickly. Calcium, phosphorus, and parathyroid hormone levels have remained within the normal range over the 18 months of treatment. For adverse effect, she experienced a rash and discomfort in the first week of treatment which resolved with ibuprofen and diphenhydramine. She also developed subcutaneous fat atrophy. Overall, in this child with a compound pathogenic variant in PIGN, off-label use of asfotase alfa has been generally well tolerated with minimal side effects and resolution of rickets, but she continues to remain skeletally fragile.

Kaposiform lymphangiomatosis: Diagnosis, pathogenesis, and treatment.

Kaposiform lymphangiomatosis (KLA) is a life-threatening rare disease that can cause substantial morbidity, mortality, and social burdens for patients and their families. Diagnosis often occurs long after initial symptoms, and there are few centers in the world with the expertise to diagnose and care for patients with the disease. KLA is a lymphatic anomaly and significant advancements have been made in understanding its pathogenesis and etiology since its first description in 2014. This review provides multidisciplinary, comprehensive, and state-of-the-art information on KLA patient presentation, diagnostic imaging, pathology, organ involvement, genetics, and pathogenesis. Finally, we describe current therapeutic approaches, important areas for research, and challenges faced by patients and their families. Further insights into the pathogenesis of KLA may advance our understanding of other vascular anomalies given that similar signaling pathways may be involved.

Bisphosphonate Therapy for Treating Osteonecrosis in Pediatric Leukemia Patients: A Systematic Review.

BACKGROUND: Despite improved outcomes in children with leukemia, complications such as osteonecrosis are common. We conducted a systematic review to investigate the role of bisphosphonates in reducing pain, improving mobility, and stabilizing lesions in pediatric leukemia survivors. METHODS: Using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines, we searched the PubMed, Embase, Cochrane, Web of Science, Scopus, CINAHL, and ClinicalTrials.gov databases. Five of 221 articles retrieved met our inclusion criteria. RESULTS: Bisphosphonates, especially when combined with dietary calcium and vitamin D supplements and physical therapy (supplements/PT) were associated with improved pain and mobility in 54% and 50% of patients, respectively. A significantly greater proportion of patients treated with bisphosphonates (83%) reported mild/moderate pain or no pain compared with those with supplements/PT alone (36%) (P<0.001). Sixty-six percent of patients treated with bisphosphonates achieved improved/full mobility compared with 27% of those treated with supplements/PT alone (P=0.02). However, 46% of patients showed progressive joint destruction despite bisphosphonate therapy. No adverse events were reported, except for acute phase reactions to intravenous therapies. CONCLUSIONS: Bisphosphonates, when combined with supplements/PT, were associated with less pain and improved mobility, but not prevention of joint destruction in pediatric leukemia patients with osteonecrosis.

Case Report: Safety and Efficacy of Denosumab in Four Children With Noonan Syndrome With Multiple Giant Cell Lesions of the Jaw.

Noonan syndrome is a genetic disorder caused by mutations in the RAS/MAPK pathway. Multiple giant cell lesions are a rare sequelae of disruptions in this pathway, termed Noonan-like multiple giant cell lesions (NL/MGCLs). Medical management of these tumors rather than surgical intervention is preferential as the lesions are benign but locally destructive and recurring. This case series describes four male pediatric patients with Noonan syndrome and multiple giant cell lesions of the jaw treated with denosumab, a monoclonal antibody to receptor activator of nuclear factor kappa B ligand (RANKL), which has been approved for the treatment of malignant giant cell tumors in adults but not evaluated for safety or efficacy in children. All four pediatric patients responded clinically and radiographically to the treatment. Adverse events occurred in a predictable pattern and included hypocalcemia and joint pain during the initiation of treatment and symptomatic hypercalcemia after the cessation of treatment. Growth was not significantly impaired in these skeletally immature patients. This case series demonstrates how a weight-adjusted denosumab dose can effectively treat NL/MGCLs and provides laboratory data for consideration of the timing of monitoring for known side effects.

Sensory nerves regulate mesenchymal stromal cell lineage commitment by tuning sympathetic tones.

The sensory nerve was recently identified as being involved in regulation of bone mass accrual. We previously discovered that prostaglandin E2 (PGE2) secreted by osteoblasts could activate sensory nerve EP4 receptor to promote bone formation by inhibiting sympathetic activity. However, the fundamental units of bone formation are active osteoblasts, which originate from mesenchymal stromal/stem cells (MSCs). Here, we found that after sensory denervation, knockout of the EP4 receptor in sensory nerves, or knockout of COX-2 in osteoblasts, could significantly promote adipogenesis and inhibit osteogenesis in adult mice. Furthermore, injection of SW033291 (a small molecule that locally increases the PGE2 level) or propranolol (a beta blocker) significantly promoted osteogenesis and inhibited adipogenesis. This effect of SW033291, but not propranolol, was abolished in conditional EP4-KO mice under normal conditions or in the bone repair process. We conclude that the PGE2/EP4 sensory nerve axis could regulate MSC differentiation in bone marrow of adult mice.

Glucocorticoids Disrupt Skeletal Angiogenesis Through Transrepression of NF-κB-Mediated Preosteoclast Pdgfb Transcription in Young Mice.

In the growing skeleton, angiogenesis is intimately coupled with osteogenesis. Chronic, high doses of glucocorticoids (GCs) are associated with decreased bone vasculature and induce osteoporosis and growth failure. The mechanism of GC-suppression of angiogenesis and relationship to osteoporosis and growth retardation remains largely unknown. Type H vessels, which are regulated by preosteoclast (POC) platelet-derived growth factor-BB (PDGF-BB), are specifically coupled with bone formation and development. We determined the effect of GCs on POC synthesis of PDGF-BB in relation to type H vessel formation, bone mass, and bone growth in the distal femur of 2-week-old young mice receiving prednisolone or vehicle for 2, 4, or 6 weeks. After 2 weeks of prednisolone, the number of POCs were unchanged while POC synthesis of PDGF-BB was reduced. Longer treatment with prednisolone reduced POCs numbers and PDGF-BB. These changes were associated with a reduction in type H vessels, bone formation rate, bone mass, and bone length at each time point. In vitro, excessive concentrations of prednisolone (10-6 M) resulted in decreased PDGF-BB concentration and POC numbers. Conditioned medium from POC cultures treated with control concentration of prednisolone (10-7 M) or recombinant PDGF-BB stimulated endothelial tube formation, whereas conditioned medium from control concentration of prednisolone-treated POC cultures neutralized by PDGF-BB antibody or excessive prednisolone inhibited endothelial tube formation. Administration of excessive prednisolone attenuated the P65 subunit of nuclear factor kappa B (NF-κB) binding to the Pdgfb promoter, resulting in lower Pdgfb transcription. Co-treatment with excessive prednisolone and the glucocorticoid receptor (GR) antagonist (RU486), GR siRNA, or TNFα rescued NF-κB binding to the Pdgfb promoter and endothelial tube formation. These results indicate that PDGF-BB synthesis in POCs is suppressed by GCs through transrepression of GR/NF-κB, thus inhibiting type H vessel formation and associated osteoporosis and growth failure. © 2020 American Society for Bone and Mineral Research.

Author Correction: Sensory innervation in porous endplates by Netrin-1 from osteoclasts mediates PGE2-induced spinal hypersensitivity in mice.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Type H blood vessels in bone modeling and remodeling.

In the mammalian skeletal system, osteogenesis and angiogenesis are intimately linked during bone growth and regeneration in bone modeling and during bone homeostasis in bone remodeling. Recent studies have expanded our knowledge about the molecular and cellular mechanisms responsible for coupling angiogenesis and bone formation. Type H vessels, termed such because of high expression of Endomucin (Emcn) and CD31, have recently been identified and have the ability to induce bone formation. Factors including platelet-derived growth factor type BB (PDGF-BB), slit guidance ligand 3 (SLIT3), hypoxia-inducible factor 1-alpha (HIF-1α), Notch, and vascular endothelial growth factor (VEGF) are involved in the coupling of angiogenesis and osteogenesis. This review summarizes the current understanding of signaling pathways that regulate type H vessels and how type H vessels modulate osteogenesis. Further studies dissecting the regulation and function of type H vessels will provide new insights into the role of bone vasculature in the metabolism of the skeleton. We also discuss considerations for therapeutic approaches targeting type H vessels to promote fracture healing, prevent pathological bone loss, osteonecrosis, osteoarthritis, and bone metastases.

Sensory innervation in porous endplates by Netrin-1 from osteoclasts mediates PGE2-induced spinal hypersensitivity in mice.

Spinal pain is a major clinical problem, however, its origins and underlying mechanisms remain unclear. Here we report that in mice, osteoclasts induce sensory innervation in the porous endplates which contributes to spinal hypersensitivity in mice. Sensory innervation of the porous areas of sclerotic endplates in mice was confirmed. Lumbar spine instability (LSI), or aging, induces spinal hypersensitivity in mice. In these conditions, we show that there are elevated levels of PGE2 which activate sensory nerves, leading to sodium influx through Nav 1.8 channels. We show that knockout of PGE2 receptor 4 in sensory nerves significantly reduces spinal hypersensitivity. Inhibition of osteoclast formation by knockout Rankl in the osteocytes significantly inhibits LSI-induced porosity of endplates, sensory innervation, and spinal hypersensitivity. Knockout of Netrin-1 in osteoclasts abrogates sensory innervation into porous endplates and spinal hypersensitivity. These findings suggest that osteoclast-initiated porosity of endplates and sensory innervation are potential therapeutic targets for spinal pain.

Multiple endocrine neoplasia type 1 presenting with concurrent insulinoma and prolactinoma in early-adolescence.

BACKGROUND: Multiple Endocrine Neoplasia Type 1 (MEN1) is a rare autosomal dominant disease that generally presents with primary hyperparathyroidism. However, initial presentation may vary and continued reevaluation of etiology of symptoms is required for appropriate diagnosis. CASE PRESENTATION: Twelve year old female presented with altered mental status that self-resolved and hypoglycemia. Laboratory evaluation revealed pituitary dysfunction with central hypothyroidism and adrenal insufficiency in the setting of hyperprolactinemia. Macroadenoma was confirmed on imaging. Despite medical treatment of pituitary hormone disorders, she continued to have significant hypoglycemia and further workup revealed hyperinsulinism. Insulinoma was identified and confirmed by endoscopic ultrasound. Hypoglycemia resolved after laproscopic enucleation of the insulinoma. CONCLUSION: Children presenting with one endocrine tumor should be investigated for other potential endocrine tumors. Multiple imaging modalities may be required to confidently identify neuroendocrine tumors for appropriate surgical intervention.

Ciliary parathyroid hormone signaling activates transforming growth factor-ß to maintain intervertebral disc homeostasis during aging.

Degenerative disc disease (DDD) is associated with intervertebral disc degeneration of spinal instability. Here, we report that the cilia of nucleus pulposus (NP) cells mediate mechanotransduction to maintain anabolic activity in the discs. We found that mechanical stress promotes transport of parathyroid hormone 1 receptor (PTH1R) to the cilia and enhances parathyroid hormone (PTH) signaling in NP cells. PTH induces transcription of integrin αvß6 to activate the transforming growth factor (TGF)-ß-connective tissue growth factor (CCN2)-matrix proteins signaling cascade. Intermittent injection of PTH (iPTH) effectively attenuates disc degeneration of aged mice by direct signaling through NP cells, specifically improving intervertebral disc height and volume by increasing levels of TGF-ß activity, CCN2, and aggrecan. PTH1R is expressed in both mouse and human NP cells. Importantly, knockout PTH1R or cilia in the NP cells results in significant disc degeneration and blunts the effect of PTH on attenuation of aged discs. Thus, mechanical stress-induced transport of PTH1R to the cilia enhances PTH signaling, which helps maintain intervertebral disc homeostasis, particularly during aging, indicating therapeutic potential of iPTH for DDD.

Oxidized phospholipids are ligands for LRP6.

Low-density lipoprotein receptor-related protein 6 (LRP6) is a co-receptor for Wnt signaling and can be recruited by multiple growth factors/hormones to their receptors facilitating intracellular signaling activation. The ligands that bind directly to LRP6 have not been identified. Here, we report that bioactive oxidized phospholipids (oxPLs) are native ligands of LRP6, but not the closely related LRP5. oxPLs are products of lipid oxidation involving in pathological conditions such as hyperlipidemia, atherosclerosis, and inflammation. We found that cell surface LRP6 in bone marrow mesenchymal stromal cells (MSCs) decreased rapidly in response to increased oxPLs in marrow microenvironment. LRP6 directly bound and mediated the uptake of oxPLs by MSCs. oxPL-LRP6 binding induced LRP6 endocytosis through a clathrin-mediated pathway, decreasing responses of MSCs to osteogenic factors and diminishing osteoblast differentiation ability. Thus, LRP6 functions as a receptor and molecular target of oxPLs for their adverse effect on MSCs, revealing a potential mechanism underlying atherosclerosis-associated bone loss.

Preservation of type H vessels and osteoblasts by enhanced preosteoclast platelet-derived growth factor type BB attenuates glucocorticoid-induced osteoporosis in growing mice.

Survival of chronic diseases in childhood is often achieved utilizing glucocorticoids, but comes with significant side effects, including glucocorticoid-induced osteoporosis (GIO). Knowledge of the mechanism of GIO is limited to the adult skeleton. We explored the effect of genetic loss and inhibition of cathepsin K (Ctsk) as a potential treatment target in a young GIO mouse model as genetic loss of cathepsin K results in a mild form of osteopetrosis secondary to impaired osteoclast bone resorption with maintenance of bone formation. We first characterized the temporal osteoclast and osteoblast progenitor populations in Ctsk-/- and wild type (WT) mice in the primary and secondary spongiosa, as sites representative of trabecular bone modeling and remodeling, respectively. In the primary spongiosa, Ctsk-/- mice had decreased numbers of osteoclasts at young ages (2 and 4 weeks) and increased osteoblast lineage cells at later age (8 weeks) relative to WT littermates. In the secondary spongiosa, Ctsk-/- mice had greater numbers of osteoclasts and osteoblast lineage cells relative to WT littermates. We next developed a young GIO mouse model with prednisolone 10 mg/m2/day injected intraperitoneally daily from 2 through 6 weeks of age. Overall, WT-prednisolone mice had lower bone volume per tissue volume, whereas Ctsk-/--prednisolone mice maintained a similar bone volume relative to Ctsk-/--vehicle controls. WT-prednisolone mice exhibited a decreased number of osteoclasts, tartrate-resistant acid phosphatase and platelet-derived growth factor type BB (PDGF-BB) co-positive cells, type H endothelial cells, and osteoblasts relative to WT-vehicle mice in both the primary and secondary spongiosa. Interestingly, Ctsk-/--prednisolone mice demonstrated a paradoxical response with increased numbers of all parameters in primary spongiosa and no change in secondary spongiosa. Finally, treatment with a cathepsin K inhibitor prevented WT-prednisolone decline in osteoclasts, osteoblasts, type H vessels, and bone volume. These data demonstrate that cells in the primary and secondary spongiosa respond differently to glucocorticoids and genetic manipulation. Inhibition of osteoclast resorption that preserves osteoclast coupling factors, such as through inhibition of cathepsin K, may be a potential preventive treatment strategy against GIO in the growing skeleton.

IGF-I induced phosphorylation of PTH receptor enhances osteoblast to osteocyte transition.

Parathyroid hormone (PTH) regulates bone remodeling by activating PTH type 1 receptor (PTH1R) in osteoblasts/osteocytes. Insulin-like growth factor type 1 (IGF-1) stimulates mesenchymal stem cell differentiation to osteoblasts. However, little is known about the signaling mechanisms that regulates the osteoblast-to-osteocyte transition. Here we report that PTH and IGF-I synergistically enhance osteoblast-to-osteocyte differentiation. We identified that a specific tyrosine residue, Y494, on the cytoplasmic domain of PTH1R can be phosphorylated by insulin-like growth factor type I receptor (IGF1R) in vitro. Phosphorylated PTH1R localized to the barbed ends of actin filaments and increased actin polymerization during morphological change of osteoblasts into osteocytes. Disruption of the phosphorylation site reduced actin polymerization and dendrite length. Mouse models with conditional ablation of PTH1R in osteoblasts demonstrated a reduction in the number of osteoctyes and dendrites per osteocyte, with complete overlap of PTH1R with phosphorylated-PTH1R positioning in osteocyte dendrites in wild-type mice. Thus, our findings reveal a novel signaling mechanism that enhances osteoblast-to-osteocyte transition by direct phosphorylation of PTH1R by IGF1R.

Transforming growth factor-ß in stem cells and tissue homeostasis.

TGF-ß 1-3 are unique multi-functional growth factors that are only expressed in mammals, and mainly secreted and stored as a latent complex in the extracellular matrix (ECM). The biological functions of TGF-ß in adults can only be delivered after ligand activation, mostly in response to environmental perturbations. Although involved in multiple biological and pathological processes of the human body, the exact roles of TGF-ß in maintaining stem cells and tissue homeostasis have not been well-documented until recent advances, which delineate their functions in a given context. Our recent findings, along with data reported by others, have clearly shown that temporal and spatial activation of TGF-ß is involved in the recruitment of stem/progenitor cell participation in tissue regeneration/remodeling process, whereas sustained abnormalities in TGF-ß ligand activation, regardless of genetic or environmental origin, will inevitably disrupt the normal physiology and lead to pathobiology of major diseases. Modulation of TGF-ß signaling with different approaches has proven effective pre-clinically in the treatment of multiple pathologies such as sclerosis/fibrosis, tumor metastasis, osteoarthritis, and immune disorders. Thus, further elucidation of the mechanisms by which TGF-ß is activated in different tissues/organs and how targeted cells respond in a context-dependent way can likely be translated with clinical benefits in the management of a broad range of diseases with the involvement of TGF-ß.

Programmed cell senescence in skeleton during late puberty.

Mesenchymal stem/progenitor cells (MSPCs) undergo rapid self-renewal and differentiation, contributing to fast skeletal growth during childhood and puberty. It remains unclear whether these cells change their properties during late puberty to young adulthood, when bone growth and accrual decelerate. Here we show that MSPCs in primary spongiosa of long bone in mice at late puberty undergo normal programmed senescence, characterized by loss of nestin expression. MSPC senescence is epigenetically controlled by the polycomb histone methyltransferase enhancer of zeste homolog 2 (Ezh2) and its trimethylation of histone H3 on Lysine 27 (H3K27me3) mark. Ezh2 maintains the repression of key cell senescence inducer genes through H3K27me3, and deletion of Ezh2 in early pubertal mice results in premature cellular senescence, depleted MSPCs pool, and impaired osteogenesis as well as osteoporosis in later life. Our data reveals a programmed cell fate change in postnatal skeleton and unravels a regulatory mechanism underlying this phenomenon.

Mechanosignaling activation of TGFß maintains intervertebral disc homeostasis.

Intervertebral disc (IVD) degeneration is the leading cause of disability with no disease-modifying treatment. IVD degeneration is associated with instable mechanical loading in the spine, but little is known about how mechanical stress regulates nucleus notochordal (NC) cells to maintain IVD homeostasis. Here we report that mechanical stress can result in excessive integrin αvß6-mediated activation of transforming growth factor beta (TGFß), decreased NC cell vacuoles, and increased matrix proteoglycan production, and results in degenerative disc disease (DDD). Knockout of TGFß type II receptor (TßRII) or integrin αv in the NC cells inhibited functional activity of postnatal NC cells and also resulted in DDD under mechanical loading. Administration of RGD peptide, TGFß, and αvß6-neutralizing antibodies attenuated IVD degeneration. Thus, integrin-mediated activation of TGFß plays a critical role in mechanical signaling transduction to regulate IVD cell function and homeostasis. Manipulation of this signaling pathway may be a potential therapeutic target to modify DDD.