An atlas of brain-bone sympathetic neural circuits in mice.

There is clear evidence that the sympathetic nervous system (SNS) mediates bone metabolism. Histological studies show abundant SNS innervation of the periosteum and bone marrow-these nerves consist of noradrenergic fibers that immunostain for tyrosine hydroxylase, dopamine beta-hydroxylase, or neuropeptide Y. Nonetheless, the brain sites that send efferent SNS outflow to the bone have not yet been characterized. Using pseudorabies (PRV) viral transneuronal tracing, we report, for the first time, the identification of central SNS outflow sites that innervate bone. We find that the central SNS outflow to bone originates from 87 brain nuclei, sub-nuclei, and regions of six brain divisions, namely the midbrain and pons, hypothalamus, hindbrain medulla, forebrain, cerebral cortex, and thalamus. We also find that certain sites, such as the raphe magnus (RMg) of the medulla and periaqueductal gray (PAG) of the midbrain, display greater degrees of PRV152 infection, suggesting that there is considerable site-specific variation in the levels of central SNS outflow to the bone. This comprehensive compendium illustrating the central coding and control of SNS efferent signals to bone should allow for a greater understanding of the neural regulation of bone metabolism, and importantly and of clinical relevance, mechanisms for central bone pain.

Ethnicity and the Prevalence of Polycystic Ovary Syndrome: The Eastern Siberia PCOS Epidemiology and Phenotype Study.

CONTEXT: Previous studies have shown that the prevalence of polycystic ovary syndrome (PCOS) may vary according to race/ethnicity, although few studies have assessed women of different ethnicities who live in similar geographic and socio-economic conditions. OBJECTIVE: To determine the prevalence of PCOS in an unselected multiethnic population of premenopausal women. DESIGN: A multicenter prospective cross-sectional study. SETTINGS: The main regional employers of Irkutsk Region and the Buryat Republic, Russia. PARTICIPANTS: During 2016-19, 1398 premenopausal women underwent a history and physical exam, pelvic ultrasound, and testing during a mandatory annual employment-related health assessment. MAIN OUTCOME MEASURES: PCOS prevalence, overall and by ethnicity in a large medically unbiased population, including Caucasian (White), Mongolic or Asian (Buryat), and mixed ethnicity individuals, living in similar geographic and socio-economic conditions for centuries. RESULTS: PCOS was diagnosed in 165/1134 (14.5%) women who had a complete evaluation for PCOS. Based on the probabilities for PCOS by clinical presentation observed in the cohort of women who had a complete evaluation we also estimated the weight-adjusted prevalence of PCOS in 264 women with an incomplete evaluation: 46.2 or 17.5%. Consequently, the total prevalence of PCOS in the population was 15.1%, higher among Caucasians and women of Mixed ethnicity compared to Asians (16.0% and 21.8% vs. 10.8%, pz <0.05). CONCLUSIONS: We observed a 15.1% prevalence of PCOS in our medically unbiased population of premenopausal women. In this population of Siberian premenopausal women of Caucasian, Asian and Mixed ethnicity living in similar geographic and socio-economic conditions, the prevalence was higher in Caucasian or Mixed than Asian women. These data highlight the need to assess carefully ethnic-dependent differences in the frequency and clinical manifestation of PCOS.

FSH, bone, belly and brain.

The pituitary gland orchestrates multiple endocrine organs by secreting tropic hormones, and therefore plays a significant role in a myriad of physiological processes, including skeletal modeling and remodeling, fat and glucose metabolism, and cognition. Expression of receptors for each pituitary hormone and the hormone itself in the skeleton, fat, immune cells, and the brain suggest that their role is much broader than the traditionally attributed functions. FSH, believed solely to regulate gonadal function is also involved in fat and bone metabolism, as well as in cognition. Our emerging understanding of nonreproductive functions of FSH, thus, opens potential therapeutic opportunities to address detrimental health consequences during and after menopause, namely, osteoporosis, obesity, and dementia. In this review, we outline current understanding of the cross-talk between the pituitary, bone, adipose tissue, and brain through FSH. Preclinical evidence from genetic and pharmacologic interventions in rodent models, and human data from population-based observations, genetic studies, and a small number of interventional studies provide compelling evidence for independent functions of FSH in bone loss, fat gain, and congnitive impairment.

Ovarian Morphology in Non-Hirsute, Normo-Androgenic, Eumenorrheic Premenopausal Women from a Multi-Ethnic Unselected Siberian Population.

Polycystic ovary syndrome (PCOS) is a highly prevalent disorder in women, and its diagnosis rests on three principal features: ovulatory/menstrual dysfunction, clinical and/or biochemical hyperandrogenism, and polycystic ovarian morphology (PCOM). Currently, data on age- and ethnicity-dependent features of PCOM remain insufficient. We aimed to estimate ethnicity- and age-dependent differences in ovarian volume (OV) and follicle number per ovary (FNPO) in a healthy, medically unbiased population of Caucasian and Asian premenopausal women, who participated in the cross-sectional Eastern Siberia PCOS epidemiology and phenotype (ESPEP) study (ClinicalTrials.gov ID: NCT05194384) in 2016-2019. The study population consisted of 408 non-hirsute, normo-androgenic, eumenorrheic premenopausal women aged 18-44 years. All participants underwent a uniform evaluation including a review of their medical history and a physical examination, blood sampling, and pelvic ultrasonography. The statistical analysis included non-parametric tests and the estimation of the upper normal limits (UNLs) by 98th percentiles for OV and FNPO. In the total study population, the upper OV percentiles did not differ by ethnicity or age group. By contrast, the UNL of FNPO was higher in Caucasian women than in Asian women, and women aged <35 years demonstrated a higher UNL of FNPO compared to older women. In summary, these data suggest that the estimation of FNPO, but not OV, should take into account the ethnicity and age of the individual in estimating the presence of PCOM.

Gene-Dose-Dependent Reduction Fshr Expression Improves Spatial Memory Deficits in Alzheimer's Mice.

Alzheimer's disease (AD) is a major progressive neurodegenerative disorder of the aging population. High post-menopausal levels of the pituitary gonadotropin follicle-stimulating hormone (FSH) are strongly associated with the onset of AD, and we have shown recently that FSH directly activates the hippocampal Fshr to drive AD-like pathology and memory loss in mice. To establish a role for FSH in memory loss, we used female 3xTg;Fshr+/+, 3xTg;Fshr+/- and 3xTg;Fshr-/- mice that were either left unoperated or underwent sham surgery or ovariectomy at 8 weeks of age. Unoperated and sham-operated 3xTg;Fshr-/- mice were implanted with 17ß-estradiol pellets to normalize estradiol levels. Morris Water Maze and Novel Object Recognition behavioral tests were performed to study deficits in spatial and recognition memory, respectively, and to examine the effects of Fshr depletion. 3xTg;Fshr+/+ mice displayed impaired spatial memory at 5 months of age; both the acquisition and retrieval of the memory were ameliorated in 3xTg;Fshr-/- mice and, to a lesser extent, in 3xTg;Fshr+/- mice- -thus documenting a clear gene-dose-dependent prevention of hippocampal-dependent spatial memory impairment. At 5 and 10 months, sham-operated 3xTg;Fshr-/- mice showed better memory performance during the acquasition and/or retrieval phases, suggesting that Fshr deletion prevented the progression of spatial memory deficits with age. However, this prevention was not seen when mice were ovariectomized, except in the 10-month-old 3xTg;Fshr-/- mice. In the Novel Object Recognition test performed at 10 months, all groups of mice, except ovariectomized 3xTg;Fshr-/- mice showed a loss of recognition memory. Consistent with the neurobehavioral data, there was a gene-dose-dependent reduction mainly in the amyloid ß40 isoform in whole brain extracts. Finally, serum FSH levels < 8 ng/mL in 16-month-old APP/PS1 mice were associated with better retrieval of spatial memory. Collectively, the data provide compelling genetic evidence for a protective effect of inhibiting FSH signaling on the progression of spatial and recognition memory deficits in mice, and lay a firm foundation for the use of an FSH-blocking agent for the early prevention of cognitive decline in postmenopausal women.

An Atlas of Brain-Bone Sympathetic Neural Circuits.

There is clear evidence that the sympathetic nervous system (SNS) mediates bone metabolism. Histological studies show abundant SNS innervation of the periosteum and bone marrow--these nerves consist of noradrenergic fibers that immunostain for tyrosine hydroxylase, dopamine beta hydroxylase, or neuropeptide Y. Nonetheless, the brain sites that send efferent SNS outflow to bone have not yet been characterized. Using pseudorabies (PRV) viral transneuronal tracing, we report, for the first time, the identification of central SNS outflow sites that innervate bone. We find that the central SNS outflow to bone originates from 87 brain nuclei, sub-nuclei and regions of six brain divisions, namely the midbrain and pons, hypothalamus, hindbrain medulla, forebrain, cerebral cortex, and thalamus. We also find that certain sites, such as the raphe magnus (RMg) of the medulla and periaqueductal gray (PAG) of the midbrain, display greater degrees of PRV152 infection, suggesting that there is considerable site-specific variation in the levels of central SNS outflow to bone. This comprehensive compendium illustrating the central coding and control of SNS efferent signals to bone should allow for a greater understanding of the neural regulation of bone metabolism, and importantly and of clinical relevance, mechanisms for central bone pain.

Absent LH signaling rescues the anxiety phenotype in aging female mice.

Clinical studies and experimental data together support a role for pituitary gonadotropins, including luteinizing hormone (LH), otherwise considered solely as fertility hormones, in age-related cognitive decline. Furthermore, rising levels of LH in post-menopausal women have been implicated in the high prevalence of mood disorders. This study was designed to examine the effect of deficient LH signaling on both cognitive and emotional behavior in 12-month-old Lhcgr-/- mice. For this, we established and validated a battery of five tests, including Dark-Light Box (DLB), Y-Maze Spontaneous Alternation, Novel Object Recognition (NOR), and contextual and cued Fear Conditioning (FCT) tests. We found that 12-month-old female wild type mice display a prominent anxiety phenotype on DLB and FCT. This phenotype was not seen in 12-month-old female Lhcgr-/- mice, indicating full phenotypic rescue. Furthermore, there was no effect of LHCGR depletion on recognition memory or working spatial memory on NOR and Y-maze testing, respectively, in 12-month-old mice, notwithstanding the absence of a basal phenotype in wild type littermates. The latter data do not exclude an effect of LH on cognition documented in previous studies. Finally, 12-month-old male mice and 3-month-old male and female mice did not consistently display deficits on any test. The data collectively document, for the first time, that loss of LH signaling reverses age-related emotional disturbances, a prelude to future targeted therapies that block LH action.

Development and biophysical characterization of a humanized FSH-blocking monoclonal antibody therapeutic formulated at an ultra-high concentration.

Highly concentrated antibody formulations are oftentimes required for subcutaneous, self-administered biologics. Here, we report the development of a unique formulation for our first-in-class FSH-blocking humanized antibody, MS-Hu6, which we propose to move to the clinic for osteoporosis, obesity, and Alzheimer's disease. The studies were carried out using our Good Laboratory Practice (GLP) platform, compliant with the Code of Federal Regulations (Title 21, Part 58). We first used protein thermal shift, size exclusion chromatography, and dynamic light scattering to examine MS-Hu6 concentrations between 1 and 100 mg/mL. We found that thermal, monomeric, and colloidal stability of formulated MS-Hu6 was maintained at a concentration of 100 mg/mL. The addition of the antioxidant L-methionine and chelating agent disodium EDTA improved the formulation's long-term colloidal and thermal stability. Thermal stability was further confirmed by Nano differential scanning calorimetry (DSC). Physiochemical properties of formulated MS-Hu6, including viscosity, turbidity, and clarity, confirmed with acceptable industry standards. That the structural integrity of MS-Hu6 in formulation was maintained was proven through Circular Dichroism (CD) and Fourier Transform Infrared (FTIR) Spectroscopy. Three rapid freeze-thaw cycles at -80 °C/25 °C or -80 °C/37 °C further revealed excellent thermal and colloidal stability. Furthermore, formulated MS-Hu6, particularly its Fab domain, displayed thermal and monomeric storage stability for more than 90 days at 4°C and 25°C. Finally, the unfolding temperature (Tm) for formulated MS-Hu6 increased by >4.80 °C upon binding to recombinant FSH, indicating highly specific ligand binding. Overall, we document the feasibility of developing a stable, manufacturable and transportable MS-Hu6 formulation at a ultra-high concentration at industry standards. The study should become a resource for developing biologic formulations in academic medical centers.

Emerging roles of brain tanycytes in regulating blood-hypothalamus barrier plasticity and energy homeostasis.

Seasonal changes in food intake and adiposity in many animal species are triggered by changes in the photoperiod. These latter changes are faithfully transduced into a biochemical signal by melatonin secreted by the pineal gland. Seasonal variations, encoded by melatonin, are integrated by third ventricular tanycytes of the mediobasal hypothalamus through the detection of the thyroid-stimulating hormone (TSH) released from the pars tuberalis. The mediobasal hypothalamus is a critical brain region that maintains energy homeostasis by acting as an interface between the neural networks of the central nervous system and the periphery to control metabolic functions, including ingestive behavior, energy homeostasis, and reproduction. Among the cells involved in the regulation of energy balance and the blood-hypothalamus barrier (BHB) plasticity are tanycytes. Increasing evidence suggests that anterior pituitary hormones, specifically TSH, traditionally considered to have unitary functions in targeting single endocrine sites, display actions on multiple somatic tissues and central neurons. Notably, modulation of tanycytic TSH receptors seems critical for BHB plasticity in relation to energy homeostasis, but this needs to be proven.

Development and Biophysical Characterization of a Humanized FSH-Blocking Monoclonal Antibody Therapeutic Formulated at an Ultra-High Concentration.

Highly concentrated antibody formulations are oftentimes required for subcutaneous, self-administered biologics. Here, we report the creation of a unique formulation for our first-in- class FSH-blocking humanized antibody, MS-Hu6, which we propose to move to the clinic for osteoporosis, obesity, and Alzheimer's disease. The studies were carried out using our Good Laboratory Practice (GLP) platform, compliant with the Code of Federal Regulations (Title 21, Part 58). We first used protein thermal shift, size exclusion chromatography, and dynamic light scattering to examine MS-Hu6 concentrations between 1 and 100 mg/mL. We found that thermal, monomeric, and colloidal stability of formulated MS-Hu6 was maintained at a concentration of 100 mg/mL. The addition of the antioxidant L-methionine and chelating agent disodium EDTA improved the formulation's long-term colloidal and thermal stability. Thermal stability was further confirmed by Nano differential scanning calorimetry (DSC). Physiochemical properties of formulated MS-Hu6, including viscosity, turbidity, and clarity, conformed with acceptable industry standards. That the structural integrity of MS-Hu6 in formulation was maintained was proven through Circular Dichroism (CD) and Fourier Transform Infrared (FTIR) spectroscopy. Three rapid freeze-thaw cycles at -80°C/25°C or -80°C/37°C further revealed excellent thermal and colloidal stability. Furthermore, formulated MS-Hu6, particularly its Fab domain, displayed thermal and monomeric storage stability for more than 90 days at 4°C and 25°C. Finally, the unfolding temperature (T m ) for formulated MS-Hu6 increased by >4.80°C upon binding to recombinant FSH, indicating highly specific ligand binding. Overall, we document the feasibility of developing a stable, manufacturable and transportable MS-Hu6 formulation at a ultra-high concentration at industry standards. The study should become a resource for developing biologic formulations in academic medical centers.

Optimizing a therapeutic humanized follicle-stimulating hormone-blocking antibody formulation by protein thermal shift assay.

Biopharmaceutical products are formulated using several Food and Drug Administration (FDA) approved excipients within the inactive ingredient limit to maintain their storage stability and shelf life. Here, we have screened and optimized different sets of excipient combinations to yield a thermally stable formulation for the humanized follicle-stimulating hormone (FSH)-blocking antibody, MS-Hu6. We used a protein thermal shift assay in which rising temperatures resulted in the maximal unfolding of the protein at the melting temperature (Tm ). To determine the buffer and pH for a stable solution, four different buffers with a pH range from 3 to 8 were screened. This resulted in maximal Tm s at pH 5.62 for Fab in phosphate buffer and at pH 6.85 for Fc in histidine buffer. Upon testing a range of salt concentrations, MS-Hu6 was found to be more stable at lower concentrations, likely due to reduced hydrophobic effects. Molecular dynamics simulations revealed a higher root-mean-square deviation with 1 mM than with 100 mM salt, indicating enhanced stability, as noted experimentally. Among the stabilizers tested, Tween 20 was found to yield the highest Tm and reversed the salt effect. Among several polyols/sugars, trehalose and sucrose were found to produce higher thermal stabilities. Finally, binding of recombinant human FSH to MS-Hu6 in a final formulation (20 mM phosphate buffer, 1 mM NaCl, 0.001% w/v Tween 20, and 260 mM trehalose) resulted in a thermal shift (increase in Tm ) for the Fab, but expectedly not in the Fc domain. Given that we used a low dose of MS-Hu6 (1 µM), the next challenge would be to determine whether 100-fold higher, industry-standard concentrations are equally stable.

Bone circuitry and interorgan skeletal crosstalk.

The past decade has seen significant advances in our understanding of skeletal homeostasis and the mechanisms that mediate the loss of bone integrity in disease. Recent breakthroughs have arisen mainly from identifying disease-causing mutations and modeling human bone disease in rodents, in essence, highlighting the integrative nature of skeletal physiology. It has become increasingly clear that bone cells, osteoblasts, osteoclasts, and osteocytes, communicate and regulate the fate of each other through RANK/RANKL/OPG, liver X receptors (LXRs), EphirinB2-EphB4 signaling, sphingolipids, and other membrane-associated proteins, such as semaphorins. Mounting evidence also showed that critical developmental pathways, namely, bone morphogenetic protein (BMP), NOTCH, and WNT, interact each other and play an important role in postnatal bone remodeling. The skeleton communicates not only with closely situated organs, such as bone marrow, muscle, and fat, but also with remote vital organs, such as the kidney, liver, and brain. The metabolic effect of bone-derived osteocalcin highlights a possible role of skeleton in energy homeostasis. Furthermore, studies using genetically modified rodent models disrupting the reciprocal relationship with tropic pituitary hormone and effector hormone have unraveled an independent role of pituitary hormone in skeletal remodeling beyond the role of regulating target endocrine glands. The cytokine-mediated skeletal actions and the evidence of local production of certain pituitary hormones by bone marrow-derived cells displays a unique endocrine-immune-skeletal connection. Here, we discuss recently elucidated mechanisms controlling the remodeling of bone, communication of bone cells with cells of other lineages, crosstalk between bone and vital organs, as well as opportunities for treating diseases of the skeleton.

Independent Skeletal Actions of Pituitary Hormones.

Over the past years, pituitary hormones and their receptors have been shown to have non-traditional actions that allow them to bypass the hypothalamus-pituitary-effector glands axis. Bone cells-osteoblasts and osteoclasts-express receptors for growth hormone, follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), adrenocorticotrophic hormone (ACTH), prolactin, oxytocin, and vasopressin. Independent skeletal actions of pituitary hormones on bone have been studied using genetically modified mice with haploinsufficiency and by activating or inactivating the receptors pharmacologically, without altering systemic effector hormone levels. On another front, the discovery of a TSH variant (TSH-ßv) in immune cells in the bone marrow and skeletal action of FSHß through tumor necrosis factor α provides new insights underscoring the integrated physiology of bone-immune-endocrine axis. Here we discuss the interaction of each pituitary hormone with bone and the potential it holds in understanding bone physiology and as a therapeutic target.

Brain atlas for glycoprotein hormone receptors at single-transcript level.

There is increasing evidence that anterior pituitary hormones, traditionally thought to have unitary functions in regulating single endocrine targets, act on multiple somatic tissues, such as bone, fat, and liver. There is also emerging evidence for anterior pituitary hormone action on brain receptors in mediating central neural and peripheral somatic functions. Here, we have created the most comprehensive neuroanatomical atlas on the expression of TSHR, LHCGR, and FSHR. We have used RNAscope, a technology that allows the detection of mRNA at single-transcript level, together with protein level validation, to document Tshr expression in 173 and Fshr expression in 353 brain regions, nuclei and subnuclei identified using the Atlas for the Mouse Brain in Stereotaxic Coordinates. We also identified Lhcgr transcripts in 401 brain regions, nuclei and subnuclei. Complementarily, we used ViewRNA, another single-transcript detection technology, to establish the expression of FSHR in human brain samples, where transcripts were co-localized in MALAT1-positive neurons. In addition, we show high expression for all three receptors in the ventricular region-with yet unknown functions. Intriguingly, Tshr and Fshr expression in the ependymal layer of the third ventricle was similar to that of the thyroid follicular cells and testicular Sertoli cells, respectively. In contrast, Fshr was localized to NeuN-positive neurons in the granular layer of the dentate gyrus in murine and human brain-both are Alzheimer's disease-vulnerable regions. Our atlas thus provides a vital resource for scientists to explore the link between the stimulation or inactivation of brain glycoprotein hormone receptors on somatic function. New actionable pathways for human disease may be unmasked through further studies.

FSH-blocking therapeutic for osteoporosis.

Pharmacological and genetic studies over the past decade have established the follicle-stimulating hormone (FSH) as an actionable target for diseases affecting millions, namely osteoporosis, obesity, and Alzheimer's disease. Blocking FSH action prevents bone loss, fat gain, and neurodegeneration in mice. We recently developed a first-in-class, humanized, epitope-specific FSH-blocking antibody, MS-Hu6, with a KD of 7.52 nM. Using a Good Laboratory Practice (GLP)-compliant platform, we now report the efficacy of MS-Hu6 in preventing and treating osteoporosis in mice and parameters of acute safety in monkeys. Biodistribution studies using 89Zr-labeled, biotinylated or unconjugated MS-Hu6 in mice and monkeys showed localization to bone and bone marrow. The MS-Hu6 displayed a ß phase t½ of 7.5 days (180 hr) in humanized Tg32 mice. We tested 217 variations of excipients using the protein thermal shift assay to generate a final formulation that rendered MS-Hu6 stable in solution upon freeze-thaw and at different temperatures, with minimal aggregation, and without self-, cross-, or hydrophobic interactions or appreciable binding to relevant human antigens. The MS-Hu6 showed the same level of "humanness" as human IgG1 in silico and was non-immunogenic in ELISpot assays for IL-2 and IFN-γ in human peripheral blood mononuclear cell cultures. We conclude that MS-Hu6 is efficacious, durable, and manufacturable, and is therefore poised for future human testing.

FSH blockade improves cognition in mice with Alzheimer's disease.

Alzheimer's disease has a higher incidence in older women, with a spike in cognitive decline that tracks with visceral adiposity, dysregulated energy homeostasis and bone loss during the menopausal transition1,2. Inhibiting the action of follicle-stimulating hormone (FSH) reduces body fat, enhances thermogenesis, increases bone mass and lowers serum cholesterol in mice3-7. Here we show that FSH acts directly on hippocampal and cortical neurons to accelerate amyloid-ß and Tau deposition and impair cognition in mice displaying features of Alzheimer's disease. Blocking FSH action in these mice abrogates the Alzheimer's disease-like phenotype by inhibiting the neuronal C/EBPß-δ-secretase pathway. These data not only suggest a causal role for rising serum FSH levels in the exaggerated Alzheimer's disease pathophysiology during menopause, but also reveal an opportunity for treating Alzheimer's disease, obesity, osteoporosis and dyslipidaemia with a single FSH-blocking agent.

Establishing Normative Values to Determine the Prevalence of Biochemical Hyperandrogenism in Premenopausal Women of Different Ethnicities from Eastern Siberia.

Androgen assessment is a key element for diagnosing polycystic ovary syndrome (PCOS), and defining a "normal" level of circulating androgens is critical for epidemiological studies. We determined the upper normal limits (UNLs) for androgens in a population-based group of premenopausal "healthy control" women, overall and by ethnicity (Caucasian and Asian), in the cross-sectional Eastern Siberia PCOS Epidemiology and Phenotype (ESPEP) Study (ClinicalTrials.gov ID: NCT05194384) conducted in 2016-2019. Overall, we identified a "healthy control" group consisting of 143 healthy premenopausal women without menstrual dysfunction, hirsutism, polycystic ovaries, or medical disorders. We analyzed serum total testosterone (TT) by using liquid chromatography with tandem mass spectrometry (LC-MS/MS), and DHEAS, sex-hormone-binding globulin (SHBG), TSH, prolactin, and 17-hydroxyprogesterone (17OHP) were assessed with an enzyme-linked immunosorbent assay (ELISA). The UNLs for the entire population for the TT, free androgen index (FAI), and DHEAS were determined as the 98th percentiles in healthy controls as follows: 67.3 (95% confidence interval (CI): 48.1, 76.5) ng/dl, 5.4 (3.5, 14.0), and 355 (289, 371) µg/dl, respectively. The study results demonstrated that the UNLs for TT and FAI varied by ethnicity, whereas the DHEAS UNLs were comparable in the ethnicities studied.

The role of PDGF-BB in the bone-vascular relationship during aging.

Cardiovascular disease (CVD) and osteoporosis often occur together, suggesting an association between CVD and bone loss. Similarly, the correlation of bone loss, atherosclerosis, and aortic calcification, especially in patients with chronic kidney disease, exemplifies a bone-vessel connection. In this issue of the JCI, Santhanam et al. investigated the role of the angiogenesis factor platelet-derived growth factor-BB (PDGF-BB) in vascular stiffening. Serum levels of bone-derived PDGF-BB differed between young and aged mice, and in mice fed a high-fat diet (HFD) compared with those fed normal chow. Experiments with genetic models led the authors to conclude that bone-derived PDGF-BB mediates the hallmark arterial stiffening of aging and metabolic stress. Notably, excessive preosteoclast-derived PDGF-BB production during aging inhibited osteoblastic bone formation and increased circulating PDGF-BB, which in turn, accelerated vascular stiffness. These findings suggest that modifying circulating PDGF-BB levels may benefit patients with CVD, osteoporosis, and other age-related diseases.

Thyrotropin, Hyperthyroidism, and Bone Mass.

Thyrotropin (TSH), traditionally seen as a pituitary hormone that regulates thyroid glands, has additional roles in physiology including skeletal remodeling. Population-based observations in people with euthyroidism or subclinical hyperthyroidism indicated a negative association between bone mass and low-normal TSH. The findings of correlative studies were supported by small intervention trials using recombinant human TSH (rhTSH) injection, and genetic and case-based evidence. Genetically modified mouse models, which disrupt the reciprocal relationship between TSH and thyroid hormone, have allowed us to examine an independent role of TSH. Since the first description of osteoporotic phenotype in haploinsufficient Tshr +/- mice with normal thyroid hormone levels, the antiosteoclastic effect of TSH has been documented in both in vitro and in vivo studies. Further studies showed that increased osteoclastogenesis in Tshr-deficient mice was mediated by tumor necrosis factor α. Low TSH not only increased osteoclastogenesis, but also decreased osteoblastogenesis in bone marrow-derived primary osteoblast cultures. However, later in vivo studies using small and intermittent doses of rhTSH showed a proanabolic effect, which suggests that its action might be dose and frequency dependent. TSHR was shown to interact with insulin-like growth factor 1 receptor, and vascular endothelial growth factor and Wnt pathway might play a role in TSH's effect on osteoblasts. The expression and direct skeletal effect of a biologically active splice variant of the TSHß subunit (TSHßv) in bone marrow-derived macrophage and other immune cells suggest a local skeletal effect of TSHR. Further studies of how locally secreted TSHßv and systemic TSHß interact in skeletal remodeling through the endocrine, immune, and skeletal systems will help us better understand the hyperthyroidism-induced bone disease.

The hepcidin regulator erythroferrone is a new member of the erythropoiesis-iron-bone circuitry.

Background: Erythroblast erythroferrone (ERFE) secretion inhibits hepcidin expression by sequestering several bone morphogenetic protein (BMP) family members to increase iron availability for erythropoiesis. Methods: To address whether ERFE functions also in bone and whether the mechanism of ERFE action in bone involves BMPs, we utilize the Erfe-/- mouse model as well as ß-thalassemic (Hbbth3/+) mice with systemic loss of ERFE expression. In additional, we employ comprehensive skeletal phenotyping analyses as well as functional assays in vitro to address mechanistically the function of ERFE in bone. Results: We report that ERFE expression in osteoblasts is higher compared with erythroblasts, is independent of erythropoietin, and functional in suppressing hepatocyte hepcidin expression. Erfe-/- mice display low-bone-mass arising from increased bone resorption despite a concomitant increase in bone formation. Consistently, Erfe-/- osteoblasts exhibit enhanced mineralization, Sost and Rankl expression, and BMP-mediated signaling ex vivo. The ERFE effect on osteoclasts is mediated through increased osteoblastic RANKL and sclerostin expression, increasing osteoclastogenesis in Erfe-/- mice. Importantly, Erfe loss in Hbbth3/+mice, a disease model with increased ERFE expression, triggers profound osteoclastic bone resorption and bone loss. Conclusions: Together, ERFE exerts an osteoprotective effect by modulating BMP signaling in osteoblasts, decreasing RANKL production to limit osteoclastogenesis, and prevents excessive bone loss during expanded erythropoiesis in ß-thalassemia. Funding: YZG acknowledges the support of the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (R01 DK107670 to YZG and DK095112 to RF, SR, and YZG). MZ acknowledges the support of the National Institute on Aging (U19 AG60917) and NIDDK (R01 DK113627). TY acknowledges the support of the National Institute on Aging (R01 AG71870). SR acknowledges the support of NIDDK (R01 DK090554) and Commonwealth Universal Research Enhancement (CURE) Program Pennsylvania.