Estrogen Withdrawal Research Articles

Estrogen Plus Estrogen Receptor Antagonists Alter Mineral Production by Osteoblasts In Vitro

In early postmenopausal women, estrogen withdrawal is associated with increased bone turnover leading to bone loss and increased risk of fracture. Recent studies have suggested that the remaining bone tissue is significantly stronger, stiffer and has an increased tissue-level mineral content. Such changes may occur to compensate for bone loss or as a direct result of estrogen deficiency. To date many details of the physiology of osteoblastic cells during estrogen deficiency are vague. In this study we test the hypothesis that osteoblastic matrix mineralisation is altered at the onset of estrogen deficiency. In vitro cell culture experiments were carried out up to 28 days to compare the mineral production of MC3T3-E1 osteoblastic cells subject to estrogen deficiency (fulvestrant), enhanced estrogen supplementation (17-β-estradiol) or a combination of both. Mineralisation was detected using von Kossa staining and was quantified with alizarin red absorbance readings. The expression of osteocalcin and osteopontin proteins, markers of osteoblast differentiation and mineralisation, was monitored using immunohistochemistry. Our results demonstrate that estrogen enhancement improves matrix mineralisation by MC3T3 cells in vitro. Furthermore this study found a significant reduction in the level of mineralisation when cells were treated with a combination of estrogen and fulvestrant. In an estrogen deficient environment mineralisation by osteoblastic cells was not altered. These findings suggest that altered tissue mineralisation following estrogen deficiency is not a direct result of estrogen deficiency on osteoblasts. Rather, we propose that altered tissue mineralisation may be a compensatory mechanism by bone to counter bone loss and reduced strength.

Activation of neurokinin 3 receptors in the median preoptic nucleus decreases core temperature in the rat.

Estrogens have pronounced effects on thermoregulation, as illustrated by the occurrence of hot flushes secondary to estrogen withdrawal in menopausal women. Because neurokinin B (NKB) gene expression is markedly increased in the infundibular (arcuate) nucleus of postmenopausal women, and is modulated by estrogen withdrawal and replacement in multiple species, we have hypothesized that NKB neurons could play a role in the generation of flushes. There is no information, however, on whether the primary NKB receptor [neurokinin 3 receptor (NK(3)R)] modulates body temperature in any species. Here, we determine the effects of microinfusion of a selective NK(3)R agonist (senktide) into the rat median preoptic nucleus (MnPO), an important site in the heat-defense pathway. Senktide microinfusion into the rat MnPO decreased core temperature in a dose-dependent manner. The hypothermia induced by senktide was similar in ovariectomized rats with and without 17β-estradiol replacement. The hypothermic effect of senktide was prolonged in rats exposed to an ambient temperature of 29.0 C, compared with 21.5 C. Senktide microinfusion also altered tail skin vasomotion in rats exposed to an ambient temperature of 29.0 but not 21.5 C. Comparisons of the effects of senktide at different ambient temperatures indicated that the hypothermia was not secondary to thermoregulatory failure or a reduction in cold-induced thermogenesis. Other than a very mild increase in drinking, senktide microinfusion did not affect behavior. Terminal fluorescent dextran microinfusion showed targeting of the MnPO and adjacent septum, and immunohistochemical studies revealed that senktide induced a marked increase in Fos-activation in the MnPO. Because MnPO neurons expressed NK(3)R-immunoreactivity, the induction of MnPO Fos by senktide is likely a direct effect. By demonstrating that NK(3)R activation in the MnPO modulates body temperature, these studies support the hypothesis that hypothalamic NKB neurons could be involved in the generation of menopausal flushes.

Maintenance of hormone responsiveness in luminal breast cancers by suppression of Notch

Luminal breast cancers express estrogen (ER) and/or progesterone (PR) receptors and respond to hormone therapies. Basal-like "triple negative" cancers lack steroid receptors but are cytokeratin (CK) 5-positive and require chemotherapy. Here we show that more than half of primary ER(+)PR(+) breast cancers contain an ER(-)PR(-)CK5(+) "luminobasal" subpopulation exceeding 1% of cells. Starting from ER(+)PR(+) luminal cell lines, we generated lines with varying luminal to luminobasal cell ratios and studied their molecular and biological properties. In luminal disease, luminobasal cells expand in response to antiestrogen or estrogen withdrawal therapies. The phenotype and gene signature of the hormone-resistant cells matches that of clinical triple negative basal-like and claudin-low disease. Luminobasal cell expansion in response to hormone therapies is regulated by Notch1 signaling and can be blocked by γ-secretase inhibitors. Our data establish a previously unrecognized plasticity of ER(+)PR(+) luminal breast cancers that, without genetic manipulation, mobilizes outgrowth of hormone-resistant basal-like disease in response to treatment. This undesirable outcome can be prevented by combining endocrine therapies with Notch inhibition.

Autophagy's Expanding Role in Development: Implantation Is Next

Autophagy's Expanding Role in Development: Implantation Is Next

Abstract 5236: Development of chronic disease model of metastatic estrogen receptor positive (ER+) breast cancer (BC)

Abstract Expression of estrogen and progesterone receptors (ER, PR) in metastatic BC (MBC) strongly predicts for estrogen-dependence and, hence, for response to estrogen withdrawal (EWD) and to pharmacological anti-estrogens (AE). However, the choice of agents and their sequence is empiric and lacks a solid scientific foundation, largely because we lack in vivo models that accurately replicate chronic human ER+ MBC. In order to generate bone(marrow) metastasis models, highly bone tropic MCF7 or T47D cells (HER2-) or BT474 (HER2+) ER+, PR+ BC cells were selected in vivo by repeated rounds of intratibia injection, harvest and reinjection in nude mice. Histologically, MCF-7- and T47D-derived tumors fill the marrow sinusoids, with minimal osteolysis. In contrast, ER+, HER2+ BT474 cells give rise to expansile osteolytic metastases associated with osteoclast activation, reminiscent of the phenotype seen in basal-like MBC models. In order to test the estrogen-dependence of bone metastases in vivo, bone tropic MCF7 cells were injected into the tibiae of oophorectomized mice. One group received estradiol (E2) supplementation, while the control group did not. Serial in vivo imaging confirmed the appearance of mixed osteolytic and -blastic bone lesions in the E2-treated group but not in the control group. In addition, we were able to detect regional metastases to the adjacent femur and fibula. At 12 weeks, E2 pellets were removed from the treatment group. E2 deprivation induced rapid resolution of lytic lesions and pathological fractures associated with dramatic callus formation. The experiment was terminated 18 weeks later. Histologically, the residual tibial tumors were epithelioid, and still expressed cytokeratin, E-cadherin, ER and PR. Primary cell lines were established from 5 of the 5 animals. Of these, 2 were still E2-dependent in vitro and ER+, PR+. Of the 3 remaining E2-independent lines, one was ER-, PR-, and one was ER+, PR-. To determine whether or not the MCF7 cells inoculated into the control mice had retained viability and tumorigenic potential even in the absence of exogenous estrogen, E2 pellets were introduced at 12 weeks after intra-tibia injection. Ten weeks later, histological analysis failed to detect any tumor growth. However, we were able to isolate a new tertiary MCF7 subclone from 1 of 5 of these mice. This cell line is E2-dependent in vitro and ER+, PR+. In conclusion, bone-tropic MCF7-derived BC clones retain E2-dependence in a bone marrow microenvironment in vivo. Skeletal metastases respond rapidly to EWD, followed by the emergence of E2-independent subclones. In addition, in the absence of E2, bone tropic MCF7 cells are able to remain dormant but viable in a bone marrow microenvironment in vivo for prolonged periods of time. Thus, this in vivo mouse model faithfully reproduces chronic ER+, PR+ MBC and will allow us, for the first time, to develop rational treatment strategies for this disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5236. doi:10.1158/1538-7445.AM2011-5236

Hormonal Manipulation Strategies in the Management of Menstrual Migraine and Other Hormonally Related Headaches

Menstrual migraine and other hormonally related headaches are common in women. Falling estrogen levels or estrogen withdrawal after periods of sustained higher levels can trigger migraine. It makes sense to target this trigger for management of hormonally related headaches, particularly when nonhormonal strategies have been unsuccessful. Decision making regarding the use of hormonal contraception and menopausal hormone therapy is complex and commonly driven by other factors, but hormonal manipulation can potentially improve the course of migraine. Providers caring for migraineurs are appropriately concerned about stroke risk. Estrogen-containing hormonal contraceptives are relatively contraindicated for women who have migraine with aura. Postmenopausal hormone therapy is acceptable for women with a history of migraine. For these women, transdermal estradiol is recommended. Estrogen replacement is important for women who undergo an early menopause, whether natural or induced. Practical strategies for hormonal manipulation in the management of migraine and other hormonally related headaches are presented.

Evaluating the Role of Serotonin on Neuropsychological Function After Breast Cancer Using Acute Tryptophan Depletion

Although cognitive dysfunction is a prevalent and disruptive problem for many breast cancer survivors (BCSs), little research has examined its etiology. One potential mechanism that remains to be explored is serotonin. Serotonin has been implicated in normal and dysfunctional cognitive processes, and serotonin levels are significantly affected by estrogen withdrawal, a common side effect of breast cancer treatment. However, no study has evaluated serotonin's role on cognitive dysfunction in BCSs. The purpose of this study was to examine the role of serotonin in cognitive dysfunction in survivors by lowering central serotonin concentrations via acute tryptophan depletion (ATD). Based on previous research in noncancer populations, we hypothesized that alterations in central serotonin levels would induce cognitive dysfunction in these women controlling for confounding characteristics such as fluctuating mood and glucose levels. Secondarily, we explored whether genetic variations in serotonin genes would partly explain ATD. Participants included 20 female BCSs, posttreatment for nonmetastatic breast cancer, who received ATD or control in a double-blind, crossover design. Cognitive performance was measured at the 5-hr tryptophan/serotonin nadir on each test day using standardized neuropsychological tests. Specific impairment was noted in episodic memory (delayed recall) and motor speed during ATD versus control. ATD did not alter new learning (immediate recall), working memory, verbal fluency, or information processing speed. Findings suggest that serotonin may play a critical role in memory consolidation and motor functioning in BCSs.

Effects of Estrogen and Testosterone on Resting Energy Expenditure in Older Men

The mechanisms by which sex hormones cause changes in body composition are unclear. Sex steroid deficiency might directly reduce energy expenditure/fat oxidation and thereby predispose to increased body fat. Alternatively, sex steroid deficiency could result in lean tissue loss and thus reduced energy expenditure. Our objective was to examine the independent and combined effects of acute testosterone and estrogen withdrawal on respiratory exchange ratio (RER) and resting energy expenditure (REE) in men. The objective of the study was to examine the independent and combined effects of acute estrogen and testosterone withdrawal on RER and REE in men. A total of 54 men aged 50-80 years, BMI range of 17-35 kg/m(2) underwent a 3-week eugonadal run-in hormone-treatment period involving suppression of endogenous sex steroids using letrozole and leuprolide acetate (Lupron) while sex steroid concentrations were maintained with transdermal testosterone (T) and estradiol (E). A second Lupron injection was then given and participants were randomized to one of the following four 3-week treatment groups: group A (-T, -E), group B (-T, +E), group C (+T, -E), and group D (+T, +E). REE and RER were measured via indirect calorimetry before and after the 3-week treatment period. Three-week suppression and/or repletion of estrogen or testosterone did not produce changes in RER or REE within or between groups. We conclude that abrupt changes in sex steroids does not change resting substrate oxidation, indicating that changes that can be observed after more prolonged periods of deficiency are most likely due to direct effects of sex steroids on body composition.

Hepatic VLDL-TG Production and MTP Gene Expression are Decreased in Ovariectomized Rats: Effects of Exercise Training

The present study was designed to investigate the effects of estrogen withdrawal and exercise training on hepatic very low density lipoprotein-triglyceride (VLDL-TG) production and on expression of genes involved in hepatic VLDL synthesis in response to lipid infusion. Female Sprague-Dawley rats underwent ovariectomy (Ovx), sham surgery (Sham), and Ovx with 17β-estradiol supplementation (OvxE2) before being subdivided into sedentary (Sed) and trained (Tr) groups for 8 weeks. Exercise training consisted of continuous running on a rodent treadmill 5 times/wk. At the end of the 8-week period, all rats in the fasted state were intravenously infused with a 20% solution of Intralipid for 3-h followed by an injection of Triton WR-1339 to block lipoprotein lipase activity. Plasma TG accumulation was subsequently measured during 90 min to estimate VLDL-TG production. An additional control group consisting of Sham-Sed rats was infused with saline (0.9% NaCl). Estrogen withdrawal resulted in higher (p<0.01) liver fat accumulation concomitantly with lower (p<0.01) VLDL-TG production and lower mRNA and protein content of hepatic microsomal triglyceride transfer protein (MTP). All of these effects in Ovx rats were corrected with estrogen supplementation. Training in Ovx rats reduced (p<0.01) liver fat accumulation and further reduced (p<0.01) hepatic VLDL-TG production along with gene expression of MTP and diacylglycerol acyltransferase-2 (DGAT-2). It is concluded that VLDL-TG synthesis and/or secretion is decreased in Ovx rats probably via MTP regulation and that this decrease may constitute one of the factors involved in hepatic fat accumulation. The training effect on reducing VLDL production was independent of the estrogenic status.

Oncogenic HER2Δ16 suppresses miR-15a/16 and deregulates BCL-2 to promote endocrine resistance of breast tumors

Tamoxifen is the most commonly prescribed therapy for patients with estrogen receptor (ER)α-positive breast tumors. Tumor resistance to tamoxifen remains a serious clinical problem especially in patients with tumors that also overexpress human epidermal growth factor receptor 2 (HER2). Current preclinical models of HER2 overexpression fail to recapitulate the clinical spectrum of endocrine resistance associated with HER2/ER-positive tumors. Here, we show that ectopic expression of a clinically important oncogenic isoform of HER2, HER2Δ16, which is expressed in >30% of ER-positive breast tumors, promotes tamoxifen resistance and estrogen independence of MCF-7 xenografts. MCF-7/HER2Δ16 cells evade tamoxifen through upregulation of BCL-2, whereas mediated suppression of BCL-2 expression or treatment of MCF-7/HER2Δ16 cells with the BCL-2 family pharmacological inhibitor ABT-737 restores tamoxifen sensitivity. Tamoxifen-resistant MCF-7/HER2Δ16 cells upregulate BCL-2 protein levels in response to suppressed ERα signaling mediated by estrogen withdrawal, tamoxifen treatment or fulvestrant treatment. In addition, HER2Δ16 expression results in suppression of BCL-2-targeting microRNAs miR-15a and miR-16. Reintroduction of miR-15a/16 reduced tamoxifen-induced BCL-2 expression and sensitized MCF-7/HER2Δ16 to tamoxifen. Conversely, inhibition of miR-15a/16 in tamoxifen-sensitive cells activated BCL-2 expression and promoted tamoxifen resistance. Our results suggest that HER2Δ16 expression promotes endocrine-resistant HER2/ERα-positive breast tumors and in contrast to wild-type HER2, preclinical models of HER2Δ16 overexpression recapitulate multiple phenotypes of endocrine-resistant human breast tumors. The mechanism of HER2Δ16 therapeutic evasion, involving tamoxifen-induced upregulation of BCL-2 and suppression of miR-15a/16, provides a template for unique therapeutic interventions combining tamoxifen with modulation of microRNAs and/or ABT-737-mediated BCL-2 inhibition and apoptosis.

Sclerostin: A gem from the genome leads to bone-building antibodies

Sclerostin: A gem from the genome leads to bone-building antibodies

Gathering of aging and estrogen withdrawal in vascular dysfunction of senescent accelerated mice

The aim of this work was to characterize a mouse model of experimental menopause and cardiovascular aging that closely reflects menopause in women. Senescence accelerated mouse (SAM)-Resistant type 1 (SAMR1, n = 30) and SAM-Prone type 8 (SAMP8, n = 30) were separated at 5 months of age into three groups: 1) sham-operated (Sham); 2) ovariectomized (Ovx); and 3) ovariectomized chronically-treated with estrogen (Ovx + E2). Contractile responses to KCl (60 mM) and thromboxane A 2 were greater in aorta from SAMP8 mice compared with SAMR1 in all groups. Neither ovariectomy nor estrogen replacement modified the contractile responses from SAMR1 mice. Conversely, in Ovx SAMP8 the increased maximal contractions were reversed by estrogen treatment. Rings with endothelium from all SAMR1 groups showed a greater relaxation to acetylcholine than SAMP8 groups. In SAMR1, endothelium-dependent relaxation was not altered in Ovx or Ovx + E2 groups. Rings from Ovx SAMP8 showed a decreased maximal response to acetylcholine compared to Sham SAMP8. Estrogen replacement restored the response to acetylcholine altered by ovariectomy. Nitric oxide inhibition by L-NAME markedly reduced acetylcholine responses in all groups, but this effect was less pronounced in SAMP8 and Ovx groups (determined by area under the curve reduction). These results indicate that SAMP8 exhibit a significant decreased endothelium-dependent and NO-mediated relaxation and increased vasoconstrictor responses that are potentiated by the lack of estrogen. Because these responses are closely in agreement with vascular dysfunction observed in menopausal women, we propose SAMP8 Ovx as a new model to concomitantly study the effects of aging and menopause in female mice.

Exercise training in ovariectomized rats stimulates estrogenic-like effects on expression of genes involved in lipid accumulation and subclinical inflammation in liver

We hypothesized that the reduction in liver fat accumulation known to occur with exercise training in ovariectomized (Ovx) rats is associated with reduced expression of genes involved in lipogenesis while favoring the expression of transcription factors regulating lipid oxidation. We also tested the hypothesis that liver fat accumulation in Ovx rats is associated with an increased gene expression of several inflammatory biomarkers and that exercise training would attenuate this response. Sprague-Dawley female rats (14 weeks of age) were randomly divided into 4 groups of sedentary sham-operated (Sham), Ovx, Ovx with 17 β-estradiol (E2) supplementation using a pellet (0.72 mg; 0.012 mg/d) with a biodegradable carrier binder, and Ovx trained with endurance exercise. Endurance exercise training consisted of continuous running on a motor-driven rodent treadmill 5 times per week for 5 weeks. Fat accumulation in liver as well as in adipose fat depots was higher ( P < .01) in Ovx than in Sham rats. This response was prevented in Ovx animals with 17 β-estradiol supplementation and with endurance exercise training. Liver gene expressions of sterol regulatory element-binding protein 1-c, stearoyl coenzyme A desaturase 1 (and its protein content), carbohydrate response element binding protein, and acetyl–coenzyme A carboxylase were increased with estrogen withdrawal ( P < .01). These responses were corrected with E2 supplementation alone as well as with training alone. Conversely, hepatic peroxisome proliferator–activated receptor α messenger RNA levels were lower ( P < .01) after estrogen removal compared with Sham rats. The lower hepatic peroxisome proliferator–activated receptor α messenger RNA levels in Ovx rats were reincreased by E2 replacement or by exercise training. Gene expression of proinflammatory cytokines including inhibitor- κB kinase β and interleukin-6, as well as protein content of nuclear factor- κB, was higher ( P < .01) in Ovx than in Sham animals. E2 supplementation or exercise training prevented the expression of the proinflammatory markers. It is concluded that exercise training reduces fat accumulation in liver of Ovx rats possibly through regulation of key molecules involved in lipogenesis and lipid oxidation. Exercise training also acts as estrogens in properly regulating the expression of inflammatory biomarkers in liver of Ovx rats.

Regulation of adult bone turnover by sex steroids

Recent reports reveal increasing complexity of mechanisms underlying the bone sparing effects of sex steroids. This review focuses on mechanisms by which sex steroids attenuate endocortical and trabecular adult bone turnover, perhaps their most important property as bone mass regulators. Clearly, estrogen withdrawal increases osteoclast number and bone resorption; however, important open questions are the extent to which osteoblasts and their precursors are involved, and the relative contributions of the RANK/RANKL/OPG system, Fas ligand and Runx2. In addition to reviewing these aspects of estrogen action, we also discuss proskeletal effects of androgens on the adult male skeleton, including aromatization to estrogens and male-specific mechanisms. Detailed understanding of skeletal site- and gender-dependent mechanisms by which sex steroids protect the adult skeleton will provide the foundation for improved risk assessment, prevention and management of osteoporosis.

Does exercise training prior to ovariectomy protect against liver and adipocyte fat accumulation in rats?

Objective To determine whether a training state protects against the metabolically deleterious effects of ovariectomy on liver and adipocyte fat accumulation in rats.Design Female rats were randomly assigned to each group (n = 8 rats/group). The animals were first either exercise-trained (Tr) for 6 weeks or kept sedentary (Sed) before being sham-operated (Sham), ovariectomized (Ovx), or ovariectomized with 17β-estradiol supplementation (OvxE2). Following surgery, sedentary rats either remained sedentary (Sed-Sed) or undertook exercise training for 6 weeks (Sed-Tr) while exercise-trained rats either became sedentary (Tr-Sed) or resumed exercise training (Tr-Tr).Results Body weight and energy intake along with intra-abdominal and subcutaneous fat pad weights and homeostasis model assessment of insulin resistance (HOMA-IR) were significantly (p < 0.01) increased in the Ovx group compared to the Sham and OvxE2 groups. Rats kept in a sedentary state after surgery showed the higher (p < 0.05) values for all of these variables whether they were trained or not before surgery (Sed-Sed and Tr-Sed), indicating no protective effect of a previous exercise-trained state. On the other hand, training conducted after surgery resulted in a lowering of fat mass and HOMA-IR whether rats had been trained or not before surgery (Sed-Tr and Tr-Tr), indicating the effectiveness of exercise training even initiated after surgery. These responses were independent of surgery. Interestingly, liver triacylglycerol concentrations followed a pattern of responses identical to fat mass with the exception that all of the responses were observed only in the Ovx group (p < 0.05).Conclusion There is no protective effect of a previous exercise-training state on ovariectomy-induced liver and adipocyte fat accumulation if rats remain sedentary after ovariectomy. However, training conducted concurrently with estrogen withdrawal has protective effects, especially on liver fat accumulation, whether or not rats were previously trained.

Post-translational Regulation of Endothelial Nitric Oxide Synthase (eNOS) by Estrogens in the Rat Vagina

Estrogens control vaginal blood flow during female sexual arousal mostly through nitric oxide (NO). Although vascular effects of estrogens are attributed to an increase in endothelial NO production, the mechanisms of endothelial NO synthase (eNOS) regulation by estrogens in the vagina are largely unknown. Our hypothesis was that estrogens regulate eNOS post-translationally in the vagina, providing a mechanism to affect NO bioavailability without changes in eNOS protein expression. We measured eNOS phosphorylation and eNOS interaction with caveolin-1 and heat shock protein 90 (HSP90) in the distal and proximal vagina of female rats at diestrus, 7 days after ovariectomy and 2 days after replacement of ovariectomized rats with estradiol-17beta (15 microg). Molecular mechanisms of eNOS regulation by estrogen in the rat vagina. We localized phospho-eNOS (Ser-1177) immunohistochemically to the endothelium lining blood vessels and vaginal sinusoids. Estrogen withdrawal decreased phosphorylation of eNOS on its positive regulatory site (Ser-1177) and increased eNOS binding to its negative regulator caveolin-1 (without affecting eNOS/HSP90 interaction), and they were both normalized by estradiol replacement. Protein expressions of phosphorylated Akt (protein kinase B) and extracellular signal-regulated protein kinase 1/2 (ERK1/2) were not affected by estrogen status, suggesting that the effect of estrogens on eNOS (Ser-1177) phosphorylation was not mediated by activated AKT or ERK1/2. eNOS phosphorylation on its negative regulatory site (Ser-114) was increased in the vagina by estrogen withdrawal and normalized by estradiol replacement, implying that the maintenance of low phosphorylation of eNOS on this site by estradiol may limit eNOS interaction with caveolin-1 and preserve the enzyme's activity. Total eNOS, inducible NOS, caveolin-1, and HSP90 protein expressions were not affected by ovariectomy or estradiol replacement in the distal or proximal vagina. These results define novel estrogen signaling mechanisms in the vagina which involve eNOS phosphorylation and eNOS-caveolin-1 interaction.

Influence of Intrinsic Aerobic Exercise Capacity on Skeletal Health

Low aerobic exercise capacity strongly associates with greater cardiovascular disease, increased incidence of metabolic syndrome, poor adaptation response to negative health environments, and weakened tissue repair. A greater capacity to sense, utilize, and optimize oxygen may represent a dominant factor in overall health and resistance to disease1-3. Bone cells are particularly responsive to their local oxygen environment, and therefore may demonstrate features as a reflection of aerobic capacity. PURPOSE: To determine the role of aerobic capacity on skeletal health, bone repair, and response to hormone withdrawal. METHODS: Experiments were performed using a rat model4 selectively bred for high (HCR) and low (LCR) intrinsic aerobic capacity that demonstrates a six-fold difference in treadmill running to exhaustion in the untrained condition. Bone morphology and mechanical properties were assessed in HCR (n = 10) and LCR (n = 9), and effect of exercise was tested in treadmill-trained LCR (LCR+TR; n = 11). Fracture repair, along with mesenchymal stem cell (MSC) proliferation, differentiation, and mineralization were assessed in HCR (n = 7) and LCR (n = 7) using femoral osteotomy and in vitro marrow cell assays. Response to estrogen withdrawal was evaluated to model postmenopausal osteoporosis (n = 12 HCR; n = 12 LCR). RESULTS: Exercise capacity of LCR+TR improved 36% above LCR (338 vs. 249 m; p < 0.05), but not to HCR levels (1731 m). HCR had smaller femora (p < 0.001), but elevated cortical tissue mineral density and Young's modulus (p < 0.01). Fracture callus size normalized by body mass was greater in HCR relative to LCR (p < 0.01), and improved healing corresponded with increased mineralization capacity of MSC's as measured in vitro. HCRs were protected against ovariectomy-induced bone loss at the femoral neck, but not at the proximal tibial metaphysis, reflecting site-specific bone loss. CONCLUSION: Results demonstrated that the intrinsic ability of bone cells to metabolize oxygen has a significant effect on phenotype, and suggest that aerobic capacity is an important regulator of bone health. (1) Gonzalez 2006 J Appl Physiol 101:1288. (2) Howlett 2008 J Appl Physiol 106:1819. (3) Morris 2009 Am J Nephrol 30:112. (4) Koch and Britton 2001 Physiol Genomics 5:45.

Parathyroid Hormone and Bone Healing

Fracture healing is a complex process, and a significant number of fractures are complicated by impaired healing and non-union. Impaired healing is prevalent in certain risk groups, such as the elderly, osteoporotics, people with malnutrition, and women after menopause. Currently, no pharmacological treatments are available. There is therefore an unmet need for medications that can stimulate bone healing. Parathyroid hormone (PTH) is the first bone anabolic drug approved for the treatment of osteoporosis, and intriguingly a number of animal studies suggest that PTH could be beneficial in the treatment of fractures and could thus be a potentially new treatment option for induction of fracture healing in humans. Furthermore, fractures in animals with experimental conditions of impaired healing such as aging, estrogen withdrawal, and malnutrition can heal in an expedited manner after PTH treatment. Interestingly, fractures occurring at both cancellous and cortical sites can be treated successfully, indicating that both osteoporotic and nonosteoporotic fractures can be the target of PTH-induced healing. Finally, the data suggest that PTH partly prevents the delay in fracture healing caused by aging. Recently, the first randomized, controlled clinical trial investigating the effect of PTH on fracture healing was published, indicating a possible clinical benefit of PTH treatment in inducing fracture healing. The aim of this article is therefore to review the evidence for the potential of PTH in bone healing, including the underlying mechanisms for this, and to provide recommendations for the clinical testing and use of PTH in the treatment of impaired fracture healing in humans.

Gender differences in the cardiac A1 adenosine receptor anti‐adrenergic effect

There have been few reports on gender differences in cardiac adenosine receptors (AR). The A1AR modulation of the male and female cardiac β‐adrenergic response was studied in isolated perfused hearts (constant perfusion pressure, paced at 420 bpm) from C57BL/6 mice, monitoring left ventricular systolic pressure (LVSP), +dP/dT and –dP/dT (n≥4). To study the effects of estrogen withdrawal, a subset of females was studied 5–7 weeks post‐ovariectomy (OvX). No differences were seen between male (M), female (F) and OvX female (OvXF) hearts in baseline hemodynamic parameters, or in the β‐adrenergic response following isoproterenol (ISO) administration. In M, 2 nM ISO increased LVSP 86±8%, +dP/dT 131± 9% and –dP/dT 147±11%. 2 nM ISO responses were similar in F and OvXF. As shown in Table 1, pretreatment with 200 nM A1AR agonist CCPA produced an enhanced blunting of the 2 nM ISO response in F as compared to M and OvXF. These observations could be explained by differences in cardiac AR gene expression, with intact females showing 25% higher A1AR and 40% lower A2aAR expression than M and OvXF. These results suggest that ovarian hormones modulate AR gene expression, contributing to gender differences in AR function. Percent change from pre‐ ISO baseline in CCPA‐Treated Hearts LVSP +dP/dT −dP/dT Male 32± 5* 52±8* 67±11* Female 6±1 12±2 16±3 OvX Female 43±7† 83±18† 71±15† p&lt;0.05 Female vs Male p&lt;0.05 Female vs. OvX Female

Fas receptor is required for estrogen deficiency-induced bone loss in mice

Bone mass is determined by bone cell differentiation, activity, and death, which mainly occur through apoptosis. Apoptosis can be triggered by death receptor Fas (CD95), expressed on osteoblasts and osteoclasts and may be regulated by estrogen. We have previously shown that signaling through Fas inhibits osteoblast differentiation. In this study we analyzed Fas as a possible mediator of bone loss induced by estrogen withdrawal. At 4 weeks after ovariectomy (OVX), Fas gene expression was greater in osteoblasts and lower in osteoclasts in ovariectomized C57BL/6J (wild type (wt)) mice compared with sham-operated animals. OVX was unable to induce bone loss in mice with a gene knockout for Fas (Fas –/– mice). The number of osteoclasts increased in wt mice after OVX, whereas it remained unchanged in Fas –/– mice. OVX induced greater stimulation of osteoblastogenesis in Fas –/– than in wt mice, with higher expression of osteoblast-specific genes. Direct effects on bone cell differentiation and apoptosis in vivo were confirmed in vitro, in which addition of estradiol decreased Fas expression and partially abrogated the apoptotic and differentiation-inhibitory effect of Fas in osteoblast lineage cells, while having no effect on Fas-induced apoptosis in osteoclast lineage cells. In conclusion, the Fas receptor has an important role in the pathogenesis of postmenopausal osteoporosis by mediating apoptosis and inhibiting differentiation of osteoblast lineage cells. Modulation of Fas effects on bone cells may be used as a therapeutic target in the treatment of osteoresorptive disorders.