Estrogen Withdrawal Research Articles

Development of Hypertensive Animal Model Using Ovariectomised Rat Fed with Short-Term 2% Cholesterol Diet

Menopause, a cessation of menstrual bleeding, is directly related to serious illness confined to cardiovascular disease (CVD) such as hypertension and stroke. The oestrogen withdrawal and consumption of high cholesterol diet has a detrimental effect on cardiovascular function and metabolism in menopausal women. The present study was aimed to develop the hypertensive rat model in the ovariectomised Sprague Dawley rats fed with 2% cholesterol diet following a short term period. Eighteen Sprague-Dawley rats were randomly divided into three groups; Sham control (sham), ovariectomized control fed with normal diet (Ovx +N), ovariectomized rats fed with 2% cholesterol diet (Ovx + Cho) for four weeks. At the end of the study, the food intake and body weight were measured. The systolic, diastolic and mean blood pressure was determined. OVX+Cho showed significant increase in physiological changes such as body weight and food intake compared to Ovx+N group. The significant increase in systolic, diastolic and mean blood pressure was observed in Ovx+Cho group. Based on the above findings, it is believed that feeding cholesterol diet for four weeks (short term) results in significant increase in the blood pressure, food intake and body weight in the ovariectomised rats.

Presenting a New, Non-Hormonally Mediated Cyclic Headache in Women: End-Menstrual Migraine.

This retrospective observational study describes what appears to be a not-uncommon yet not previously typified headache disorder, "end-menstrual migraine" (EMM) named for its proximate occurrence with the terminal days of menstrual bleeding each month. Our menstrual migraine (MM) clinic's database was queried for patients with migraine, regular menses, and the locally used diagnostic code of EMM, signifying the patient's assertion that she had migraine at the end of menses as well as calendar data confirming that association. Serum ferritin levels were available for all subjects with EMM. Among the most recently seen 119 women in the clinic, 85 had both regular menses and a diagnosis of migraine, and were thus appropriate for evaluation. In this selected population, 30 women (35.3%) were confirmed to have EMM. Twenty-eight of those 30 patients (93.3%) had ferritin levels that fell below the generally accepted lower desirable limit of 50 ng/mL (mean = 21.9 ng/mL) - half of whom fell below 18 ng/mL, the established minimum levels for women. EMM is a common complaint in women evaluated for menstrual-related migraine (MRM), yet these migraines occur many days after the estrogen withdrawal that precipitates MRM. The authors do not believe that EMM is hormonally mediated, but rather that it is causally related to menstrual blood loss, resulting in a brief relative anemia with consequent migraine. Further study is needed to substantiate this association, search for confounders, and evaluate response to iron therapy.

Detection of bone marrow changes related to estrogen withdrawal in rats with a tabletop stray-field NMR scanner.

Osteoporosis is characterized by a decrease in bone mineral density (BMD). A preliminary stage of the disease is progressive bone marrow adiposity, caused by imbalance between osteogenesis and adipogenesis in the marrow. Detection of osteoporosis relies on the quantification of BMD with techniques such as dual-energy X-ray absorptiometry. This work aimed to detect bone marrow changes in an experimental model of osteopenia using a low-field tabletop NMR scanner. An experiment was performed on 32 female rats, 3 months old, 16 of which were ovariectomized (OVX) and 16 were sham-operated (sham). The femur and tibia from both hind limbs were isolated and underwent ex vivo NMR scans at four time points after the OVX and sham operations. NMR scans were complemented by BMD measurements and histology. Significant changes in the bone marrow of ovariectomized rats, relative to sham operated rats, were observed after 3.5 and 4.5 months. Bone marrow adiposity was detected by significant changes in T1 and T2 relaxation times, and in the diffusion coefficient. This study suggests a potential detection of changes to the bone marrow using a tabletop NMR device. Clinical translation may facilitate screening, early detection of bone weakening as a result of estrogen withdrawal, and monitoring of treatment efficacy. Magn Reson Med 78:860-870, 2017. © 2016 International Society for Magnetic Resonance in Medicine.

Modulation of nociceptive threshold by combined hormonal contraceptives in women with oestrogen-withdrawal migraine attacks: a pilot study

BackgroundMenstrually-related headache and headaches associated with oestrogen withdrawal are common conditions, whose pathophysiology has not been completely elucidated. In this study we evaluated the influence of combined hormonal contraceptives (CHC) on pain threshold in women presenting migraine attacks during hormone-free interval.FindingsEleven women with migraine attacks recurring exclusively during the oestrogen-withdrawal period were studied with the nociceptive flexion reflex, a neurophysiological assessment of the pain control systems, during the third week of active treatment and during the hormone-free interval.During the hormone-free interval, nociceptive withdrawal reflex threshold was significantly lower (12.8 ± 8.0 mA) as compared to the third week of hormonal treatment (15.6 ± 6.6 mA) (p = 0.02). No change was observed in the pain perceived and in the temporal summation.ConclusionsOestrogen withdrawal may mediate an increased sensitivity to somatosensory stimuli in women with migraine attacks recurring during the hormone-free interval.

Short-Term Estrogen Withdrawal Increases Adiposity in Healthy Men.

T deprivation increases risk of insulin resistance in men, but whether this risk is independent of changes in body composition is unknown. Further, the metabolic roles of T and its metabolite estradiol have not been clearly defined in men. This study sought to establish the effects of selective sex steroid withdrawal on insulin sensitivity in healthy men. This was a double-blinded, placebo-controlled, randomized trial at an academic medical center of 56 healthy men, 19-55 years of age. Subjects received the GnRH antagonist acyline plus one of the following: placebo gel (Castrate), 1.25 g testosterone gel (Low T/E), 5 g testosterone gel (Normal T/E), or 5 g testosterone gel with letrozole (Normal T/Low E) daily for 4 weeks. Body composition and glucose tolerance were assessed at baseline and end of treatment. Insulin sensitivity was quantified by the Matsuda index. Predicted circulating sex steroid concentrations were achieved in all treatment groups. The time-by-group interaction for Matsuda index did not achieve significance in overall repeated measures ANOVA (baseline vs week 4; P = .16). A significant time-by-group interaction was observed for fat mass (P = .003), with changes in fat mass attributable predominantly to estrogen exposure in linear regression analysis (P = .016). A time-by-group interaction also was observed for lean mass (P = .03) and influenced by androgen exposure (P = .003). Short-term sex steroid withdrawal in healthy men causes adverse changes in body composition. These findings support the role of estradiol as a determinant of adiposity in men.

Overweight postmenopausal women with different plasma estradiol concentrations present with a similar pattern of energy expenditure and substrate oxidation rate before and after a fatty meal challenge

Menopause-related withdrawal of ovarian estrogens is associated with reduced energy metabolism and overall impairment of substrate oxidation. Estradiol's withdrawal after menopause is associated with a reduction in energy metabolism and impaired substrate oxidation, which contributes to weight gain and visceral fat accumulation. Here we aimed to investigate the association between plasma estradiol concentrations and energy expenditure (EE)/substrate oxidation in a group of overweight postmenopausal women before and after a fatty meal challenge. Women were divided into three groups according to their plasma estradiol concentrations (E2): group 1 - E2≤39, group 2 - 40≤E2≤59, and group 3 - E2≥60pg/mL. VO2 and VCO2 volumes were collected following indirect calorimetry 5h following a single lipid overload meal (1100kcal, 72% of fat). For comparisons between groups and within the same group, a linear regression model with mixed effects was applied (P<0.05). Forty-four women aged 55±0.7 years-old, 8±1.1 years following menopause, with a BMI of 30.5±0.5kg/m2, and 41.9±0.7% of body fat were enrolled the study. Plasma E2 concentrations were: group 1 - 30.4±1.9, group 2 - 46.9±1.5, and group 3 - 91.3±12.0pg/mL (P<0.0001). EE at baseline and in the resting state was 1320±24.3kcal/d, and increased to 1440±27.0kcal/d 30min following ingestion of the fatty meal (P<0.0001), and rose again to an average of 1475±30.3kcal/d at the completion of experiment (P<0.0001). Carbohydrate oxidation (Chox) was 0.155±0.01g/min at resting, maintained as 0.133±0.00g/min 30min after ingestion of the fatty meal, and was 0.123±0.01g/min at the end of the testing period. Lipid oxidation (Lipox) was 0.041±0.003g/min at resting, increasing to 0.054±0.003g/min at 30min (P=0.01), and reaching 0.063±0.003g/min at the end of the experiment (P<0.0001). There was no difference between groups for EE, Chox or Lipox. Our data suggest that EE and substrate oxidation were modulated following a lipid-meal challenge equally in all groups and this did not differ with plasma E2 concentrations.

Sex hormones in women with and without migraine: Evidence of migraine-specific hormone profiles.

To compare daily sex hormone levels and rates of change between women with history of migraine and controls. History of migraine, daily headache diaries, and daily hormone data were collected in ovulatory cycles of pre- and early perimenopausal women in the Study of Women's Health Across the Nation. Peak hormone levels, average daily levels, and within-woman day-to-day rates of decline over the 5 days following each hormone peak were calculated in ovulatory cycles for conjugated urinary estrogens (E1c), pregnanediol-3-glucuronide, luteinizing hormone, and follicle-stimulating hormone. Comparisons were made between migraineurs and controls using 2-sample t tests on the log scale with results reported as geometric means. The sample included 114 women with history of migraine and 223 controls. Analyses of within-woman rates of decline showed that E1c decline over the 2 days following the luteal peak was greater in migraineurs for both absolute rate of decline (33.8 [95% confidence interval 28.0-40.8] pg/mgCr vs 23.1 [95% confidence interval 20.1-26.6] pg/mgCr, p = 0.002) and percent change (40% vs 30%, p < 0.001). There was no significant difference between migraineurs and controls in absolute peak or daily E1c, pregnanediol-3-glucuronide, luteinizing hormone, and follicle-stimulating hormone levels. Secondary analyses demonstrated that, among migraineurs, the rate of E1c decline did not differ according to whether a headache occurred during the cycle studied. Migraineurs are characterized by faster late luteal phase E1c decline compared to controls. The timing and rate of estrogen withdrawal before menses may be a marker of neuroendocrine vulnerability in women with migraine.

11,12-Epoxyecosatrienoic acids mitigate endothelial dysfunction associated with estrogen loss and aging: Role of membrane depolarization

ObjectiveEndothelial dysfunction, including upregulation of inflammatory adhesion molecules and impaired vasodilatation, is a key element in cardiovascular disease. Aging and estrogen withdrawal in women are associated with endothelial inflammation, vascular stiffness and increased cardiovascular disease. Epoxyecosatrienoic acids (EETs), the products of arachidonic acid metabolism mediated by cytochrome P450 (CYP) 2J, 2C and other isoforms, are regulated by soluble epoxide hydrolase (sEH)-catalyzed conversion into less active diols. We hypothesized that 11,12-EETs would reduce the endothelial dysfunction associated with aging and estrogen loss. Approach/resultsWhen stabilized by an sEH inhibitor (seHi), 11,12-EET at a physiologically low dose (0.1nM) reduced cytokine-stimulated upregulation of adhesion molecules on human aorta endothelial cells (HAEC) and monocyte adhesion under shear flow through marked depolarization of the HAEC when combined with TNFα. Mechanistically, neither 11,12-EETs nor 17β-estradiol (E2) at physiologic concentrations prevented activation of NFκB by TNFα. E2 at physiological concentrations reduced sEH expression in HAEC, but did not alter CYP expression, and when combined with TNFα depolarized the cell. We also examined vascular dysfunction in adult and aged ovariectomized Norway brown rats (with and without E2 replacement) using an ex-vivo model to analyze endothelial function in an intact segment of artery. sEHi and 11,12-EET with or without E2 attenuated phenylephrine induced constriction and increased endothelial-dependent dilation of aortic rings from ovariectomized rats. ConclusionsIncreasing 11,12-EETs through sEH inhibition effectively attenuates inflammation and may provide an effective strategy to preserve endothelial function and prevent atherosclerotic heart disease in postmenopausal women.

Involvement of receptor-interacting protein 140 in estrogen-mediated osteoclasts differentiation, apoptosis, and bone resorption.

Estrogen withdrawal following menopause results in an increase of osteoclasts formation and bone resorption, which is one of the most important mechanisms of postmenopausal osteoporosis. Recently, growing evidence has suggested that receptor-interacting protein 140 was implicated in estrogen-regulated metabolic disease, including fat metabolism and lipid metabolism. However, little is known regarding the role of receptor-interacting protein 140 in the regulation of bone metabolic by estrogen. In the present study, Western blotting disclosed that estrogen brings a significant increasing expression of receptor-interacting protein 140 in osteoclasts, but not in osteoblasts and bone marrow mesenchymal stem cells. Furthermore, analysis of TRAP staining and bone resorption assay showed that depletion of receptor-interacting protein 140 could significantly alleviate the inhibitory effects of estrogen on osteoclasts formation and bone resorption activity. Moreover, estrogen could induce osteoclasts apoptosis by increasing receptor-interacting protein 140 expression through the Fas/FasL pathway. Taken together, receptor-interacting protein 140 might be a critical player in estrogen-mediated osteoclastogenesis and bone resorption.

Osteocyte Apoptosis Caused by Hindlimb Unloading is Required to Trigger Osteocyte RANKL Production and Subsequent Resorption of Cortical and Trabecular Bone in Mice Femurs

Osteocyte apoptosis is essential to activate bone remodeling in response to fatigue microdamage and estrogen withdrawal, such that apoptosis inhibition in vivo prevents the onset of osteoclastic resorption. Osteocyte apoptosis has also been spatially linked to bone resorption owing to disuse, but whether apoptosis plays a similar controlling role is unclear. We, therefore, 1) evaluated the spatial and temporal effects of disuse from hindlimb unloading (HLU) on osteocyte apoptosis, receptor activator of NF-κB ligand (RANKL) expression, bone resorption, and loss in mouse femora, and 2) tested whether osteocyte apoptosis was required to activate osteoclastic activity in cortical and trabecular bone by treating animals subjected to HLU with the pan-caspase apoptosis inhibitor, QVD (quinolyl-valyl-O-methylaspartyl-[-2,6-difluorophenoxy]-methylketone). Immunohistochemistry was used to identify apoptotic and RANKL-producing osteocytes in femoral diaphysis and distal trabecular bone, and µCT was used to determine the extent of trabecular bone loss owing to HLU. In both cortical and trabecular bone, 5 days of HLU increased osteocyte apoptosis significantly (3- and 4-fold, respectively, p < 0.05 versus Ctrl). At day 14, the apoptotic osteocyte number in femoral cortices declined to near control levels but remained elevated in trabeculae (3-fold versus Ctrl, p < 0.05). The number of osteocytes producing RANKL in both bone compartments was also significantly increased at day 5 of HLU (>1.5-fold versus Ctrl, p < 0.05) and further increased by day 14. Increases in osteocyte apoptosis and RANKL production preceded increases in bone resorption at both endocortical and trabecular surfaces. QVD completely inhibited not only the HLU-triggered increases in osteocyte apoptosis but also RANKL production and activation of bone resorption at both sites. Finally, µCT studies revealed that apoptosis inhibition completely prevented the trabecular bone loss caused by HLU. Together these data indicate that osteocyte apoptosis plays a central and controlling role in triggering osteocyte RANKL production and the activation of new resorption leading to bone loss in disuse. © 2016 American Society for Bone and Mineral Research.

Abstract A08: A patient-derived xenograft model to study the effects of postmenopausal obesity on endocrine therapy resistance

Abstract Introduction: Obesity affects more than one-third of adults in the U.S. and increases the risk for and worsens outcomes of individuals with cancer. Obese women have a greater risk of postmenopausal hormone-receptor positive breast cancer and are more likely to relapse after endocrine therapy. The prevailing dogma is that elevated circulating estrogen, produced by aromatase in abundant adipose tissue, drives breast cancer formation and progression. While estrogen is clearly an important factor in the obesity-breast cancer relationship, increasing the dose of aromatase inhibitors provides no added benefit to obese women with breast cancer. A switch from hormone- to growth factor-dependence has been implicated in the development of endocrine therapy resistance. Hypothesis and Study Objective: We hypothesized that obese, postmenopausal women have elevated growth factor production in adipose tissue, which supports the progression of breast tumors that were previously dependent on estrogen. The purpose of our study was to develop a transplant-competent mouse model of obesity and postmenopausal breast cancer using estrogen receptor (ER) positive breast cancer patient-derived xenografts (PDX) from peri- and postmenopausal women (ages 45-71). Methods: Rag1-null mice on the C57Bl/6 background were fed either a low fat (LFD) or high fat (HFD) diet for 10 weeks. Thermoneutral housing was used to facilitate development of the obese phenotype. Animal weights and percent body fat were monitored. After weight separation (approximately 10 weeks), mice were ovariectomized and implanted with an ER+ estrogen-dependent PDX derived from a 54 year old woman bilaterally into the orthotopic site (inguinal mammary fat pad). Mice were given slow release estrogen pellets and tumor growth was monitored. After 8 weeks of tumor growth, a subset of animals received estrogen withdrawal (EWD) to mimic the menopausal transition. Results: HFD-fed mice weighed more, and had significantly increased body fat percentages, as well as subcutaneous, and gonadal fat pad weights, compared to LFD-fed mice. After EWD, tumors regressed in LFD-fed mice; however, the HFD prevented tumor regression in the absence of estrogen. Circulating estrogen levels in both LFD and HFD EWD groups were not significantly different. Tumor cellularity and Ki67 were greater in HFD- compared to LFD-fed mice in the absence of estrogen. Phospho-protein analysis of tumors suggested increased human epidermal growth factor receptor 2 (HER2) and fibroblast growth factor receptor (FGFR) signaling in HFD-EWD versus LFD-EWD mice. Conclusions: We have developed a novel, transplant-competent mouse model of obesity and menopause using a specialized HFD and thermoneutral housing. Using this model, we determined that HFD supports the growth of a previously estrogen-dependent breast cancer PDX after EWD, potentially through increased signaling through HER2 and FGFR pathways. Our mouse model, in combination with PDX breast tumors, will offer novel insights into the relationships between obesity, menopause, and ER+ breast cancer progression. Citation Format: Elizabeth Wellberg, Britta Jacobsen, Peter Kabos, Paul MacLean, Carol Sartorius. A patient-derived xenograft model to study the effects of postmenopausal obesity on endocrine therapy resistance. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr A08.

Preclinical studies and clinical evaluation of compounds from the genus Epimedium for osteoporosis and bone health.

The morbidity and mortality associated with fractures due to osteoporosis or "porous bone" contributes significantly to global healthcare costs and will increase exponentially with ageing populations. In menopausal women, the onset of menopause and rapid estrogen withdrawal leads to osteoporotic fractures. Healthy bone requires the coordinated remodeling function of osteoclasts, osteoblasts, and osteocytes in the basic bone multicellular unit, regulated by estrogen, RANKL/OPG, ROS, growth factors, and other kinase signaling pathways. Anti-osteoporotic drugs in current use such as hormone replacement therapy, selective estrogen receptor modulators, and bisphosphonates are designed to target these pathways, but all have their limitations. Extracts of the dried aerial parts of the traditional Chinese medicinal plant Epimedium (Berberidaceae) has long been used for bone health. Some nine Epimedium prenylflavonoid compounds have been reported to target estrogen signaling and other bone morphogenesis pathways in mesenchymal stem cell, osteoblast, and osteoclast cell lineages. Epimedium prenylflavonoids and enriched extracts can exert beneficial effects on bone health in estrogen-deficient and other osteoporosis animal models. The development of sensitive and rapid mass chromatographic techniques to quantify compounds extracted from Epimedium, including icariin and icaritin, has been used to standardize production and to study the pharmacokinetics and metabolism of Epimedium in animal models and humans. Recent clinical trials have reported positive effects on bone health, suggesting that compounds or extracts of Epimedium have the potential to be developed as agents, alone or in combination with other drugs, to prevent or delay the onset of osteoporosis and reduce the risk of hip fractures.

Expression and functional roles of estrogen receptor GPR30 in human intervertebral disc

Estrogen withdrawal, a characteristic of female aging, is associated with age-related intervertebral disc (IVD) degeneration. The function of estrogen is mediated by two classic nuclear receptors, estrogen receptor (ER)-α and -β, and a membrane bound G-protein-coupled receptor 30 (GPR30). To date, the expression and function of GPR30 in human spine is poorly understood. This study aimed to evaluate GPR30 expression in IVD, and its role in estrogen-related regulation of proliferation and apoptosis of disc nucleus pulposus (NP) cells. GPR30 expression was examined in 30 human adult NP and 9 fetal IVD. Results showed that GPR30 was expressed in NP cells at both mRNA and protein levels. In human fetal IVD, GPR30 protein was expressed in the NP at 12–14 weeks gestation, but was undetectable at 8–11 weeks. The effect of 17β-estradiol (E2) on GPR30-mediated proliferation and interleukin-1β (IL-1β)-induced apoptosis of NP cells was investigated. Cultured NP cells were treated with or without E2, GPR30 antagonist G36, and ER antagonist ICI 182,780. NP cell viability was tested by MTS assay. Apoptosis was determined by flow cytometry using fluorescence labeled annexin-V, TUNEL assay and immumnocytochemical staining of activated caspase-3. E2 enhanced cell proliferation and prevented IL-1β-induced cell death, but the effect was partially blocked by G36 and completely abrogated by a combination of ICI 182,780 and G36. This study demonstrates that GPR30 is expressed in human IVD to transmit signals triggering E2-induced NP cell proliferation and protecting against IL-1β-induced apoptosis. The effects of E2 on NP cells require both GPR30 and classic estrogen receptors.

Postpartum estrogen withdrawal impairs hippocampal neurogenesis and causes depression- and anxiety-like behaviors in mice

Postpartum estrogen withdrawal is known to be a particularly vulnerable time for depressive symptoms. Ovariectomized adult mice (OVX-mice) treated with hormone-simulated pregnancy (HSP mice) followed by a subsequent estradiol benzoate (EB) withdrawal (EW mice) exhibited depression- and anxiety-like behaviors, as assessed by forced swim, tail suspension and elevated plus-maze, while HSP mice, OVX mice or EB-treated OVX mice (OVX/EB mice) did not. The survival and neurite growth of newborn neurons in hippocampal dentate gyrus were examined on day 5 after EW. Compared with controls, the numbers of 28-day-old BrdU+ and BrdU+/NeuN+ cells were increased in HSP mice but significantly decreased in EW mice; the numbers of 10-day-old BrdU+ cells were increased in HSP mice and OVX/EB mice; and the density of DCX+ fibers was reduced in EW mice and OVX mice. The phosphorylation of hippocampal NMDA receptor (NMDAr) NR2B subunit or Src was increased in HSP mice but decreased in EW mice. NMDAr agonist NMDA prevented the loss of 28-day-old BrdU+ cells and the depression- and anxiety-like behaviors in EW mice. NR2B inhibitor Ro25-6981 or Src inhibitor dasatinib caused depression- and anxiety-like behaviors in HSP mice with the reduction of 28-day-old BrdU+ cells. The hippocampal BDNF levels were reduced in EW mice and OVX mice. TrkB receptor inhibitor K252a reduced the density of DCX+ fibers in HSP mice without the reduction of 28-day-old BrdU+ cells, or the production of affective disorder. Collectively, these results indicate that postpartum estrogen withdrawal impairs hippocampal neurogenesis in mice that show depression- and anxiety-like behaviors.

Decreased bioavailability of nitric oxide in aorta from ovariectomized senescent mice. Role of cyclooxygenase

This study investigates the effects of aging and/or ovariectomy on vascular reactivity to thromboxane A2 (TXA2) receptor stimulation with U46619, and the modulation by nitric oxide (NO) and cyclooxygenase (COX) in aorta from female senescence-accelerated mice (SAMP8) and from senescence resistant mice (SAMR1). Five-month-old female SAMR1 and SAMP8 were divided into three groups: sham-operated, ovariectomized and ovariectomized plus estradiol. Twenty-eight days after surgery, thoracic aortic rings were mounted for isometric recording of tension and concentration–response curves for U46619 (10−10–3×10−7M) were performed in the absence and in the presence of the NO synthase inhibitor NG-nitro-l-arginine methyl ester (L-NAME, 10−4M) and/or COX inhibitor indomethacin (10−5M). Vascular superoxide production was detected by dihydroethidium staining on sections of thoracic aorta. NO bioavailability in response to U46619 was suppressed by estrogen withdrawn in young and senescent mice and was restored by the administration of estradiol. In the presence of indomethacin, contractions to U46619 decreased in all groups indicating an aging- and estrogen-dependent modulation of contractile prostanoids. The simultaneous incubation of L-NAME and indomethacin did not change the maximal responses and sensitivities to TXA2 in any group in comparison with untreated aortic segments. The superoxide generation induced by TXA2 was greater in aorta from SAMP8 than in SAMR1. Moreover, in ovariectomized groups superoxide production was further increased and treatment with 17β-estradiol reverted the effects of the ovariectomy. Inhibition of COX with indomethacin prevented the U46619-induced increase in superoxide formation. Our results indicate that NO bioavailability in response to TP receptor activation is both estrogen- and aging-dependent. TXA2 induced contractions are partially mediated by COX activation. Both aging and ovariectomy enhanced COX-dependent component of the TXA2-induced contraction. It is noteworthy that in the absence of estrogen, COX inhibition induces an increase of NO bioavailability. Therefore, in senescent female mice with an experimental menopause, TP-receptor stimulation is responsible for COX activation and enhanced superoxide generation, which may result in reduced NO bioavailability. These effects were reversed by estrogen administration.

Dynamics of hepatic and intestinal cholesterol and bile acid pathways: The impact of the animal model of estrogen deficiency and exercise training.

Plasma cholesterol level is determined by a complex dynamics that involves transport lipoproteins which levels are tightly dependent on how the liver and the intestine regulate cholesterol and biliary acid metabolism. Regulation of cholesterol and biliary acids by the liver and the intestine is in turn coupled to a large array of enzymes and transporters that largely influence the inflow and the outflow of cholesterol and biliary acids through these organs. The activity of the key regulators of cholesterol and biliary acids may be influenced by several external factors such as pharmacological drugs and the nutritional status. In recent years, more information has been gathered about the impact of estrogens on regulation of cholesterol in the body. Exposure to high levels of estrogens has been reported to promote cholesterol gallstone formation and women are twice as likely as men to develop cholesterol gallstones. The impact of estrogen withdrawal, such as experienced by menopausal women, is therefore of importance and more information on how the absence of estrogens influence cholesterol regulation is started to come out, especially through the use of animal models. An interesting alternative to metabolic deterioration due to estrogen deficiency is exercise training. The present review is intended to summarize the present information that links key regulators of cholesterol and biliary acid pathways in liver and intestine to the absence of estrogens in an animal model and to discuss the potential role of exercise training as an alternative.

Neutrophils Derived from Ezh2 -/- Progenitor Cells Demonstrate Aberrant Erythroid Lineage Gene Expression

Abstract Introduction: Ezh2 is the catalytic component of the polycomb repressive complex 2, which methylates lysine 27 of histone H3 (H3K27). Loss of function mutations in EZH2 are found in 6% of MDS patients and are independently associated with worse overall survival compared to patients with wildtype EZH2 (Bejar R. et al., 2011 and 2012). Our group has described that neutrophils derived from Ezh2-/- mice have functional defects (Perez-Ladaga et al., 2013), including decreased phagocytosis, aberrant migration and overproduction of reactive oxygen species (ROS). To determine how loss of Ezh2 might contribute to these functional deficits, we performed gene expression profiling on immortalized myeloid cell lines capable of neutrophilic differentiation. Methods: Bone marrow from Ezh2 null (Ezh2-/-) and littermate control mice (WT) were transduced with HOXB8 fused to the estrogen receptor ligand-binding domain to produce immortalized myeloid progenitor cells. Removal of estrogen from the media allows these cells differentiate into mature neutrophils (Wang G.G., 2006). RNA from progenitor and mature neutrophils (WT and Ezh2-/-) was extracted each condition in duplicate and subjected to gene expression profile (Affymetrix). Transcriptome analysis was conducted with TAC software from Affymetrix and gene set comparisons between the different phenotypes were analyzed with Gene Set Enrichment Analysis (GSEA). Rescue by lentiviral re-introduction of Ezh2 into Ezh2-/- cells is currently ongoing. Results: Estrogen withdrawal causes differentiation of WT and Ezh2-/- lines into mature neutrophils after six days. Interestingly, WT neutrophils lose Ezh2 mRNA and protein expression as soon as three days after estrogen withdrawal. WT mature neutrophils lack Ezh2 and trimethyl-H3K27 (me3H3K27), showing similar amounts as Ezh2-/- derived neutrophils. Gene expression profiling of 65956 transcripts demonstrated that 1953 of them were differentially expressed between WT and Ezh2-/- mature neutrophils. Nearly 65% of these genes were upregulated in Ezh2-/- derived neutrophils when compared to WT. As Ezh2 levels in mature neutrophils are similar in both conditions, gene expression differences are likely due to EZH2 and me3H3K27 differences in the progenitor state. Among the differentially expressed genes, the transcription factor GATA1 was found upregulated in Ezh2-/- derived neutrophils, a result confirmed by qPCR. GATA1 regulates the expression of hundreds of genes and is essential for erythropoiesis. GATA1 target erythroid genes were also found upregulated in Ezh2-/- derived neutrophils when compared to WT, while no significant differences in neutrophil gene expression were detected. Similarly, GSEA analysis of Ezh2-/- vs. WT confirmed strong enrichment for erythroid associated expression programs. A Heme Metabolism Signature based on a panel of 182 genes showed a strong correlation with Ezh2-/- derived neutrophils (Figure 1A). GSEA was used to examine possible mechanisms behind the functional defects previously reported in Ezh2-/- derived neutrophils such as overproduction of ROS and impaired migration. A gene set based on 192 genes encoding proteins involved in oxidative phosphorylation demonstrated a significant correlation between this pathway signature and Ezh2-/- derived neutrophils (Figure 1B).On the other hand, GSEA showed a positive correlation between WT differentiated neutrophils and a panel of 115 genes involved in leukocyte transendothelial migration (Figure 1C). Conclusion: Our results show that HOXB8-ER immortalized myeloid progenitor cells are able to produce mature neutrophils even in absence of Ezh2. The loss of Ezh2 in myeloid progenitor cells is associated with the differential expression of 1953 genes in mature neutrophils, including the upregulation of genes involved in erythroid differentiation programs and oxidative phosphorylation, and the downregulation of genes involved in leukocyte migration. Ongoing rescue experiments re-introducing Ezh2 into Ezh2-/- progenitor cells are being performed to determine if this restores normal neutrophil functions and silences the aberrant erythroid gene expression in Ezh2-/- derived neutrophils. Our findings may help explain how Ezh2 loss causes neutrophil dysfunction and contributes to the adverse prognosis associated with EZH2 mutations in MDS patients. Disclosures Orkin: Editas Inc.: Consultancy. Ebert:genoptix: Consultancy, Patents &amp; Royalties; Celgene: Consultancy; H3 Biomedicine: Consultancy. Bejar:Alexion: Other: ad hoc advisory board; Celgene: Consultancy, Honoraria; Genoptix Medical Laboratory: Consultancy, Honoraria, Patents &amp; Royalties: MDS prognostic gene signature.

Missense Mutations in LRP5 Associated with High Bone Mass Protect the Mouse Skeleton from Disuse- and Ovariectomy-Induced Osteopenia.

The low density lipoprotein receptor-related protein-5 (LRP5), a co-receptor in the Wnt signaling pathway, modulates bone mass in humans and in mice. Lrp5 knock-out mice have severely impaired responsiveness to mechanical stimulation whereas Lrp5 gain-of-function knock-in and transgenic mice have enhanced responsiveness to mechanical stimulation. Those observations highlight the importance of Lrp5 protein in bone cell mechanotransduction. It is unclear if and how high bone mass-causing (HBM) point mutations in Lrp5 alter the bone-wasting effects of mechanical disuse. To address this issue we explored the skeletal effects of mechanical disuse using two models, tail suspension and Botulinum toxin-induced muscle paralysis, in two different Lrp5 HBM knock-in mouse models. A separate experiment employing estrogen withdrawal-induced bone loss by ovariectomy was also conducted as a control. Both disuse stimuli induced significant bone loss in WT mice, but Lrp5 A214V and G171V were partially or fully protected from the bone loss that normally results from disuse. Trabecular bone parameters among HBM mice were significantly affected by disuse in both models, but these data are consistent with DEXA data showing a failure to continue growing in HBM mice, rather than a loss of pre-existing bone. Ovariectomy in Lrp5 HBM mice resulted in similar protection from catabolism as was observed for the disuse experiments. In conclusion, the Lrp5 HBM alleles offer significant protection from the resorptive effects of disuse and from estrogen withdrawal, and consequently, present a potential mechanism to mimic with pharmaceutical intervention to protect against various bone-wasting stimuli.

Neoflavonoid dalbergiphenol from heartwood of Dalbergia sissoo acts as bone savior in an estrogen withdrawal model for osteoporosis.

Dalbergiphenol (DGP) is a neoflavonoid isolated from heartwood of Dalbergia sissoo. Effects of DGP on skeletal health remain to be elucidated. The objective of the present study was to investigate the biological effects of DGP on bone loss in ovariectomized mice. Adult BALB/c mice were ovariectomized and administered DGP (1 and 5 mg/kg/d) or 17β-estradiol (E2) orally for 6 weeks. The sham group and the ovariectomy (OVX) + vehicle group served as controls. Eight female BALB/c mice were taken for each group. Uterine estrogenicity, bone microarchitecture, biomechanical strength, new bone formation (based on bone formation rate and mineral apposition rate), and skeletal expression of osteogenic and resorptive gene markers were studied. OVX resulted in a marked increase in body weight and a decrease in femoral and vertebral trabecular bone volume that were prevented by DGP or E2 treatment. DGP treatment increased bone biomechanical strength and new bone formation rate in ovariectomized mice, comparable with E2 treatment. However, increase in uterine weight and estrogenicity were observed in E2-treated ovariectomized mice, but not in response to DGP treatment. Treatment with DGP increased messenger RNA expression of runt-related transcription factor 2, osterix, and collagen type I, and decreased messenger RNA expression of tartrate-resistant acid phosphatase and the osteoprotegerin-to-receptor activator of nuclear factor-κB ligand ratio in the femur of ovariectomized mice. Overall findings suggest that DGP treatment can effectively prevent OVX-induced increase in bone loss and decrease in bone strength possibly by increasing osteoblastic activities and by decreasing osteoclastic activities.

Wnt16 Is Associated with Age-Related Bone Loss and Estrogen Withdrawal in Murine Bone.

Genome Wide Association Studies suggest that Wnt16 is an important contributor to the mechanisms controlling bone mineral density, cortical thickness, bone strength and ultimately fracture risk. Wnt16 acts on osteoblasts and osteoclasts and, in cortical bone, is predominantly derived from osteoblasts. This led us to hypothesize that low bone mass would be associated with low levels of Wnt16 expression and that Wnt16 expression would be increased by anabolic factors, including mechanical loading. We therefore investigated Wnt16 expression in the context of ageing, mechanical loading and unloading, estrogen deficiency and replacement, and estrogen receptor α (ERα) depletion. Quantitative real time PCR showed that Wnt16 mRNA expression was lower in cortical bone and marrow of aged compared to young female mice. Neither increased nor decreased (by disuse) mechanical loading altered Wnt16 expression in young female mice, although Wnt16 expression was decreased following ovariectomy. Both 17β-estradiol and the Selective Estrogen Receptor Modulator Tamoxifen increased Wnt16 expression relative to ovariectomy. Wnt16 and ERβ expression were increased in female ERα-/- mice when compared to Wild Type. We also addressed potential effects of gender on Wnt16 expression and while the expression was lower in the cortical bone of aged males as in females, it was higher in male bone marrow of aged mice compared to young. In the kidney, which we used as a non-bone reference tissue, Wnt16 expression was unaffected by age in either males or females. In summary, age, and its associated bone loss, is associated with low levels of Wnt16 expression whereas bone loss associated with disuse has no effect on Wnt16 expression. In the artificially loaded mouse tibia we observed no loading-related up-regulation of Wnt16 expression but provide evidence that its expression is influenced by estrogen receptor signaling. These findings suggest that while Wnt16 is not an obligatory contributor to regulation of bone mass per se, it potentially plays a role in influencing pathways associated with regulation of bone mass during ageing and estrogen withdrawal.