High-dose E2 Research Articles

Roles of toll-like receptor 4 and immunomodulatory hormones in bovine endometritis

The aim of the study is to explore the role of Toll-like receptor 4 (TLR4) and immunomodulatory hormones in bovine endometritis. The bovine endometrial epithelial cell model (BenEpC) was used in the study. After contamination by Escherichia coli, BenEpCs were randomly divided into several groups according to the different concentration of 17-β estradiol (E2) treatment and the different stimulation, lipopolysaccharide (LPS) or lipoteichoic acid (LTA). The expressions of TLR4 and COX-2 mRNA were determined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Prostaglandin F (PGF) and prostaglandin E (PGE) levels were evaluated by radioimmunoassay. A significant difference was observed in TLR4 mRNA expression of bacterial contaminated BEnEpCs among low, middle and high-dose E2 treatment group (p < 0.05). Meanwhile, there was also a statistical difference in the relative changes of theprostaglandin E2 (PGE2) and COX-2 mRNA expression in BEnEpCs among low, middle and high-dose E2 treatment group (p < 0.05). Furthermore, the most significant increase of PGE2and Cyclooxygenase-2 (COX-2) mRNA expression was achieved in the LPS-stimulated BEnEpCs compared with those stimulated with LTA. Our findings suggested that TLR4 was activated in bovine endometritis, and then led to more prostaglandin production as regulation of local inflammatory response. However, this response was dependent on and influenced by the immunomodulatory hormones. Key words: Bovine endometritis, lipopolysaccharide, toll-like receptor 4, 17-β estradiol.

Increased fat deposition in injured skeletal muscle is regulated by sex-specific hormones

Sex differences in skeletal muscle regeneration are controversial; comparisons of regenerative events between sexes have not been rigorously defined in severe injury models. We comprehensively quantified inflammation and muscle regeneration between sexes and manipulated sex-specific hormones to determine effects on regeneration. Cardiotoxin injury was induced in intact, castrated and ovariectomized female and male mice; ovariectomized mice were replaced with low- or high-dose 17-β estradiol (E(2)) or progesterone (P4). Extent of injury was comparable between intact mice, but females were more efficient in removal of necrotic debris, despite similar tissue levels of inflammatory cells and chemokines. Myofiber size during regeneration was equivalent between intact mice and after castration or ovariectomy (OVX) but was decreased (P < 0.001) in ovariectomized mice with high-dose E(2) replacement. Intermuscular adipocytes were absent in uninjured muscle, whereas adipocyte area was increased among regenerated myofibers in all groups. Interestingly, intermuscular fat was greater (P = 0.03) in intact females at day 14 compared with intact males. Furthermore, castration increased (P = 0.01) and OVX decreased adipocyte accumulation. After OVX, E(2), but not P4, replacement decreased (P ≤ 0.03) fat accumulation. In conclusion, sex-dependent differences in regeneration consisted of more efficient removal of necrosis and increased fat deposition in females with similar injury, inflammation, and regenerated myofiber size; high-dose E(2) decreased myofiber size and fat deposition. Adipocyte accumulation in regenerating muscle was influenced by sex-specific hormones. Recovery following muscle injury was different between males and females, and sex-specific hormones contributed to these differences, suggesting that sex-specific treatments could be beneficial after injury.

Prevention of menstrual migraine with perimenstrual transdermal 17-β-estradiol: a randomized, placebo-controlled, double-blind crossover study

The effect of treatment with percutaneous E(2) (100 μg/24 h) during 2 weeks perimenstrually on the number and severity of menstrual migraine attacks was studied in 27 women in a randomized, placebo-controlled, double-blind, crossover trial. We were not able to demonstrate any difference between E(2) supplementation and placebo on the number or severity of migraine attacks, but both regimens showed significant effects compared with before treatment. CLINICAL TRIAL IDENTIFICATION NUMBER: NCT00204074.

Effects of short-term hormonal replacement on learning and on basal forebrain ChAT and TrkA content in ovariectomized rats

It has been proposed that sex steroid hormones improve performance in some cognitive tasks by regulating the basal forebrain cholinergic function. However, the molecular basis of such influence still remains unknown. Current study analyzed the performance of ovariectomized rats in an autoshaping learning task after a short-term treatment with 17β-estradiol (E2: 4 and 40 μg/kg) and/or progesterone (P4: 4 mg/kg). These results were correlated with basal forebrain choline acetyltransferase (ChAT) and TrkA protein content. The high dose of E2 enhanced both acquisition in the autoshaping task and the content of ChAT and TrkA. P4 treatment increased ChAT and TrkA content without affecting performance of rats in the autoshaping learning task. Interestingly, the continuous and simultaneous administration of E2 plus P4 did not significantly modify behavioral and biochemical evaluated parameters. These results address the influence of both E2 and P4 on cholinergic and TrkA activity and suggest that the effects of ovarian hormones on cognitive performance involve basal forebrain cholinergic neurons.

The effect of oestradiol-17β on the motility, viability and the acrosomal status of bull sperm

The aim of this study was to test the effect of oestradiol-17β (E2) on the motility, viability and the acrosomal status of bull sperm in vitro. Pooled semen from Holstein bulls were incubated in the presence of 2, 4, and 8 μg E2/mL for 24 h. Semen was also incubated in media without E2. During the incubation, the number of motile, viable sperm and the number of sperm possessing lost/damaged acrosomes, cytoplasmic droplets and coiled tails were counted at 0, 4, 18 and 24 h of incubation. Addition of 2 μg E2/mL at 18 h of incubation increased the total motility over the control. The number of forward progressing sperm was increased by the supplementation of 2 and 8 μg E2/mL over the control group at the 4 h incubation. Lower doses of E2 (2 and 4 μg/mL) did not affect viability of sperm, but a high dose of E2 (8 μg/mL) caused reductions in viability at 4 and 24 h of incubation. The number of sperm cells with lost acrosomes was significantly high in control group at 24 h of incubation. The number of sperm cells possessing proximal and distal cytoplasmic droplets and the number of sperm cells bearing coiled tails were not altered by any of the treatments. A small dose of E2 (2 μg/mL) had a beneficial effect on the motility and acrosome integrity of bull sperm in vitro. Higher dose of E2 (8 μg/mL) had a detrimental effect on viability. Keywords: Bovine males, estradiol, acrosome, motility, spermatozoa, in vitro incubation

2-(4-Hydroxyphenyl)-Benzo[b]thiophen-6-ol, an Estrogen-Like Compound, Induces Apoptosis in T47D/PKCα Breast Cancer Cells.

Abstract Background: Tamoxifen (TAM) treatment failure is a major obstacle encountered in the clinical setting. Our lab has previously shown that constitutive overexpression of PKCα imparts a TAM resistant/ hormone independent phenotype in the T47D:A18 breast cancer cell line. Furthermore 17β-estradiol (E2) inhibits tumor growth in vivo as well as inhibits colony formation when cells are grown in 3D Matrigel (Zhang, 2009). Before TAM treatment was introduced, breast cancer patients received high-dose E2 and diesthystilbesterol (DES) treatment. DES treated patients may have a survival advantage over TAM treated patients in the long term (Peethambaram,1999). Others have shown that E2 can have inhibitory effects on MCF-7 cells both in vitro and in vivo (Lewis-Wambi, 2009). Taken together, this suggests an estrogenic compound may be efficacious for TAM-resistant breast cancer. The present study was designed to evaluate the effect of an estrogenic compound, 2-(4-hydroxyphenyl)-benzo[b]thiophen-6-ol (BTC), related to the SERMs raloxifene and arzoxifene, on the T47D:A18/PKCα breast cancer cell line.Methods: To determine if BTC can activate estrogen regulated genes we preformed an ERE-luciferase reporter assay 24 hours after BTC treatment. 3D Matrigel colony formation assay was performed to determine if BTC could inhibit colony formation. Colonies were stained and counted on day 10. To determine if apoptosis was occurring, cells were grown in Matrigel and the TUNEL Assay was performed on Day 6.Results: T47D:A18/PKCα cells showed a 31-fold induction in ERE-luciferase reporter activity when treated with BTC (10-7M) compared to 7-fold induction when treated with E2. In the T47D:A18/Neo parental cell line, BTC induced a 40-fold induction whereas estradiol induced a 96-fold induction. In the Matrigel colony formation assay T47D:A18/PKCα cells formed significantly fewer colonies when treated with BTC. Conversely, in the T47D:A18/Neo parental cell line BTC enhanced colony formation similar to E2 treatment. Our results indicate that BTC was able to induce significant apoptosis on day 6 (P= 0.038) as determined by the TUNEL Assay. Therefore BTC acts similarly to estradiol by inhibiting T47D:A18/PKCα colony formation and inducing apoptosis when cells are grown in Matrigel.Conclusion: These findings suggest that BTC is a potential lead compound in the treatment of PKCα overexpressing breast cancer. Furthermore, BTC was shown to be a potent inducer of the cytoprotective enzyme NQO1 in the Hepa 1c1c7 cell system as well as a strong activator of antioxidant responsive elements (Yu, 2007) indicating it may have the added benefit of chemoprotective effects which are absent in estradiol treatment. Further understanding of the mechanism in which BTC induces apoptosis in the T47D:A18/PKCα cell line may result in a clinical advantage to estradiol treatment including fewer side effects. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5140.

The effects of 17β-estradiol on various reproductive parameters in the hermaphrodite fish Kryptolebias marmoratus

The effect of a single injection of 17β-estradiol (E2) was evaluated in the hermaphrodite fish Kryptolebias marmoratus. The fish [average body weight (BW), 0.15 ± 0.01 g] were injected with either two concentrations of E2 (1 and 100 μg/g BW) once intraperitoneally. They were sampled at intervals of 7, 15, and 30 days after a single E2 injection. Gonadosomatic index (GSI), hepatosomatic index (HSI), the frequency of gonadal development, number of ovulated eggs, and plasma steroids levels were measured. The transcript abundances of vitellogenin (VTG) and estrogen receptors (ERα and β) mRNA in the liver were also analyzed using quantitative real time polymerase chain reaction (real time PCR). GSI and the frequency of mature oocytes in the 100-μg E2-exposed group decreased compared to that of the control group during the experiment, and the number of ovulated eggs in the 100-μg E2-exposed group was lower when compared to the other groups. However, plasma E2 and 11-ketotestosterone (11-KT) levels were not significantly different between the experimental groups. On the other hand, plasma testosterone level and VTG mRNA abundance in the 100-μg E2-exposed group were significantly lower than the control group after 30 days. These results indicate that E2 stimulation at high concentration interferes with reproductive phenomena through delayed response. In addition, HSI in the 100-μg E2-exposed group and ERα mRNA abundance in the 1-μg E2-exposed group were significantly higher than the control group at 7 days after E2 injection, although there was no significant difference in HSI and ERα mRNA between all groups at 30 days. These results indicate temporal responses in reproductive parameters following high-dose E2 exposure in the hermaphrodite fish K. marmoratus.

17β-Estradiol modulates local cardiac renin-angiotensin system to prevent cardiac remodeling in the DOCA-salt model of hypertension in rats

Ventricular remodeling can play a detrimental role in the progression of cardiovascular diseases, leading to heart failure. The current study was designed to investigate the effects of 17β-estradiol (E2) on cardiac remodeling. Cardiac fibrosis and hypertrophy were examined in deoxycorticosterone acetate (DOCA)-salt treated rats with chronic, six-week administration of two different doses of E2. Bilaterally ovariectomized (Ovex) female Sprague–Dawley rats were randomly assigned to one of the following groups: Ovex-control; Ovex-DOCA; Ovex-DOCA + low-dose E2 (1.66 μg/day); or Ovex-DOCA + high-dose E2 (2.38 μg/day). All DOCA-treated rats were uninephrectomized and drinking water was replaced by 0.15 M NaCl solution for the remainder of the study period. DOCA-salt treatment resulted in a significant increase in blood pressure, which was not altered by estrogen replacement. Histological examinations revealed marked cardiac remodeling (both ventricular hypertrophy and interstitial fibrosis) with DOCA treatment, which was attenuated in animals receiving estrogen therapy. Western blot analysis demonstrated increased cardiac levels of angiotensin converting enzyme (ACE) with DOCA treatment, which was attenuated by E2 replacement. Furthermore, increased levels of cardiac angiotensin converting enzyme 2 (ACE2) protein were observed in animals receiving high-dose E2 replacement. These findings suggest that physiologically relevant estrogen replacement therapy has blood pressure-independent cardioprotective effects, which are possibly mediated through modulation of the cardiac renin-angiotensin system.

Estradiol-Induced Regression in T47D:A18/PKCα Tumors Requires the Estrogen Receptor and Interaction with the Extracellular Matrix

Several breast cancer tumor models respond to estradiol (E(2)) by undergoing apoptosis, a phenomenon known to occur in clinical breast cancer. Before the application of tamoxifen as an endocrine therapy, high-dose E(2) or diethystilbesterol treatment was successfully used, albeit with unfavorable side effects. It is now recognized that such an approach may be a potential endocrine therapy option. We have explored the mechanism of E(2)-induced tumor regression in our T47D:A18/PKCalpha tumor model that exhibits autonomous growth, tamoxifen resistance, and E(2)-induced tumor regression. Fulvestrant, a selective estrogen receptor (ER) down-regulator, prevents T47D:A18/PKCalpha E(2)-induced tumor growth inhibition and regression when given before or after tumor establishment, respectively. Interestingly, E(2)-induced growth inhibition is only observed in vivo or when cells are grown in Matrigel but not in two-dimensional tissue culture, suggesting the requirement of the extracellular matrix. Tumor regression is accompanied by increased expression of the proapoptotic FasL/FasL ligand proteins and down-regulation of the prosurvival Akt pathway. Inhibition of colony formation in Matrigel by E(2) is accompanied by increased expression of FasL and short hairpin RNA knockdown partially reverses colony formation inhibition. Classic estrogen-responsive element-regulated transcription of pS2, PR, transforming growth factor-alpha, C3, and cathepsin D is independent of the inhibitory effects of E(2). A membrane-impermeable E(2)-BSA conjugate is capable of mediating growth inhibition, suggesting the involvement of a plasma membrane ER. We conclude that E(2)-induced T47D:A18/PKCalpha tumor regression requires participation of ER-alpha, the extracellular matrix, FasL/FasL ligand, and Akt pathways, allowing the opportunity to explore new predictive markers and therapeutic targets.

Low dose but not high dose of 17â‐estradiol prevents cardiac fibrosis in DOCA‐salt model of hypertension in rats

The current study was designed to investigate the effects of 17ß‐estradiol (E2) on cardiac remodeling. To address this issue, we evaluated cardiac fibrosis and hypertrophy in deoxycorticosterone acetate (DOCA)‐salt treated rats with chronic, six‐week administration of two different doses of E2. Female SD rats were ovariectomized bilaterally (Ovex). Hypertension was induced by subcutaneous implantation of DOCA‐pellet in uninephrectomized animals, which were later maintained on 1% saline. The rats were randomly assigned to one of the following groups: Ovex control; Ovex + DOCA; Ovex + DOCA + E2 (low dose); or Ovex + DOCA + E2 (high dose). Blood pressure measurement showed a significant increase in DOCA treated rats over a six week period, which was not altered by estrogen replacement. No difference in cardiac function was observed among the different groups when assessed using isolated rat heart Langendorff apparatus. Both doses of estrogen attenuated cardiac hypertrophy observed in the DOCA treated animals. However, when measuring cardiac fibrosis induced by DOCA treatment, only the low dose E2 therapy showed a protective effect. The high dose E2 therapy failed to exhibit anti‐fibrotic effects. These findings suggest that estrogen replacement therapy may have blood‐pressure independent cardio‐protective effects that are dose‐dependent.

Estradiol promotes pituitary cell proliferation and gonadotroph differentiation at different doses and with different mechanisms in chick embryo

Estrogens are known to play a role in the feedback regulation of pituitary gonadotropin secretion in adults. However, it is still unknown whether estrogens are involved in promoting pituitary development. In this study, we selected chick embryo as the animal model and microinjected different doses of estradiol (E2) at stage E27–28, which was when endogenous E2 was not detected. First, the results demonstrated that E2 at different doses promoted pituitary cell proliferation and gonadotroph differentiation. Lower doses of E2 had a more significant effect on cell proliferation, while higher doses of E2 were required for luteinizing hormone (LH) secreting cell differentiation. Furthermore, the levels of early growth response protein 1 (Egr-1) and GATA2 mRNAs were also elevated with E2 treatment at a higher dose than that required to increase the level of proliferating cell nuclear antigen (PCNA) in vitro. To investigate whether estrogen receptors (ERs) mediate these effects of estradiol, the ER antagonist ICI 182,780 was added, and the results showed that ICI 182,780 did not modify the enhancing effects of E2 on cell proliferation; however, it inhibited the stimulatory effect of E2 on LH secreting cell differentiation. These results suggest that E2 at different doses promotes pituitary cell proliferation and gonadotroph differentiation with different mechanisms. Our results are important to further understanding of the physiological and pharmacological functions and related mechanisms of estrogens and their receptors, although the related mechanism need to be elucidated in future studies.

Estradiol and Tamoxifen Mediate Rescue of the Dominant-Negative Effects of Estrogen Response Element-Binding Protein in Vivo and in Vitro

Biological responses to estrogens are dependent on the integrated actions of proteins, including the estrogen receptor (ER)-alpha, that regulate the transcription of estrogen response element (ERE)-containing target genes. We have identified a naturally occurring ERE antagonist, termed an ERE-binding protein (BP). To verify that ERE-BP can induce estradiol (E(2)) resistance in vivo, we generated transgenic mice that overexpress this protein in breast tissue. Female transgenic mice with high levels of ERE-BP were unable to lactate, and we hypothesized that this effect was dependent on the relative levels of ERE-BP and ERalpha ligand. To test this hypothesis, wild-type and ERE-BP-expressing female mice were implanted with capsules containing E(2), the selective estrogen receptor modulator tamoxifen, or placebo. Histological analysis of nonlactating mammary glands showed a 4.5-fold increase in gland branch number and 3.7-fold increase in ducts in ERE-BP mice treated with E(2) (7.5 mg, 21 d) compared with placebo-treated ERE-BP mice. Wild-type mice showed a 5.3-fold increase in branches and 1.4-fold increase in ducts under the same conditions. Similar results were obtained with tissue from lactating mice, in which tamoxifen also increased mammary gland branch number. Studies using ERE-BP-expressing MCF-7 breast cells showed that high doses of E(2) (1000 nM) restored normal ERalpha-chromatin interaction in these cells, whereas tamoxifen was able to achieve this effect at a dose of 10 nM. These data highlight the importance of ERE-BP as an attenuator of normal ERalpha signaling in vivo and further suggest that ERE-BP is a novel target for modulation by selective estrogen receptor modulators.

Dose‐response effect of estrogen on the kidney and heart

Recent clinical trials indicate that estrogen replacement therapy (ERT) may increase the risk of cardiovascular diseases. Here we studied whether the adverse effects of ERT are related to the doses used. Ovariectomized (OVX) mice received 17β‐estrodial (E2) at 0.001 (VL, very low), 0.42 (L, low), 4.2 (M, moderate) or 28.3 μg/d (H, high) for 60 days. E2 at moderate and high doses increased plasma estrogen levels by about 4.5‐fold. This was associated with ascites, nephrydrosis, proteinuria and fluid retention in uterus horns. Low dose of E2 restored plasma estrogen and uterus weight to levels similar to normal controls and no fluid retention or renal damage were found in this group of mice. Moderate and high doses of E2 also increased cardiac ANP mRNA expression and PKC and PI3K phospholyration. Aside from diastolic function, blood pressure and cardiac function were not altered by ERT at any given dose. These results suggest that ERT dosage could be an important determinant for a beneficial or detrimental outcome.

Sex change of adult initial-phase male wrasse, Halichoeres trimaculatus by estradiol-17β treatment

Sex steroids are considered major regulators of sex change processes in fish. Estrogen depletion is shown to be crucial for female–male sex change initiation; however, its role in male–female sex change is largely unknown. In the present study, we examined the effects of estradiol-17β (E2) treatments on testes of initial-phase (IP) males of the three-spot wrasse (Halichoeres trimaculatus), which naturally do not undergo male–female sex change. Sexually mature IP males were fed a diet containing E2 (low, 20μg/g feed; high, 200μg/g feed) for 6 or 12 weeks, and changes in gonadal structures were examined. Percentage of sex change varied with the dosage of E2 and the duration of treatment. All individuals treated with high-dose E2 for 6 weeks had ovaries with many immature oocytes; whereas 75% of individuals treated with low-dose of E2 for 6 weeks and sampled on the 12th week had ovaries with yolky oocytes and an ovarian cavity indicating a typical mature ovary. No testicular tissue was observed in sex-reversed gonads in both treatment groups. Contrary to the previous assumptions, present results suggest that IP male wrasses have the potential to undergo male–female sex change in response to exogenous estrogen. How the presence or absence of estrogen creates sexual plasticity in gonadal germ and somatic cells remains to be clarified.

Development of Primordial Follicles in the Hamster: Role of Estradiol-17β

The role of E2 on primordial follicle formation was examined by treating neonatal hamsters with 1 or 2 microg estradiol cypionate (ECP) at age postnatal d 1 (P1) and P4 or by in vitro culture of embryonic d 15 (E15) ovaries with 1, 5, or 10 ng/ml estradiol-17beta (E2). The specificity of E2 action was examined by ICI 182,780. One microgram of ECP maintained serum levels of E2 within the physiological range, significantly reduced apoptosis, and stimulated the formation and development of primordial follicles. In contrast, 2 microg ECP increased serum E2 levels to 400 pg/ml and had significantly less influence on primordial follicle formation. In vivo, ICI 182,780 significantly increased apoptosis and caused a modest reduction in primordial follicle formation. The formation and development of primordial follicles in vitro increased markedly with 1 ng/ml E2, and the effect was blocked by ICI 182,780. Higher doses of E2 had no effect on primordial follicle formation but significantly up-regulated apoptosis, which was blocked by ICI 182,780. CYP19A1 mRNA expression occurred by E13 and increased with the formation of primordial follicles. P4 ovaries synthesized E2 from testosterone, which increased further by FSH. Both testosterone and FSH maintained ovarian CYP19A1 mRNA, but FSH up-regulated the expression. These results suggest that neonatal hamster ovaries produce E2 under FSH control and that E2 action is essential for the survival and differentiation of somatic cells and the oocytes leading to the formation and development of primordial follicles. This supportive action of E2 is lost when hormone levels increase above a threshold.

17Beta-Estradiol Triggers Postspawning in Spermatogenically Active Gilthead Seabream (Sparus aurata L.) Males1

The testis is a tightly controlled dynamic tissue. In mammals, there is growing evidence that estrogen plays a role in the regulation of testicular functions. In teleosts, high levels of 17beta-estradiol (E2) in serum correlate with the end of spermatogenesis, spawning, and the initiation of postspawning stages when spermatogonia are the main cell types in the testis. Moreover, E2 modulates leukocyte functions in several teleost species. We hypothesized, therefore, that E2 would induce the infiltration of acidophilic granulocytes and cause a resumption of testicular cell proliferation in spermatogenically active gilthead seabream males. Several studies of this species have reported that supraphysiological doses of E2 are needed to induce histological and developmental changes in males. In fact, as gilthead seabream is a protandrous hermaphrodite teleost, long exposures (6-14 wk) to high doses of E2 result in feminization of the males. Taking all this into account, we sharply increased E2 levels during short times by i.p. injecting E2 diluted in coconut oil as the vehicle and sampled the fish after 7, 13, and 18 days to assess the effects that E2 had on spermatogenesis. It was observed that E2 levels in plasma increased, while 11-ketotestosterone (11-KT) and testosterone (T) levels remained unaltered. However, 11-KT and T levels strongly increased in control fish 18 days postinjection. The most relevant result of our study was that E2 accelerates the final events of spermatogenesis, inhibits the proliferation of spermatogonia in early stages, and induces some of the processes that usually occur during postspawning, such as the infiltration of acidophilic granulocytes and the apoptosis of primary spermatogonia. Strikingly, neither the shedding of spermatozoa nor an increase in the proliferative rate of spermatogonia stem cells was observed, probably because of the lack of other necessary stimuli, such as the increase in T levels that takes place during normal postspawning.

Vascular Endothelial Cadherin Regulates Vascular Permeability: Implications for Ovarian Hyperstimulation Syndrome

Ovarian hyperstimulation syndrome (OHSS) is an iatrogenic complication of treatment with fertility drugs. It is characterized by increased vascular permeability and simultaneous overexpression of vascular endothelial growth factor (VEGF) in ovarian cells. We tested the hypothesis that the endothelium and endothelial cell-to-cell junctions are downstream targets of VEGF during OHSS pathogenesis. We investigated the potential involvement of vascular endothelial (VE)-cadherin, an interendothelial adhesion molecule, in the capillary hyperpermeability in OHSS. Human endothelial cells from umbilical veins (HUVEC) were used as an in vitro model of OHSS. Cell cultures were treated with varying doses of estradiol (E2), human chorionic gonadotropin (hCG), VEGF, and antihuman VEGF antibodies, either alone or in combination, and the effect on VE-cadherin release was evaluated at different time points. Permeability assays were performed using fluoresceinisothiocyanate-labeled albumin, and actin filaments rearrangement was evaluated by fluorescent microscopy. Culturing of HUVEC with high doses of E2 produced no significant changes in VE-cadherin concentration, but hCG and VEGF produced a significant increase in VE-cadherin release. Time-course experiments showed that VE-cadherin was secreted 12 h after VEGF addition. Antihuman VEGF antibodies prevented these changes. Permeability assays demonstrated that, although E2 did not alter the arrangement of HUVEC in vitro, hCG and VEGF caused changes in the actin fibers indicative of increased capillary permeability. VEGF also induced an increase in paracellular permeability of HUVEC at the same doses used in the previous experiments. Adhesion molecules like VE-cadherin may play a role in the development and progression of increased capillary permeability in severe OHSS.

Dose and Temporal Pattern of Estrogen Exposure Determines Neuroprotective Outcome in Hippocampal Neurons: Therapeutic Implications

To address controversies of estrogen therapy, in vitro models of perimenopause and prevention vs. treatment modes of 17beta-estradiol (E(2)) exposure were developed and used to assess the neuroprotective efficacy of E(2) against beta-amyloid-1-42 (Abeta(1-42))-induced neurodegeneration in rat primary hippocampal neurons. Low E(2) (10 ng/ml) exposure exerted neuroprotection in each of the perimenopausal temporal patterns, acute, continuous, and intermittent. In contrast, high E(2) (200 ng/ml) was ineffective at inducing neuroprotection regardless of temporal pattern of exposure. Although high E(2) alone was not toxic, neurons treated with high-dose E(2) resulted in greater Abeta(1-42)-induced neurodegeneration. In prevention vs. treatment simulations, E(2) was most effective when present before and during Abeta(1-42) insult. In contrast, E(2) treatment after Abeta(1-42) exposure was ineffective in reversing Abeta-induced degeneration, and exacerbated Abeta(1-42)-induced cell death when administered after Abeta(1-42) insult. We sought to determine the mechanism by which high E(2) exacerbated Abeta(1-42)-induced neurodegeneration by investigating the impact of low vs. high E(2) on Abeta(1-42)-induced dysregulation of calcium homeostasis. Results of these analyses indicated that low E(2) significantly prevented Abeta(1-42)-induced rise in intracellular calcium, whereas high E(2) significantly increased intracellular calcium and did not prevent Abeta(1-42)-induced calcium dysregulation. Therapeutic benefit resulted only from low-dose E(2) exposure before, but not after, Abeta(1-42)-induced neurodegeneration. These data are relevant to impact of perimenopausal E(2) exposure on protection against neurodegenerative insults and the use of estrogen therapy to prevent vs. treat Alzheimer's disease. Furthermore, these data are consistent with a healthy cell bias of estrogen benefit.

Chronic Treatment with Physiological Doses of Estradiol Affects the GH-IGF-1 Axis and Fat Metabolism in Young and Middle-aged Ovariectomized Rats

Estrogen deficiency may be partly responsible for the metabolic syndrome and the condition may be reversible with hormone replacement therapy. However, after the heart and estrogen/progestin replacement study and the women's health initiative study the prospect of HRT on CVD has changed dramatically. As the pituitary and the liver are targets for estrogen action we assessed the effect of ovariectomy (OVX) and long-term (3 months) estradiol (E2) treatment by means of subcutaneously (s.c.) implanted silastic capsules on pituitary and liver function in young and middle-aged female rats. Our results demonstrate that triglyceride serum levels increased, whereas insulin-like growth factor-1 (IGF-1), high density lipoprotein cholesterol (HDL), and glucose levels decreased during the transition from young to middle-age. E2 treatment increased dose-independently growth hormone (GH) secretion. IGF-1 levels were increased upon OVX in middle-aged rats and the high dose of E2 decreased IGF-1 concentrations in both age groups. Cholesterol concentrations increased after OVX and were attenuated by E2 administration in middle-aged rats. Both, low density lipoprotein cholesterol (LDL) and HDL levels raised after castration and both parameters decreased in response to E2 in young and middle-aged rats. Glucose serum concentrations decreased after E2 treatment in all animals. Taken together, our results clearly demonstrate that the pituitary and the liver metabolism of middle-aged rats remain susceptible to the influence of OVX and E2 treatment.

Regulation of components of the brain and cardiac renin-angiotensin systems by 17β-estradiol after myocardial infarction in female rats

The present study tested the hypothesis that 17beta-estradiol (E2) inhibits increases in angiotensin-converting enzyme (ACE) and ANG II type 1 receptor (AT1R) in the brain and heart after myocardial infarction (MI) and, thereby, inhibits development of left ventricular (LV) dysfunction after MI. Age-matched female Wistar rats were treated as follows: 1) no surgery (ovary intact), 2) ovariectomy + subcutaneous vehicle treatment (OVX + Veh), or 3) OVX + subcutaneous administration of a high dose of E2 (OVX + high-E2). After 2 wk, rats were randomly assigned to coronary artery ligation (MI) and sham operation groups and studied after 3 wk. E2 status did not affect LV function in sham rats. At 2-3 wk after MI, impairment of LV function was similar across MI groups, as measured by echocardiography and direct LV catheterization. LV ACE mRNA abundance and activity were increased severalfold in all MI groups compared with respective sham animals and to similar levels across MI groups. In most brain nuclei, ACE and AT1R densities increased after MI. Unexpectedly, compared with the respective sham groups the relative increase was clearest (20-40%) in OVX + high-E2 MI rats, somewhat less (10-15%) in ovary-intact MI rats, and least (< 10-15%) in OVX + Veh MI rats. However, because in the sham group brain ACE and AT1R densities increased in the OVX + Veh rats and decreased in the OVX + high-E2 rats compared with the ovary-intact rats, actual ACE and AT1R densities in most brain nuclei were modestly higher (< 20%) in OVX + Veh MI rats than in the other two MI groups. Thus E2 does not inhibit upregulation of ACE in the LV after MI and amplifies the percent increases in ACE and AT1R densities in brain nuclei after MI, despite E2-induced downregulation in sham rats. Consistent with these minor variations in the tissue renin-angiotensin system, during the initial post-MI phase, E2 appears not to enhance or hinder the development of LV dysfunction.