Medicolegal Review: Essure Lawsuits and Legal Strategies Adverse to Gynecologists.

The minimally invasive Essure procedure for hysteroscopic sterilization is an ongoing target for litigation. Although efficacious, this device has been scrutinized by the US Food and Drug Administration (FDA) owing to alleged complications. Patients affected by these potential complications are filing lawsuits against Bayer, the manufacturer of Essure. Many of these lawsuits have been barred by preemption, a legal doctrine that limits what can be required of a product by state lawsuits once the FDA approves it; however, in the lawsuits that have been allowed to proceed, the manufacturer has used a legal strategy termed the "learned intermediary doctrine" in an effort to shift blame to the gynecologist to absolve itself of liability. The learned intermediary only requires that a manufacturer inform the gynecologist of the risks associated with the device, and the gynecologist, in turn, must notify the patients through adequate informed consent. To incorporate the necessary components of informed consent, a gynecologist should include what a reasonable practitioner would consider pertinent to the discussion, as well as what a prudent patient would want to know to make a treatment decision. This disclosure entails explaining the risks, benefits, and alternatives, which should be clearly documented in the medical records. Understanding the importance of proper documentation and the legal strategies used in suits will help gynecologists lessen liability exposure when using a medical device, such as Essure, that is being targeted for litigation.

Medicolegal Review of Methotrexate Administration to Viable Intrauterine Pregnancies.

Objective: To examine 3 legal cases in which physicians prescribed methotrexate to women with a viable intrauterine pregnancy, presumed to be ectopic, resulting in adverse fetal outcomes. Study Design: We conducted an electronic literature search for legal cases using the keywords "methotrexate" and "pregnancy" in the LexisNexis legal research engine as well as an Internet-wide search using the additional keyword "verdict." We manually searched the resultant list of identified cases and categorized the studies identified in the search by verdict, award amount, and outcome of the embryo exposed to methotrexate. Results: The monetary awards are typically greater when the embryo exposed to methotrexate lives and requires continuous medical and custodial care as compared to when the fetus dies in utero or shortly after birth. Conclusion: Physicians who, with all good intentions, prescribe methotrexate to women with a viable pregnancy, presumed to be ectopic, could find them-selves liable for an adverse fetal outcome. For the benefit of patients, their unborn offspring, and the liability exposure of the physician, it is important to be very cautious when prescribing methotrexate.

Postpartum Teenagers' Views on Providing Contraception in School-Based Health Clinics.

BACKGROUND: To determine characteristics of teen pregnancies in southeast Texas and the opinions of postpartum teenagers with regard to having contraceptive services available in high school clinics. METHODS: A cross-sectional study of postpartum teenagers interviewed during their hospital stay. RESULTS: Of 404 postpartum teenagers interviewed, 86% had unplanned pregnancies. Approximately 53% of respondents first had intercourse at less than 16 years of age. Of the 130 teenagers who had used contraception prior to pregnancy, 85% became pregnant because they were unable to visit the clinic to obtain a contraceptive refill or replacement. In multivariate modeling, factors associated with using contraceptives prior to pregnancy included black race (p < .001) and more than 1 previous pregnancy (p < .001). Variables associated with having an unplanned pregnancy included having discussed contraceptives at home or school (p = 0.049). Of the 404 postpartum teenagers surveyed, 223 (82%) were in favor of having contraceptive services offered in high school clinics. CONCLUSIONS: Contraceptive education is not sufficient to prevent teenage pregnancy. Increase in access is critical as teenagers with previous pregnancies were more likely to use contraception, likely due to their interaction with the medical community during the antecedent pregnancy. One possible solution is to bring contraceptive services to the teenagers, by offering them at school based health systems. A majority of teenagers surveyed in this study supported this proposal.

Competitive Binding Assay for the G-Protein-Coupled Receptor 30 (GPR30) or G-Protein-Coupled Estrogen Receptor (GPER).

The role of 2-methoxyestradiol is becoming a major area of investigation because of its therapeutic utility, though its mechanism is not fully explored. Recent studies have identified the G-protein-coupled receptor 30 (GPR30, GPER) as a high-affinity membrane receptor for 2-methoxyestradiol. However, studies aimed at establishing the binding affinities of steroid compounds for specific targets are difficult, as the tracers are highly lipophilic and often result in nonspecific binding in lipid-rich membrane preparations with low-level target receptor expression. 2-Methoxyestradiol binding studies are essential to elucidate the underlying effects of this novel estrogen metabolite and to validate its targets; therefore, this competitive receptor-binding assay protocol was developed in order to assess the membrane receptor binding and affinity of 2-methyoxyestradiol.

Enmeshed in Controversy: Use of Vaginal Mesh in the Current Medicolegal Environment.

UNLABELLED: Vaginal mesh has been a valuable tool in the treatment of stress urinary incontinence and pelvic organ prolapse. As our knowledge of the long-term outcomes and complications of this product has evolved, however, vaginal mesh has become the subject of legal scrutiny. Therefore, it is imperative that physicians understand pertinent litigation techniques to optimize their informed consent and documentation processes and protect themselves. OBJECTIVES: Our objective is to familiarize physicians who use vaginal mesh with how law suits involving transvaginal mesh are construed. We also describe the current medicolegal environment surrounding the use of these products. METHODS: The food and drug administration public safety communications, food and drug administration Manufacturer and User Facility Device Experience database, and LexisNexis legal search engine were used to review data relevant to current vaginal mesh litigation. This information was used to create a medicolegal review. RESULTS: Litigation involving transvaginal mesh follows 3 paths. The first consists of claims against the manufacture of transvaginal mesh with allegations, such as design defects, failure to warn, and misrepresentation. The second is a defensive legal strategy called the learned intermediary doctrine, used by manufacturers to shift liability from themselves to surgeons. The manufacturers claim that the duty to inform patients of potential complications lies with the surgeon. The third involves claims by patients against surgeons for lack of informed consent, alleging that they were not sufficiently informed of potential complications associated with transvaginal mesh before insertion. CONCLUSIONS: To lessen the liability, a surgeon using transvaginal mesh should inform patients of potential complications associated with the products and document informed consent in their medical records.

The BRCA1 tumor suppressor binds to inositol 1,4,5-trisphosphate receptors to stimulate apoptotic calcium release.

The inositol 1,4,5-trisphosphate receptor (IP3R) is a ubiquitously expressed endoplasmic reticulum (ER)-resident calcium channel. Calcium release mediated by IP3Rs influences many signaling pathways, including those regulating apoptosis. IP3R activity is regulated by protein-protein interactions, including binding to proto-oncogenes and tumor suppressors to regulate cell death. Here we show that the IP3R binds to the tumor suppressor BRCA1. BRCA1 binding directly sensitizes the IP3R to its ligand, IP3. BRCA1 is recruited to the ER during apoptosis in an IP3R-dependent manner, and, in addition, a pool of BRCA1 protein is constitutively associated with the ER under non-apoptotic conditions. This is likely mediated by a novel lipid binding activity of the first BRCA1 C terminus domain of BRCA1. These findings provide a mechanistic explanation by which BRCA1 can act as a proapoptotic protein.

Dystroglycan in human fetal membranes decreases in later gestation and with spontaneous membrane rupture.

BACKGROUND/AIMS: The transmembrane protein dystroglycan (DG) is known to anchor the cell membrane to the extracellular matrix, and is susceptible to cleavage by matrix metalloproteinases. This study tested the hypothesis that changes in DG abundance in fetal membranes (FM) occur late in gestation, with spontaneous rupture of membranes (SROM), and during labor. METHODS: FM were collected from a prospective cohort consisting of four groups of patients (term labor, term unlabored, preterm labor, and preterm unlabored). FM were subjected to immunohistochemical staining using antibodies specific for α- and ß-DG subunits, and staining intensity was graded by a blinded pathologist. RESULTS: α- and ß-DG staining was significantly decreased at term and after SROM (p < 0.05), but not in the presence of labor. CONCLUSIONS: Decreased DG intensity was seen in FM of patients at term and with SROM, but no change was observed with labor.

Simvastatin potently induces calcium-dependent apoptosis of human leiomyoma cells.

Statins are drugs commonly used for the treatment of high plasma cholesterol levels. Beyond these well known lipid-lowering properties, they possess broad-reaching effects in vivo, including antitumor effects. Statins inhibit the growth of multiple tumors. However, the mechanisms remain incompletely understood. Here we show that simvastatin inhibits the proliferation of human leiomyoma cells. This was associated with decreased mitogen-activated protein kinase signaling and multiple changes in cell cycle progression. Simvastatin potently stimulated leiomyoma cell apoptosis in a manner mechanistically dependent upon apoptotic calcium release from voltage-gated calcium channels. Therefore, simvastatin possesses antitumor effects that are dependent upon the apoptotic calcium release machinery.

Trends in the national distribution of laparoscopic hysterectomies from 2003 to 2010.

STUDY OBJECTIVE: The purpose of this analysis was to compare the trends in undergoing laparoscopic hysterectomy (versus abdominal or vaginal hysterectomy) based on patient age, race, median income and insurance type, from 2003 to 2010. DESIGN: Retrospective study (Canadian Task Force classification II-3). SETTING: National sample of hospital admissions after hysterectomy. PATIENTS: Health Cost and Utilization Project-Nationwide Inpatient Sample database was used to review records of women who underwent hysterectomy for either menorrhagia or leiomyoma from 2003-2010. INTERVENTION: The predicted probability of undergoing laparoscopic hysterectomy was determined for each year according to patient age, race, median income, and insurance type. The slopes of these values (i.e. the trend) was compared for each subgroup (i.e. black, white, Asian, etc.) in these categories. MAIN RESULTS: A total of 530, 154 cases were included in this study. Total number of hysterectomies decreased by 39% from 60,364 to 36,835 from 2003 to 2010. The percent of hysterectomies that were laparoscopic increased from 11% in 2003 to 29% in 2010. All groups analyzed experienced an increase in predicted probability of undergoing a laparoscopic hysterectomy. Of all women undergoing hysterectomy, the probability of undergoing a laparoscopic hysterectomy remained highest for women who were less than 35 years old, white, with the highest median income, and with private insurance from 2003-2010. The slope was significantly greater for (1) white females versus all other races analyzed (p<0.01), (2) females in the highest income quartile versus females in the lowest income quartile (p<0.01) and (3) females with private insurance versus females with Medicaid (p<0.01) or Medicare (p<0.01). CONCLUSIONS: There remains a gap in distribution of laparoscopic hysterectomies with regards to age, race, median income and insurance type that does not seem to be closing, despite the increased availability of laparoscopic hysterectomies.

2-methoxyestradiol binding of GPR30 down-regulates angiotensin AT(1) receptor.

Controlling angiotensin AT1 receptor function has been shown to be protective for many pathophysiological disorders. Although estrogen metabolite, 2-methoxyestradiol (2ME2) can down-regulate angiotensin AT1 receptor expression independently of nuclear receptors, no specific cellular targets have been identified. This study was focused on identification and validation of a cellular target responsible for 2ME2-mediated angiotensin AT1 receptor down-regulation in a continuously passaged rat liver epithelial cell line. Cell membranes were isolated and used to determine 2ME2 specific binding. Cell membranes exposed to [(3)H]2ME2 showed specific saturable binding, which was found to be pertussis toxin (PTx) sensitive. Under similar conditions, G-protein coupled receptor 30 (GPR30) agonist (G1) and antagonist (G15) inhibited 2ME2 specific binding. In these cells GPR30 was found localized to endoplasmic reticulum (ER) membranes. In intact cells, G1 down-regulated angiotensin AT1 receptor expression and this effect was reversed by G15. Furthermore, 2ME2 mediated activation of epidermal growth factor receptor (EGFR) followed by ERK1/2 phosphorylation, an essential signaling step in angiotensin AT1 receptor down-regulation, was abrogated by G15, suggesting that this signal is GPR30 dependent. Additionally, EGF was found to independently down-regulate angiotensin AT1 receptor in an ERK1/2-dependent manner. In summary, our results demonstrate for the first time that 2ME2 down-regulation of angiotensin AT1 receptor is dependent on ER membrane-associated GRP30. Moreover, this effect is facilitated by GPR30 dependent transactivation of EGFR and ERK1/2 phosphorylation. This study provides further understanding of the physiological significance of 2ME2 and its role in modulating angiotensin AT1 receptor expression.

Intervention to improve follow-up for abnormal Papanicolaou tests: a randomized clinical trial.

OBJECTIVE: To evaluate the effect of a theory-based, culturally targeted intervention on adherence to follow-up among low-income and minority women who experience an abnormal Pap test. METHOD: 5,049 women were enrolled and underwent Pap testing. Of these, 378 had an abnormal result and 341 (90%) were randomized to one of three groups to receive their results: Intervention (I): culturally targeted behavioral and normative beliefs + knowledge/skills + salience + environmental constraints/barriers counseling; Active Control (AC): nontargeted behavioral and normative beliefs + knowledge/skills + salience + environmental constraints/barriers counseling; or Standard Care Only (SCO). The primary outcome was attendance at the initial follow-up appointment. Secondary outcomes included delay in care, completion of care at 18 months, state anxiety (STAI Y-6), depressive symptoms (CES-D), and distress (CDDQ). Anxiety was assessed at enrollment, notification of results, and 7-14 days later with the CDDQ and CES-D. RESULTS: 299 women were included in intent-to-treat analyses. Adherence rates were 60% (I), 54% (AC), and 58% (SCO), p = .73. Completion rates were 39% (I) and 35% in the AC and SCO groups, p = .77. Delay in care (in days) was (M ± SD): 58 ± 75 (I), 69 ± 72 (AC), and 54 ± 75 (SCO), p = .75. Adherence was associated with higher anxiety at notification, p < .01 and delay < 90 days (vs. 90+) was associated with greater perceived personal responsibility, p < .05. Women not completing their care (vs. those who did) had higher CES-D scores at enrollment, p < .05. CONCLUSIONS: A theory-based, culturally targeted message was not more effective than a nontargeted message or standard care in improving behavior.

Disparities in use of laparoscopic hysterectomies: a nationwide analysis.

STUDY OBJECTIVE: To determine patient and hospital characteristics that were associated with undergoing laparoscopic hysterectomy compared with abdominal hysterectomy. DESIGN: Canadian Task Force Classification II-3. METHODS: In this retrospective cohort study, we analyzed the 2010 Healthcare Cost and Utilization Project Nationwide Inpatient Sample database. All women who underwent laparoscopic or abdominal hysterectomy for either menorrhagia or leiomyoma were included based on International Classification of Diseases, Ninth Revision coding. A linear model with binomial distribution and logit link function was used to determine patient and hospital characteristics associated with hysterectomy approach. MAIN RESULTS: A total of 32 436 patients were included in this study. Of these, 32% patients underwent laparoscopic hysterectomies, and 67% underwent abdominal hysterectomies. With regard to patient characteristics, women younger than 35 years old were more likely to undergo laparoscopic hysterectomy when compared with each of the other age categories (p < .001). White women were more likely to undergo laparoscopic hysterectomy than black women, Hispanic women, or women classified as "other" races (p < .001 for all comparisons). With regard to median income, patients from the lowest national quartile were less likely to undergo laparoscopic hysterectomy when compared with each of the other 3 national quartiles for income (p = .01, p < .001, p = .001, respectively). Payment by private insurance was associated with laparoscopic hysterectomy when compared with payment by Medicare or payment by insurance category "other" (p < .001 for both). With regard to hospital characteristics, hospitals in the Northeast were more likely to have laparoscopic hysterectomies than hospitals in the Midwest or South (p < .001 for both comparisons); urban hospitals were more likely than rural hospitals (p < .001); teaching hospitals were more likely than nonteaching hospitals (p < .001); and government-owned hospitals were less likely than private, nonprofit or private, investor owned (p < .001 for both comparisons). CONCLUSIONS: Despite the increased popularity of and training in laparoscopic hysterectomies, there remains an obvious disparity in its delivery with regard to patient and hospital characteristics. Further investigation is needed on the etiology of this disparity and interventions that may alleviate it.

Analysis of tubal patency after essure placement.

STUDY OBJECTIVE: To evaluate tubal patency after hysteroscopic sterilization using the Essure microinsert (Conceptus Inc, San Carlos, CA). DESIGN: A retrospective longitudinal cohort study. DESIGN CLASSIFICATION: II-3. SETTING: Patients undergoing hysteroscopic sterilization in the outpatient clinic of a university-based hospital in Southeast Texas from July 2009 to November 2011. PATIENTS: Two hundred twenty-nine women (ages 21-44 yrs, 71% Hispanic) desiring sterilization with a history of regular menses, demonstrated prior fertility (≥1 live birth), and the ability to use an alternative contraceptive method for at least 90 days after coil placement were included. Twenty six patients in this cohort were excluded because of failure to perform a hysterosalpingogram (HSG), tubal perforation, severe dyspareunia, a history of ectopic pregnancy, tubal surgery, or cervical intraepithelial neoplasia. INTERVENTIONS: Not applicable. MEASUREMENTS AND MAIN RESULTS: HSGs were assessed for microinsert location and tubal occlusion. Two hundred three patients were included for analysis. After the successful bilateral hysteroscopic placement of Essure microinserts in fallopian tubes, all patients returned for the first follow-up HSG a mean of 103 ± 38 days after the procedure. Patients with fallopian tube patency at the initial HSG returned for second and/or third HSGs as needed at 192 ± 45 and 291 ± 97 days, respectively. Correct device placement was confirmed in 100% of cases at the first HSG. The tubal patency rates at the 90-day and 180-day HSGs were 16.1% (95% confidence interval, 7.4%-31.7%) and 5.8% (95% CI, 1.2%-24.4%), respectively. These rates were estimated by the accelerated failure time model with log normal distribution and interval censored time to event. The 16.1% 90-day tubal patency rate is significantly different from the 8% rate reported by Cooper et al in the 2003 multicenter phase III pivotal trial (p <.001). CONCLUSION: Our data indicate that hysteroscopic sterilization with Essure results in a higher initial tubal patency rate than previously reported. Multivariate analyses are needed to identify factors associated with an increased risk of postprocedure tubal patency.

Interplay between EGR1 and SP1 is critical for 13-cis retinoic acid-mediated transcriptional repression of angiotensin type 1A receptor.

Recently, we have demonstrated that 13-cis retinoic acid (13cRA) downregulates rat angiotensin type 1A receptor (Agtr1a) gene transcription through a MAP kinase (ERK1/2)-dependent mechanism in rat liver epithelial and aortic smooth muscle cells. However, the exact mechanism remained unknown. In this study, we determined the signaling intermediates activated by ERK1/2 involved in 13cRA-mediated Agtr1a downregulation. Rat Agtr1a chloramphenicol acetyltransferase (CAT) promoter construct containing a sequence -2541 and -1836 bp upstream of the start site demonstrated reduced CAT activity; this region possesses a specificity protein 1 (SP1) consensus sequence (5'-TGGGGCGGGGCGGGG-3'). Mobility shift analysis using untreated nuclear extracts in the presence of mithramycin A suggests that the trans-acting factor binding to this cis-acting element is SP1. 13cRA significantly reduced specific binding without any change in SP1 protein expression. Studies showed that 13cRA treatment maximally phosphorylates ERK1/2 within 5-10 min, which translocates to the nucleus, activating early growth response protein 1 (Egr1) mRNA expression at 20 min followed by de novo protein synthesis, leading to an EGR1/SP1 interaction. siRNA silencing of Egr1 restored Agtr1a mRNA and protein expression in 13cRA-treated cells, and Sp1 silencing results in complete loss of Agtr1a expression. Our study suggests that 13cRA-mediated activation of ERK1/2, through EGR1, is capable of disrupting SP1, the requisite trans-activator for Agtr1a expression, providing a novel paradigm in Agtr1a gene transcription.

Structure-Function Of The Tumor Suppressor BRCA1.

BRCA1, a multi-domain protein, is mutated in a large percentage of hereditary breast and ovarian cancers. BRCA1 is most often mutated in three domains or regions: the N-terminal RING domain, exons 11-13, and the BRCT domain. The BRCA1 RING domain is responsible for the E3 ubiquitin ligase activity of BRCA1 and mediates interactions between BRCA1 and other proteins. BRCA1 ubiquitinates several proteins with various functions. The BRCA1 BRCT domain binds to phosphoproteins with specific sequences recognized by both BRCA1 and ATM/ATR kinases. Structural studies of the RING and BRCT domains have revealed the molecular basis by which cancer causing mutations impact the functions of BRCA1. While no structural data is available for the amino acids encoded by exons 11-13, multiple binding sites and functional domains exist in this region. Many mutations in exons 11-13 have deleterious effects on the function of these domains. In this mini-review, we examine the structure-function relationships of the BRCA1 protein and the relevance to cancer progression.

Functional role of sodium glucose transporter in high glucose-mediated angiotensin type 1 receptor downregulation in human proximal tubule cells.

Previously, we have demonstrated human angiotensin type 1 receptor (hAT(1)R) promoter architecture with regard to the effect of high glucose (25 mM)-mediated transcriptional repression in human proximal tubule epithelial cells (hPTEC; Thomas BE, Thekkumkara TJ. Mol Biol Cell 15: 4347-4355, 2004). In the present study, we investigated the role of glucose transporters in high glucose-mediated hAT(1)R repression in primary hPTEC. Cells were exposed to normal glucose (5.5 mM) and high glucose (25 mM), followed by determination of hyperglycemia-mediated changes in receptor expression and glucose transporter activity. Exposure of cells to high glucose resulted in downregulation of ANG II binding (4,034 ± 163.3 to 1,360 ± 154.3 dpm/mg protein) and hAT(1)R mRNA expression (reduced 60.6 ± 4.643%) at 48 h. Under similar conditions, we observed a significant increase in glucose uptake (influx) in cells exposed to hyperglycemia. Our data indicated that the magnitude of glucose influx is concentration and time dependent. In euglycemic cells, inhibiting sodium-glucose cotransporters (SGLTs) with phlorizin and facilitative glucose transporters (GLUTs) with phloretin decreased glucose influx by 28.57 ± 0.9123 and 54.33 ± 1.202%, respectively. However, inhibiting SGLTs in cells under hyperglycemic conditions decreased glucose influx by 53.67 ± 2.906%, while GLUT-mediated glucose uptake remained unaltered (57.67 ± 3.180%). Furthermore, pretreating cells with an SGLT inhibitor reversed high glucose-mediated downregulation of the hAT(1)R, suggesting an involvement of SGLT in high glucose-mediated hAT(1)R repression. Our results suggest that in hPTEC, hyperglycemia-induced hAT(1)R downregulation is largely mediated through SGLT-dependent glucose influx. As ANG II is an important modulator of hPTEC transcellular sodium reabsorption and function, glucose-mediated changes in hAT(1)R gene expression may participate in the pathogenesis of diabetic renal disease.

Monopolar intracochlear pulse trains selectively activate the inferior colliculus.

Previous cochlear implant studies using isolated electrical stimulus pulses in animal models have reported that intracochlear monopolar stimulus configurations elicit broad extents of neuronal activation within the central auditory system-much broader than the activation patterns produced by bipolar electrode pairs or acoustic tones. However, psychophysical and speech reception studies that use sustained pulse trains do not show clear performance differences for monopolar versus bipolar configurations. To test whether monopolar intracochlear stimulation can produce selective activation of the inferior colliculus, we measured activation widths along the tonotopic axis of the inferior colliculus for acoustic tones and 1,000-pulse/s electrical pulse trains in guinea pigs and cats. Electrical pulse trains were presented using an array of 6-12 stimulating electrodes distributed longitudinally on a space-filling silicone carrier positioned in the scala tympani of the cochlea. We found that for monopolar, bipolar, and acoustic stimuli, activation widths were significantly narrower for sustained responses than for the transient response to the stimulus onset. Furthermore, monopolar and bipolar stimuli elicited similar activation widths when compared at stimulus levels that produced similar peak spike rates. Surprisingly, we found that in guinea pigs, monopolar and bipolar stimuli produced narrower sustained activation than 60 dB sound pressure level acoustic tones when compared at stimulus levels that produced similar peak spike rates. Therefore, we conclude that intracochlear electrical stimulation using monopolar pulse trains can produce activation patterns that are at least as selective as bipolar or acoustic stimulation.

Tannic acid down-regulates the angiotensin type 1 receptor through a MAPK-dependent mechanism.

In the present study, we investigated the effects of tannic acid (TA), a hydrolysable polyphenol, on angiotensin type 1 receptor (AT1R) expression in continuously passaged rat liver epithelial cells. Under normal conditions, exposure of cells to TA resulted in the down-regulation of AT1R-specific binding in concentrations ranging from 12.5-100 µg/ml (7.34-58.78 µm) over a time period of 2-24 h with no change in receptor affinity to angiotensin II (AngII). The inhibitory effect of TA on AT1R was specific and reversible. In TA-treated cells, we observed a significant reduction in AngII-mediated intracellular calcium signaling, a finding consistent with receptor down-regulation. Under similar conditions, TA down-regulated AT1R mRNA expression without changing the rate of mRNA degradation, suggesting that TA's effect is mediated through transcriptional inhibition. Cells expressing recombinant AT1R without the native promoter show no change in receptor expression, whereas a pCAT reporter construct possessing the rat AT1R promoter was significantly reduced in activity. Furthermore, TA induced the phosphorylation of MAPK p42/p44. Pretreatment of the cells with a MAPK kinase (MEK)-specific inhibitor PD98059 prevented TA-induced MAPK phosphorylation and down-regulation of the AT1R. Moreover, there was no reduction in AngII-mediated intracellular calcium release upon MEK inhibition, suggesting that TA's observed inhibitory effect is mediated through MEK/MAPK signaling. Our findings demonstrate, for the first time, that TA inhibits AT1R gene expression and cellular response, suggesting the observed protective effects of dietary polyphenols on cardiovascular conditions may be, in part, through inhibition of AT1R expression.

Pharmacologic effects of 2-methoxyestradiol on angiotensin type 1 receptor down-regulation in rat liver epithelial and aortic smooth muscle cells.

BACKGROUND: Delayed onset of cardiovascular disease (CVD) in female patients is not well understood, but could be due in part to the protective effect of estrogen before menopause. Experimental studies have identified the angiotensin type 1 receptor (AT1R) as a key factor in the progression of CVD. OBJECTIVE: We examined the effects of the estrogen metabolite 2-methoxyestradiol (2ME2) on AT1R expression. METHODS: Rat liver cells were exposed to 2ME2 for 24 hours, and angiotensin II (AngII) binding and AT1R mRNA expressions were assessed. RESULTS: In the presence of 2ME2, cells exhibited significant down-regulation of AngII binding that was both dose and time dependent, independent of estrogen receptors (ERα/ERß). Down-regulation of AngII binding was AT1R specific, with no change in receptor affinity. Under similar conditions, we observed lower expression of AT1R mRNA, significant inhibition of AngII-mediated increase in intracellular Ca(2+), and increased phosphorylation of ERK1/2. Pretreatment of cells with the MEK inhibitor PD98059 prevented 2ME2-induced ERK1/2 phosphorylation and down-regulation of AT1R expression, which suggests that the observed inhibitory effect is mediated through ERK1/2 signaling intermediates. Similar analyses in stably transfected CHO (Chinese hamster ovary) cell lines with a constitutively active cytomegalovirus promoter showed no change in AT1R expression, which suggests that 2ME2-mediated effects are through transcriptional regulation. The effects of 2ME2 on AT1R down-regulation through ERK1/2 were consistently reproduced in primary rat aortic smooth muscle cells. CONCLUSIONS: Because AT1R has a critical role in the control of CVD, 2ME2-induced changes in receptor expression may provide beneficial effects to the cardiovascular and other systems.