Richard H. Karas

The protective effects of estrogen on the cardiovascular system.

Estrogen has direct and indirect effects on the cardiovascular system that are mediated by the estrogen receptors ER-alpha and ER-beta. The direct effects of estrogen occur through rapid nongenomic and longer-term genomic pathways. The rapid effects of estrogen are mediated by ERs and result in the activation of endothelial nitric oxide synthase, leading to arterial vasodilation. Longer-term effects involve changes in gene and protein expression, modulating the response to injury and atherosclerosis. Estrogen also indirectly influences serum lipoprotein and triglyceride profiles, and the expression of coagulant and fibrinolytic proteins. Advanced atherosclerosis and certain progestins, however, may attenuate some of the protective effects of estrogen.

Estrogen receptor a mediates the nongenomic activation of endothelial nitric oxide synthase by estrogen

Estrogen is an important vasoprotective molecule that causes the rapid dilation of blood vessels by activating endothelial nitric oxide synthase (eNOS) through an unknown mechanism. In studies of intact ovine endothelial cells, 17beta-estradiol (E2) caused acute (five-minute) activation of eNOS that was unaffected by actinomycin D but was fully inhibited by concomitant acute treatment with specific estrogen receptor (ER) antagonists. Overexpression of the known transcription factor ERalpha led to marked enhancement of the acute response to E2, and this was blocked by ER antagonists, was specific to E2, and required the ERalpha hormone-binding domain. In addition, the acute response of eNOS to E2 was reconstituted in COS-7 cells cotransfected with wild-type ERalpha and eNOS, but not by transfection with eNOS alone. Furthermore, the inhibition of tyrosine kinases or mitogen-activated protein (MAP) kinase kinase prevented the activation of eNOS by E2, and E2 caused rapid ER-dependent activation of MAP kinase. These findings demonstrate that the short-term effects of estrogen central to cardiovascular physiology are mediated by ERalpha functioning in a novel, nongenomic manner to activate eNOS via MAP kinase-dependent mechanisms.

A transcriptionally active DNA-binding site for human p53 protein complexes.

Recent studies have demonstrated transcriptional activation domains within the tumor suppressor protein p53, while others have described specific DNA-binding sites for p53, implying that the protein may act as a transcriptional regulatory factor. We have used a reiterative selection procedure (CASTing: cyclic amplification and selection of targets) to identify new specific binding sites for p53, using nuclear extracts from normal human fibroblasts as the source of p53 protein. The preferred consensus is the palindrome GGACATGCCCGGGCATGTCC. In vitro-translated p53 binds to this sequence only when mixed with nuclear extracts, suggesting that p53 may bind DNA after posttranslational modification or as a complex with other protein partners. When placed upstream of a reporter construct, this sequence promotes p53-dependent transcription in transient transfection assays.

Assessment of peripheral vascular endothelial function with finger arterial pulse wave amplitude.

BACKGROUND Abnormalities in pulse wave amplitude (PWA) have been described in subjects with atherosclerosis and may be a marker of future cardiac events. We evaluated the relationship between changes in PWA of the finger and peripheral endothelial function. METHODS We performed measurements of PWA with a novel finger plethysmograph (peripheral arterial tonometry [PAT]) and compared the findings with a simultaneous noninvasive measurement of peripheral endothelial function with brachial artery ultrasound scanning (BAUS) in 89 subjects. The PAT hyperemia ratio was defined as the ratio of PWA during reactive hyperemia relative to the baseline. Flow-mediated dilation (FMD) was defined by BAUS as the ratio of the brachial artery diameter during reactive hyperemia relative to the baseline. Sixty-eight subjects underwent exercise myocardial perfusion imaging (ExMPI). RESULTS Fifty-four men and 35 women were examined. There was a linear relationship between the PAT hyperemia ratio and FMD during the same episode of reactive hyperemia (r = 0.55, P <.0001). Subjects in the lowest FMD quartile had the lowest PAT hyperemia ratio, whereas subjects in the highest FMD quartile had the highest PAT hyperemia ratio (P <.001 for trend). Similar to BAUS, the PAT hyperemia ratio was more impaired in subjects with cardiovascular risk factors and in subjects with ExMPI studies that were indicative of coronary artery disease. CONCLUSIONS Assessment of PWA with PAT demonstrates patterns of abnormality similar to that of BAUS assessment of FMD. PWA during reactive hyperemia is influenced by factors known to affect endothelial function, including cardiovascular risk factors and coronary artery disease. These findings support the concept that analysis of PWA with PAT during reactive hyperemia may be used to study peripheral vascular endothelial function.

Human vascular smooth muscle cells contain functional estrogen receptor.

BACKGROUND The decreased incidence of coronary artery disease observed in postmenopausal women given estrogen (E2) replacement demonstrates an atheroprotective effect of E2 that is generally believed to be mediated by indirect, E2-induced changes in cardiovascular risk factor profiles. We hypothesized that the atheroprotective effect of E2 may be in part mediated by a direct effect of E2 on vascular smooth muscle cells (VSMCs). Therefore, a series of experiments was performed to determine whether human VSMCs contain a competent E2 receptor, a ligand-activated transcription factor known to mediate E2-induced effects in nonvascular cells. METHODS AND RESULTS Ribonuclease protection assays, with a probe derived from the human E2 receptor, were used to demonstrate E2-receptor mRNA in human saphenous vein VSMCs. To show that VSMCs contain E2-receptor protein as well as message, immunoblotting and immunofluorescence studies with a monoclonal anti-E2-receptor antibody were performed, and E2-receptor protein was detected by both methods. Transient transfection assays using a specific E2-responsive reporter system were used next to determine whether the VSMC E2 receptor is capable of E2-induced transcriptional transactivation. Initial studies using mammary artery-derived VSMCs resulted in a 2.4-fold increase in reporter activity in response to 10(-7) mol/L E2. Subsequent studies using saphenous vein VSMCs demonstrated increasing levels of reporter activation as the concentration of E2 was increased from 10(-9) mol/L (1.3-fold increase; SEM, 0.07; P = .05, n = 3) to 10(-7) mol/L (1.6-fold increase; SEM, 0.04; P = .002, n = 6). The specificity of the E2-induced transactivation of the reporter gene was shown by dose-dependent inhibition of transactivation by the pure E2 antagonist ICI 164,384 and by enhancement of the transactivation by simultaneous overexpression of the E2 receptor. CONCLUSIONS We have demonstrated for the first time that human VSMCs express E2-receptor mRNA and protein and that the E2 receptor in VSMCs is capable of estrogen-dependent gene activation. These data suggest a mechanism by which estrogen may directly alter VSMC function.

Postmenopausal hormone therapy: An endocrine society scientific statement

OBJECTIVE Our objective was to provide a scholarly review of the published literature on menopausal hormonal therapy (MHT), make scientifically valid assessments of the available data, and grade the level of evidence available for each clinically important endpoint. PARTICIPANTS IN DEVELOPMENT OF SCIENTIFIC STATEMENT: The 12-member Scientific Statement Task Force of The Endocrine Society selected the leader of the statement development group (R.J.S.) and suggested experts with expertise in specific areas. In conjunction with the Task Force, lead authors (n = 25) and peer reviewers (n = 14) for each specific topic were selected. All discussions regarding content and grading of evidence occurred via teleconference or electronic and written correspondence. No funding was provided to any expert or peer reviewer, and all participants volunteered their time to prepare this Scientific Statement. EVIDENCE Each expert conducted extensive literature searches of case control, cohort, and randomized controlled trials as well as meta-analyses, Cochrane reviews, and Position Statements from other professional societies in order to compile and evaluate available evidence. No unpublished data were used to draw conclusions from the evidence. CONSENSUS PROCESS A consensus was reached after several iterations. Each topic was considered separately, and a consensus was achieved as to content to be included and conclusions reached between the primary author and the peer reviewer specific to that topic. In a separate iteration, the quality of evidence was judged using the GRADE (Grading of Recommendations, Assessment, Development, and Evaluation) system in common use by The Endocrine Society for preparing clinical guidelines. The final iteration involved responses to four levels of additional review: 1) general comments offered by each of the 25 authors; 2) comments of the individual Task Force members; 3) critiques by the reviewers of the Journal of Clinical Endocrinology & Metabolism; and 4) suggestions offered by the Council and members of The Endocrine Society. The lead author compiled each individual topic into a coherent document and finalized the content for the final Statement. The writing process was analogous to preparation of a multiauthored textbook with input from individual authors and the textbook editors. CONCLUSIONS The major conclusions related to the overall benefits and risks of MHT expressed as the number of women per 1000 taking MHT for 5 yr who would experience benefit or harm. Primary areas of benefit included relief of hot flashes and symptoms of urogenital atrophy and prevention of fractures and diabetes. Risks included venothrombotic episodes, stroke, and cholecystitis. In the subgroup of women starting MHT between ages 50 and 59 or less than 10 yr after onset of menopause, congruent trends suggested additional benefit including reduction of overall mortality and coronary artery disease. In this subgroup, estrogen plus some progestogens increased the risk of breast cancer, whereas estrogen alone did not. Beneficial effects on colorectal and endometrial cancer and harmful effects on ovarian cancer occurred but affected only a small number of women. Data from the various Womens Health Initiative studies, which involved women of average age 63, cannot be appropriately applied to calculate risks and benefits of MHT in women starting shortly after menopause. At the present time, assessments of benefit and risk in these younger women are based on lower levels of evidence.

Cardiac myocytes and fibroblasts contain functional estrogen receptors

Gender‐based differences found in cardiovascular diseases raise the possibility that estrogen may have direct effects on cardiac tissue. Therefore we investigated whether cardiac myocytes and fibroblasts express functional estrogen receptors. Immunofluorescence demonstrated estrogen receptor protein expression in both female and male rat cardiac myocytes and fibroblasts. Nuclear translocation of the estrogen receptor protein was observed after stimulation of cardiomyocytes with 17β‐estradiol (E2). Cells transfected with an estrogen‐responsive reporter plasmid showed that treatment with E2 induced a significant increase in reporter activity. Furthermore, E2 induced a significant increase in expression of the estrogen receptors α and β, progesterone receptor and connexin 43 in cardiac myocytes. Cardiac myocytes and fibroblasts contain functional estrogen receptors and estrogen regulates expression of specific cardiac genes. These data suggest that gender‐based differences in cardiac diseases may in part be due to direct effects of estrogen on the heart.

Regulator of G-protein signaling-2 mediates vascular smooth muscle relaxation and blood pressure

Nitric oxide (NO) inhibits vascular contraction by activating cGMP-dependent protein kinase I-α (PKGI-α), which causes dephosphorylation of myosin light chain (MLC) and vascular smooth muscle relaxation. Here we show that PKGI-α attenuates signaling by the thrombin receptor protease-activated receptor-1 (PAR-1) through direct activation of regulator of G-protein signaling-2 (RGS-2). NO donors and cGMP cause cGMP-mediated inhibition of PAR-1 and membrane localization of RGS-2. PKGI-α binds directly to and phosphorylates RGS-2, which significantly increases GTPase activity of Gq, terminating PAR-1 signaling. Disruption of the RGS-2–PKGI-α interaction reverses inhibition of PAR-1 signaling by nitrovasodilators and cGMP. Rgs2−/− mice develop marked hypertension, and their blood vessels show enhanced contraction and decreased cGMP-mediated relaxation. Thus, PKGI-α binds to, phosphorylates and activates RGS-2, attenuating receptor-mediated vascular contraction. Our study shows that RGS-2 is required for normal vascular function and blood pressure and is a new drug development target for hypertension.

Estrogen Receptor-α Mediates the Protective Effects of Estrogen Against Vascular Injury

Blood vessel cells express the 2 known estrogen receptors, &agr; and &bgr; (ER&agr;, ER&bgr;), which are thought to mediate estrogen inhibition of vascular injury and atherosclerosis, but the relative role of ER&agr; and ER&bgr; in these events is controversial. Estrogen inhibits the vascular injury response to the same extent in ovariectomized female wild-type mice and in the original single gene knockout mice for ER&agr; (ER&agr;KOChapel Hill [ER&agr;KOCH]) and ER&bgr; (ER&bgr;KOChapel Hill [ER&bgr;KOCH]). In double gene knockout mice generated by crossing these animals (ER&agr;,&bgr;KOCH), estrogen no longer inhibits medial thickening after vascular injury, but still inhibits vascular smooth muscle cell proliferation and increases uterine weight. The partial retention of estrogen responsiveness in ER&agr;,&bgr;KOCH mice could be due either to the presence of a novel, unidentified estrogen receptor or to functional expression of an estrogen receptor-&agr; splice variant in the parental ER&agr;KOCH mice. To distinguish between these possibilities, we studied recently generated mice fully null for estrogen receptor &agr; (ER&agr;KOStrasbourg [ER&agr;KOSt]) and examined the effect of estrogen on the response to vascular injury. In the present study, we show that after vascular injury in ovariectomized ER&agr;KOSt mice, estrogen has no detectable effect on any measure of vascular injury, including medial area, proteoglycan deposition, or smooth muscle cell proliferation. These data demonstrate that estrogen receptor-&agr; mediates the protective effects of estrogen on the response to vascular injury.

Estrogen and progestogen use in postmenopausal women: July 2008 position statement of The North American Menopause Society

Objective:To update for both clinicians and the lay public the evidence-based position statement published by The North American Menopause Society (NAMS) in March 2007 regarding its recommendations for menopausal hormone therapy (HT) for postmenopausal women, with consideration for the therapeutic benefit-risk ratio at various times through menopause and beyond. Design:An Advisory Panel of clinicians and researchers expert in the field of womens health was enlisted to review the March 2007 NAMS position statement, evaluate new evidence through an evidence-based analysis, and reach consensus on recommendations. The Panels recommendations were reviewed and approved by the NAMS Board of Trustees as an official NAMS position statement. The document was provided to other interested organizations to seek their endorsement. Results:Current evidence supports a consensus regarding the role of HT in postmenopausal women, when potential therapeutic benefits and risks around the time of menopause are considered. This paper lists all these areas along with explanatory comments. Conclusions that vary from the 2007 position statement are highlighted. Addenda include a discussion of risk concepts, a new component not included in the 2007 paper, and a recommended list of areas for future HT research. A suggested reading list of key references is also provided. Conclusions:Recent data support the initiation of HT around the time of menopause to treat menopause-related symptoms; to treat or reduce the risk of certain disorders, such as osteoporosis or fractures in select postmenopausal women; or both. The benefit-risk ratio for menopausal HT is favorable close to menopause but decreases with aging and with time since menopause in previously untreated women.