Estrogen actions in the cardiovascular system.

This brief review summarizes the current state of the field for estrogen receptor actions in the cardiovascular system and the cardiovascular effects of hormone replacement therapy (HRT). It is organized into three parts: a short Introduction and overview of the current view of how estrogen works on blood vessels; a summary of the current status of clinical information regarding HRT and cardiovascular effects; and an update on state-of-the-art mouse models of estrogen action using estrogen receptor knockout mice.

Association between arterial stiffness and variations in oestrogen-related genes.

Increased arterial stiffness and wave reflection have been identified as cardiovascular disease risk factors. In light of significant sex differences and the moderate heritability of vascular function measures, we hypothesized that variation in the genes coding for oestrogen receptors alpha (ESR1) and beta (ESR2) and aromatase (CYP19A1) is associated with aortic stiffness and pressure wave reflection as measured by non-invasive arterial tonometry. In all, 1261 unrelated Framingham Offspring Study participants who attended the seventh examination cycle (mean age 62+/-10 years, 52% women) and had arterial tonometry and genotyping data were included in the study. Analysis of covariance was used to assess the association of polymorphisms with forward wave amplitude, augmented pressure, augmentation index (AI), carotid-femoral pulse wave velocity and mean arterial pressure with adjustment for potential confounders. In the sex-pooled analysis, those homozygous for the minor allele at any of four ESR1 variants that were in strong linkage disequilibrium ((TA)(n), rs2077647, rs2234693 and rs9340799) had on an average 18% higher augmented pressure and 16% greater AI compared with carriers of one or two major alleles (P=0.0002-0.01). A similar magnitude of association was detected in those homozygous for the common allele at two ESR2 single-nucleotide polymorphisms (P=0.007-0.02). Our results are consistent with the hypothesis that variation in ESR1 and ESR2, but not CYP19A1, is associated with an increased wave reflection that may contribute to associations between these variants and adverse clinical events demonstrated earlier. Our findings will need to be replicated in additional cohorts.

Uvulopalatopharyngoplasty for sleep apnea in mentally retarded obese 14-year-old: an anaesthetic challenge.

Anaesthetic management of patients with obstructive sleep apnea for upper airway surgery has always been a challenging task. We report our anaesthetic approach for a young, mentally retarded obese patient with documented obstructive sleep apnea undergoing uvulopalatopharyngoplasty. The therapeutic intervention before, during and after operation is discussed.

Activation of estrogen receptor beta is a prerequisite for estrogen-dependent upregulation of nitric oxide synthases in neonatal rat cardiac myocytes.

Physiological effects of estrogen on myocardium are mediated by two intracellular estrogen receptors, ERalpha and ERbeta, that regulate transcription of target genes through binding to specific DNA target sequences. To define the role of ERbeta in the transcriptional activation of both endothelial (eNOS) and inducible nitric oxide synthase (iNOS) in cardiac myocytes, we used the complete ER-specific antagonist R,R-tetrahydrochrysene (R,R-THC). R,R-THC inhibited activation of iNOS/eNOS promoter-luciferase reporter constructs (iNOS/eNOS-Luc) in a dose-dependent fashion in COS7 cells selectively transfected with ERbeta, but failed to influence ERalpha-mediated increase of iNOS/ eNOS-Luc. In neonatal rat cardiomyocytes transfected with eNOS-Luc or iNOS-Luc, incubation with 17betaestradiol (E2, 10(-8) M) for 24 h stimulated expression of eNOS and iNOS. R,R-THC (10(-5) M) completely inhibited this effect. Furthermore, eNOS and iNOS protein expression in cardiac myocytes induced by E2 was completely blocked by R,R-THC as shown by immunoblot analysis. Taken together, these results show that ERbeta mediates transcriptional activation of eNOS and iNOS by E2.

Different levels of repressor activity assign redundant and specific roles to Nkx6 genes in motor neuron and interneuron specification.

Specification of neuronal fate in the vertebrate central nervous system depends on the profile of transcription factor expression by neural progenitor cells, but the precise roles of such factors in neurogenesis remain poorly characterized. Two closely related transcriptional repressors, Nkx6.2 and Nkx6.1, are expressed by progenitors in overlapping domains of the ventral spinal cord. We provide genetic evidence that differences in the level of repressor activity of these homeodomain proteins underlies the diversification of interneuron subtypes, and provides a fail-safe mechanism during motor neuron generation. A reduction in Nkx6 activity further permits V0 neurons to be generated from progenitors that lack homeodomain proteins normally required for their generation, providing direct evidence for a model in which progenitor homeodomain proteins direct specific cell fates by actively suppressing the expression of transcription factors that direct alternative fates.

Effects of estrogen on the vascular injury response in estrogen receptor alpha, beta (double) knockout mice.

The two known estrogen receptors, ERalpha and ERbeta, mediate the effects of estrogen in all target tissues, including blood vessels. We have shown previously that estrogen inhibits vascular injury response to the same extent in female wild-type (WT), ERalpha knockout (ERalphaKO(CH)), and ERbeta knockout (ERbetaKO(CH)) mice. We generated mice harboring disruptions of both ERalpha and ERbeta genes (ERalpha,betaKO(CH)) by breeding and studied the effect of 17beta-estradiol (E2) on vascular injury responses in ovariectomized female ERalpha,betaKO(CH) mice and WT littermates. E2 inhibited increases in vascular medial area following injury in the WT mice but not in the ERalpha,betaKO(CH) mice, demonstrating for the first time that the two known estrogen receptors are necessary and sufficient to mediate estrogen inhibition of a component of the vascular injury response. Surprisingly, as in WT littermates, E2 still significantly increased uterine weight and inhibited vascular smooth muscle cell (VSMC) proliferation following injury in the ERalpha,betaKO(CH) mice. These data support that the role of estrogen receptors differs for specific components of the vascular injury response in the ERalpha,betaKO(CH) mice. The results leave unresolved whether E2 inhibition of VSMC proliferation in ERalpha,betaKO(CH) mice is caused by a receptor-independent mechanism, an unidentified receptor responsive to estrogen, or residual activity of the ERalpha splice variant reported previously in the parental ERalphaKO(CH) mice. These possibilities may be resolved by studies of mice in which ERalpha has been fully disrupted (ERalphaKO(St)), which are in progress.

A complex role for the progesterone receptor in the response to vascular injury.

Clinical studies of hormone replacement therapy to prevent cardiovascular diseases have heightened interest in the cardiovascular effects of progestins. However, the role of the progesterone receptor (PR) in vascular biology has not been studied in vivo. We studied ovariectomized female PR knockout (PRKO) mice and their wild-type (WT) littermates using the mouse carotid artery injury model. Placebo-treated PRKO mice showed significantly greater vascular medial hypertrophy and vascular smooth muscle cell (VSMC) proliferation in response to vascular injury than did WT mice. Progesterone had no significant effect in the PRKO mice, but worsened the response to injury in WT mice. VSMCs cultured from PRKO mouse aortae were markedly hyperproliferative, and their growth was not affected by progesterone. In contrast to the in vivo findings, progesterone inhibited proliferation of WT-derived VSMCs. Furthermore, reintroduction of PR into PRKO-derived VSMCs using adenoviral methods restored progesterone-mediated inhibition of proliferation to these cells. This effect was reversed by the PR antagonist, RU 486. Thus, the effects of PR and progesterone differ markedly between cultured VSMCs and intact blood vessels. These data demonstrate a direct role for the PR in regulating the response to vascular injury and VSMC proliferation.

High-density lipoprotein binding to scavenger receptor-BI activates endothelial nitric oxide synthase.

Atherosclerosis is the primary cause of cardiovascular disease, and the risk for atherosclerosis is inversely proportional to circulating levels of high-density lipoprotein (HDL) cholesterol. However, the mechanisms by which HDL is atheroprotective are complex and not well understood. Here we show that HDL stimulates endothelial nitric oxide synthase (eNOS) in cultured endothelial cells. In contrast, eNOS is not activated by purified forms of the major HDL apolipoproteins ApoA-I and ApoA-II or by low-density lipoprotein. Heterologous expression experiments in Chinese hamster ovary cells reveal that scavenger receptor-BI (SR-BI) mediates the effects of HDL on the enzyme. HDL activation of eNOS is demonstrable in isolated endothelial-cell caveolae where SR-BI and eNOS are colocalized, and the response in isolated plasma membranes is blocked by antibodies to ApoA-I and SR-BI, but not by antibody to ApoA-II. HDL also enhances endothelium- and nitric-oxide-dependent relaxation in aortae from wild-type mice, but not in aortae from homozygous null SR-BI knockout mice. Thus, HDL activates eNOS via SR-BI through a process that requires ApoA-I binding. The resulting increase in nitric-oxide production might be critical to the atheroprotective properties of HDL and ApoA-I.

Using a computer to organize digital photographs.

A facial plastic surgeon's photographic files are a potential storehouse of learning material. Manual review of before-and-after photographs is time consuming and self-limiting. Computers can facilitate the analysis, storage, and review of photographs and thereby serve as an aid to learning from the results of our surgery. This article describes a personal approach to automating this process. The surgeon classifies the photographs, and the office staff performs the typing and filing. As the speed and resources of computers rapidly expand, this process will become faster, simpler, and more popular.

Plasma membrane estrogen receptors are coupled to endothelial nitric-oxide synthase through Galpha(i).

Estrogen causes rapid endothelial nitric oxide (NO) production because of the activation of plasma membrane-associated estrogen receptors (ER) coupled to endothelial NO synthase (eNOS). In the present study, we determined the role of G proteins in eNOS activation by estrogen. Estradiol-17beta (E(2), 10(-8) m) and acetylcholine (10(-5) m) caused comparable increases in NOS activity (15 min) in intact endothelial cells that were fully blocked by pertussis toxin (Ptox). In addition, exogenous guanosine 5'-O-(2- thiodiphosphate) inhibited E(2)-mediated eNOS stimulation in isolated endothelial plasma membranes, and Ptox prevented enzyme activation by E(2) in COS-7 cells expressing ERalpha and eNOS. Coimmunoprecipitation studies of plasma membranes from COS-7 cells transfected with ERalpha and specific Galpha proteins demonstrated E(2)-stimulated interaction between ERalpha and Galpha(i) but not between ERalpha and either Galpha(q) or Galpha(s); the observed ERalpha-Galpha(i) interaction was blocked by the ER antagonist ICI 182,780 and by Ptox. E(2)-stimulated ERalpha-Galpha(i) interaction was also demonstrable in endothelial cell plasma membranes. Cotransfection of Galpha(i) into COS-7 cells expressing ERalpha and eNOS yielded a 3-fold increase in E(2)-mediated eNOS stimulation, whereas cotransfection with a protein regulator of G protein signaling, RGS4, inhibited the E(2) response. These findings indicate that eNOS stimulation by E(2) requires plasma membrane ERalpha coupling to Galpha(i) and that activated Galpha(i) mediates the requisite downstream signaling events. Thus, novel G protein coupling enables a subpopulation of ERalpha to initiate signal transduction at the cell surface. Similar mechanisms may underly the nongenomic actions of other steroid hormones.

In vivo studies of the gamma subunit of retinal cGMP-phophodiesterase with a substitution of tyrosine-84.

The inhibitory rod cGMP phosphodiesterase gamma subunit (PDEgamma) is a major component of the photoresponse and is required to support rod integrity. Pdeg(tm1)/Pdeg(tm1) mice (which lack PDEgamma owing to a targeted disruption of the Pdeg gene) suffer from a very rapid and severe photoreceptor degeneration. The Y84G (Tyr(84)-->Gly) allele of PDEgamma has previously been shown in experiments carried out in vitro to reduce the regulatory control of the PDE catalytic core (PDEalphabeta) exerted by the wild-type gamma subunit. To determine the effects of this mutation on in vivo function, the murine opsin promoter was used to direct expression to the photoreceptors of +/Pdeg(tm1) mice of a mutant Y84G and a wild-type PDEgamma control transgene. The transgenic mice were crossed with Pdeg(tm1)/Pdeg(tm1) mice to generate animals able to synthesize only the transgenic PDEgamma. Our results showed that wild-type PDEgamma and Y84G transgenes could complement the Pdeg(tm1)/Pdeg(tm1) mutant for photoreceptor survival. The mutation caused a significant biochemical defect in PDE activation by transducin. However, the Y84G mutation did not fully eliminate the control of PDEgamma on the PDE catalytic core in vivo; the expression of the mutant subunit was associated with only a 10-fold reduction in the amplitude of the a-wave and a 1.5-fold decrease in the b-wave of the corneal electroretinogram. Unexpectedly, the mutation caused a much 'milder' phenotype in vivo than was predicted from the biochemical assays in vitro.

Heart failure etiology affects peripheral vascular endothelial function after cardiac transplantation.

OBJECTIVES: The goal of this study was to examine the effect of heart failure etiology on peripheral vascular endothelial function in cardiac transplant recipients. BACKGROUND: Peripheral vascular endothelial dysfunction occurs in patients with heart failure of either ischemic or nonischemic etiology. The effect of heart failure etiology on peripheral endothelial function after cardiac transplantation is unknown. METHODS: Using brachial artery ultrasound, endothelium-dependent, flow-mediated dilation (FMD) was assessed in patients with heart failure with either nonischemic cardiomyopathy (n = 10) or ischemic cardiomyopathy (n = 7), cardiac transplant recipients with prior nonischemic cardiomyopathy (n = 10) or prior ischemic cardiomyopathy (n = 10) and normal controls (n = 10). RESULTS: Patients with heart failure with either ischemic cardiomyopathy or nonischemic cardiomyopathy had impaired FMD (3.6 +/- 1.0% and 5.1 +/- 1.2%, respectively, p = NS) compared with normal subjects (13.9 +/- 1.3%, p < 0.01 compared with either heart failure group). In transplant recipients with antecedent nonischemic cardiomyopathy, FMD was markedly higher than that of heart failure patients with nonischemic cardiomyopathy (13.0 +/- 2.4%, p < 0.001) and similar to that of normal subjects (p = NS). However, FMD remained impaired in transplant recipients with prior ischemic cardiomyopathy (5.5 +/- 1.5%, p = 0.001 compared with normal, p = 0.002 vs. transplant recipients with previous nonischemic cardiomyopathy). CONCLUSIONS: Peripheral vascular endothelial function is normal in cardiac transplant recipients with antecedent nonischemic cardiomyopathy, but remains impaired in those with prior ischemic cardiomyopathy. In contrast, endothelial function is uniformly abnormal for patients with heart failure, regardless of etiology. These findings indicate that cardiac transplantation corrects peripheral endothelial function for patients without ischemic heart disease, but not in those with prior atherosclerotic coronary disease.

Evaluation of three somatic genetic biomarkers as indicators of low dose radiation effects in clean-up workers of the Chernobyl nuclear reactor accident.

The goals of this study were to assess three biomarkers of genetic effect for their individual and collective ability to detect and estimate radiation exposure in Russian Chernobyl clean-up workers. Work assignments were planned to limit dose to 0.25 Gy. The three biomarkers employed were chromosome translocations detectcd in lynmphocytes by florescence in situ hybridisation (FISH), and mutation at two genes, glycophorin A (GPA) in red blood cells detected by flow cytometry and hypoxanthine phosphoribosyltransferase (HPRT) in lymphocytes detected by selective cell culture. Samples were Obtained from 1992 to 2000. The time between exposure at Chernobyl and sample acquisition was > or =5 years. The lymphocyte assays detected an elevation over controls in average outcomes it clean-up workers: translocation rates were 46% higher when adjusted for age and smoking and HPRT mutant frequencies were were 16% higher when adjusted for age. The G PA assay did not detect an exposure effect. The results indicate that measuring frequency of translocations by FISH is preferred for low dose radiation, retrospective biochemistry.

Genetic ablation and restoration of the olfactory topographic map.

In the olfactory sensory system, neurons expressing a given odorant receptor project with precision to two of 1800 spatially invariant glomeruli creating a topographic map within the olfactory bulb. Olfactory sensory neurons have a half-life of about 90 days and are continually renewing. This poses the problem of how this precise spatial map is maintained throughout the life of the organism. We have developed a genetic approach to effect the synchronous ablation of subpopulations of neurons expressing a given receptor. The axons of newly generated neurons can then be followed as they enter the brain and converge on glomerular targets during adult life. The observation that following neuronal cell killing, the spatial map is faithfully restored, demonstrates that the information necessary for the establishment of the sensory map persists throughout the life of the organism.

Nongenomic, ER-mediated activation of endothelial nitric oxide synthase: how does it work? What does it mean?

The administration of estrogens in animals tends to inhibit the development of atherosclerosis. [Therefore], attempts are being made in human males to prevent further progress of the disease in cases of angina or previous coronary thrombosis by long-range estrogen therapy. This work is in its infancy. It is hoped that, with further research, compounds might be found that eliminate the undesirable action of such hormones and yet retain its beneficial effects on arteries (Samuel A. Levine, 1958).