Mitochondrial Effects of Estrogen Are Mediated by Estrogen Receptor α in Brain Endothelial Cells

Department of Pharmacology, School of Medicine, University of California, Irvine, CA 92697-4625, USA.
Journal of Pharmacology and Experimental Therapeutics (Impact Factor: 3.86). 07/2008; 325(3):782-90. DOI: 10.1124/jpet.107.134072
Source: PubMed

ABSTRACT Mitochondrial reactive oxygen species (ROS) and endothelial dysfunction are key contributors to cerebrovascular pathophysiology. We previously found that 17beta-estradiol profoundly affects mitochondrial function in cerebral blood vessels, enhancing efficiency of energy production and suppressing mitochondrial oxidative stress. To determine whether estrogen specifically affects endothelial mitochondria through receptor mechanisms, we used cultured human brain microvascular endothelial cells (HBMECs). 17beta-Estradiol treatment for 24 h increased mitochondrial cytochrome c protein and mRNA; use of silencing RNA for estrogen receptors (ERs) showed that this effect involved ERalpha, but not ERbeta. Mitochondrial ROS were determined by measuring the activity of aconitase, an enzyme with an iron-sulfur center inactivated by mitochondrial superoxide. 17beta-Estradiol increased mitochondrial aconitase activity in HBMECs, indicating a reduction in ROS. Direct measurement of mitochondrial superoxide with MitoSOX Red showed that 17beta-estradiol, but not 17alpha-estradiol, significantly decreased mitochondrial superoxide production, an effect blocked by the ER antagonist, ICI-182,780 (fulvestrant). Selective ER agonists demonstrated that the decrease in mitochondrial superoxide was mediated by ERalpha, not ERbeta. The selective estrogen receptor modulators, raloxifene and 4-hydroxy-tamoxifen, differentially affected mitochondrial superoxide production, with raloxifene acting as an agonist but 4-hydroxy-tamoxifen acting as an estrogen antagonist. Changes in superoxide by 17beta-estradiol could not be explained by changes in manganese superoxide dismutase. Instead, ERalpha-mediated decreases in mitochondrial ROS may depend on the concomitant increase in mitochondrial cytochrome c, previously shown to act as an antioxidant. Mitochondrial protective effects of estrogen in cerebral endothelium may contribute to sex differences in the occurrence of stroke and other age-related neurodegenerative diseases.

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Available from: Vincent Procaccio, Jun 13, 2015
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    • "This is in line with other results showing that neuroprotection involves attenuation of NADPH oxidase activation and superoxide production via an ERα-mediated nongenomic signaling cascade (Zhang et al., 2009). In human brain microvascular endothelial cells physiological levels of 17β-Estradiol but not its enantiomer 17α-Estradiol suppressed mitochondrial superoxide production mediated through ERα receptor (Razmara et al., 2008). The failure of 17α-Estradiol, which does not activate ER but shares antioxidant properties, underlines the irrelevance of estradiol's direct antioxidant activity at physiological levels. "
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    ABSTRACT: Introduction 17β-estradiol (E2) is neuroprotective in experimental models of stroke. While some postulate a mainly antioxidative action due to E2’s free C3 hydroxyl group at its A-ring, others suggest that neuroprotection is mediated by a hormonal, receptor mediated effect. The aim of the current study was to clarify this issue by testing whether E2 analogues lacking hormonal activity are also neuroprotective following cerebral ischemia. Material & Methods Focal cerebral ischemia was induced in male C57/BL6 mice by laser-Doppler-controlled endovascular occlusion of the middle cerebral artery for 40 min. Mice received either 1) memantine, a NMDA-receptor antagonist, as a positive control, 2) E2 (1400 µg/kg b.w.), or 3) 2,4,6-trimethylphenol (TMP), an E2 analogue without hormonal activity (1400, 140, or 14 µg/kg b.w.). Motor function was tested 3 h and 24 h after ischemia. Thereafter mice were sacrificed and brain damage was quantified by histomorphometry. Results Treatment with memantine or E2 significantly reduced infarct volume by >40% and significantly improved neurological function while treatment with TMP had no effect. Conclusion E2 is equally neuroprotective as antagonization of NMDA receptors while E2 analogues without hormonal activity are not neuroprotective. Therefore the current data suggest that the neuroprotection activity of E2 is independent of its free-radical scavenging properties.
    Brain Research 11/2014; 1589. DOI:10.1016/j.brainres.2014.08.029 · 2.83 Impact Factor
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    • "The role of endothelial cells is important in mediating the effects of circulating as well as locally-synthesized steroids during fetal brain development. The cerebral blood vessels are affected by sex steroids in different ways (Krause et al., 2011), and the hormones may alter endothelial functions via specific vascular receptors (Razmara et al., 2008). Since steroids play an important role in neuroprotection qualitative PCR and immunocytochemical analysis for ER-a and b investigation were performed. "
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    ABSTRACT: Immortalized cell lines from fetal brain are an experimental model for studying the in vitro molecular pathways regulating neural cell differentiation and the development of neural networks. The procedures are described to obtain an established cell line from the 90-day old fetal sheep hypothalamus. Viral oncogene LT-SV40 transformation was used to isolate a stable cell line (ENOS-01) that was characterized immunocytochemically. Immortalized cells can be classified as an endothelial cell line of hypothalamic microvasculature. Furthermore, mRNA expression and immunocytochemical of estrogen receptors α and β were also evaluated. Since it is known that cerebral vessels are directly targeted by sex steroids, our established cell line represents an alternative system to study estradiol/receptor interactions during brain development. Our in vitro model can provide a tool to investigate the complex relationships among the cell types forming the blood-brain barrier, which is known to be involved in the pathogenesis of sheep transmissible neurological diseases.
    Research in Veterinary Science 11/2012; 94(3). DOI:10.1016/j.rvsc.2012.10.022 · 1.51 Impact Factor
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    • "A recent study (Krause et al. 2011) has established that cerebral blood vessels are affected by sex steroids in various ways. Estrogens and androgens can alter vascular tone, endothelial functions, oxidative stress and inflammatory responses via specific vascular receptors (Razmara et al. 2008). Moreover, endothelial cells in cerebral arteries also express enzymes involved in the metabolism of sex steroids (Gonzales et al. 2007). "
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    ABSTRACT: Estrogens diversely affect various physiological processes by genomic or non-genomic mechanisms, in both excitable and non-excitable cells. Additional to the trophic effects of estrogens promoting cell growth and differentiation, recent experimental evidence highlights their involvement in the regulation of intracellular Ca(2+) homeostasis. The effects of estrogens on excitable cells are well documented. However, these steroids also influence numerous physiological events in non-excitable cells, such as fibroblasts or vascular endothelial cells. We have focused our attention on an immortalized endothelial-like cell line derived from fetal bovine cerebellum. Estradiol (E(2)) effects on intracellular Ca(2+) homeostasis were tested by varying the exposure time to the hormone (8, 24, 48 h). Calcium measurements were performed with genetically encoded Ca(2+) probes (Cameleons) targeted to the main subcellular compartments involved in intracellular Ca(2+) homeostasis (cytosol, endoplasmic reticulum, mitochondria). Mitochondrial Ca(2+) uptake significantly decreased after 48-h exposure to E(2), whereas cytosolic and endoplasmic reticulum responses were unaffected. The effect of E(2) on mitochondrial Ca(2+) handling was blocked by ICI 182,780, a pure estrogen receptor antagonist, suggesting that the effect was estrogen-receptor-mediated. To evaluate whether the decrease of Ca(2+) uptake affected mitochondrial membrane potential (ΔΨm), cells were monitored in the presence of tetra-methyl-rhodamine-methylester; no significant changes were seen between cells treated with E(2) and controls. To investigate a mechanism of action, we assessed the possibile involvement of the permeability transition pore (PTP), an inner mitochondrial membrane channel influencing energy metabolism and cell viability. We treated cells with CyclosporinA (CsA), which binds to the matrix chaperone cyclophilin-D and regulates PTP opening. CsA reversed the effects of a 48-h treatment with E(2), suggesting a possible transcriptional modulation of proteins involved in the mitochondrial permeability transition process.
    Cell and Tissue Research 07/2012; 350(1):109-18. DOI:10.1007/s00441-012-1460-2 · 3.33 Impact Factor
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