Vascular actions of estrogens: functional implications.

The impact of estrogen exposure in preventing or treating cardiovascular disease is controversial. But it is clear that estrogen has important effects on vascular physiology and pathophysiology, with potential therapeutic implications. Therefore, the goal of this review is to summarize, using an integrated approach, current knowledge of the vascular effects of estrogen, both in humans and in experimental animals. Aspects of estrogen synthesis and receptors, as well as general mechanisms of estrogenic action are reviewed with an emphasis on issues particularly relevant to the vascular system. Recent understanding of the impact of estrogen on mitochondrial function suggests that the longer lifespan of women compared with men may depend in part on the ability of estrogen to decrease production of reactive oxygen species in mitochondria. Mechanisms by which estrogen increases endothelial vasodilator function, promotes angiogenesis, and modulates autonomic function are summarized. Key aspects of the relevant pathophysiology of inflammation, atherosclerosis, stroke, migraine, and thrombosis are reviewed concerning current knowledge of estrogenic effects. A number of emerging concepts are addressed throughout. These include the importance of estrogenic formulation and route of administration and the impact of genetic polymorphisms, either in estrogen receptors or in enzymes responsible for estrogen metabolism, on responsiveness to hormone treatment. The importance of local metabolism of estrogenic precursors and the impact of timing for initiation of treatment and its duration are also considered. Although consensus opinions are emphasized, controversial views are presented to stimulate future research.

Hormonal modulation of endothelial NO production.

Since the discovery of endothelium-derived relaxing factor and the subsequent identification of nitric oxide (NO) as the primary mediator of endothelium-dependent relaxations, research has focused on chemical and physical stimuli that modulate NO levels. Hormones represent a class of soluble, widely circulating chemical factors that impact production of NO both by rapid effects on the activity of endothelial nitric oxide synthase (eNOS) through phosphorylation of the enzyme and longer term modulation through changes in amount of eNOS protein. Hormones that increase NO production including estrogen, progesterone, insulin, and growth hormone do so through both of these common mechanisms. In contrast, some hormones, including glucocorticoids, progesterone, and prolactin, decrease NO bioavailability. Mechanisms involved include binding to repressor response elements on the eNOS gene, competing for co-regulators common to hormones with positive genomic actions, regulating eNOS co-factors, decreasing substrate for eNOS, and increasing production of oxygen-derived free radicals. Feedback regulation by the hormones themselves as well as the ability of NO to regulate hormonal release provides a second level of complexity that can also contribute to changes in NO levels. These effects on eNOS and changes in NO production may contribute to variability in risk factors, presentation of and treatment for cardiovascular disease associated with aging, pregnancy, stress, and metabolic disorders in men and women.

[Mechanism of estrogen-mediated neuroprotection: regulation of mitochondrial function].

Numerous studies show the neuroprotective effects of estrogen, but the underlying mechanism still remains unclear. Recent studies indicate that mitochondria are critically involved in estrogen-mediated neuroprotection. Mitochondria are the main sources of cellular energy and reactive oxygen species (ROS), they play an important role in signaling transduction and cellular life-death decisions. Estrogen exerts multiple effects on mitochondria under physiological and/or pathological conditions, these effects may include modulating ATP and ROS production, preserving mitochondria membrane potential, maintaining calcium homeostasis, and regulating mitochondrial gene and protein expression, etc. In this paper, we discussed the neuroprotective effects of estrogen, particularly focused on the underlying mechanisms related to mitochondria.

Mitochondrial effects of estrogen are mediated by estrogen receptor alpha in brain endothelial cells.

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.

Androgenic/estrogenic balance in the male rat cerebral circulation: metabolic enzymes and sex steroid receptors.

Tissues from males can be regulated by a balance of androgenic and estrogenic effects because of local metabolism of testosterone and expression of relevant steroid hormone receptors. As a critical first step to understanding sex hormone influences in the cerebral circulation of males, we investigated the presence of enzymes that metabolize testosterone to active products and their respective receptors. We found that cerebral blood vessels from male rats express 5alpha-reductase type 2 and aromatase, enzymes responsible for conversion of testosterone into dihydrotestosterone (DHT) and 17beta-estradiol, respectively. Protein levels of these enzymes, however, were not modulated by long-term in vivo hormone treatment. We also showed the presence of receptors for both androgens (AR) and estrogens (ER) from male cerebral vessels. Western blot analysis showed bands corresponding to the full-length AR (110 kDa) and ERalpha (66 kDa). Long-term in vivo treatment of orchiectomized rats with testosterone or DHT, but not estrogen, increased AR levels in cerebral vessels. In contrast, ERalpha protein levels were increased after in vivo treatment with estrogen but not testosterone. Fluorescent immunostaining revealed ERalpha, AR, and 5alpha-reductase type 2 in both the endothelial and smooth muscle layers of cerebral arteries, whereas aromatase staining was solely localized to the endothelium. Thus, cerebral vessels from males are target tissues for both androgens and estrogen. Furthermore, local metabolism of testosterone might balance opposing androgenic and estrogenic influences on cerebrovascular as well as brain function in males.

Influence of sex steroid hormones on cerebrovascular function.

The cerebral vasculature is a target tissue for sex steroid hormones. Estrogens, androgens, and progestins all influence the function and pathophysiology of the cerebral circulation. Estrogen decreases cerebral vascular tone and increases cerebral blood flow by enhancing endothelial-derived nitric oxide and prostacyclin pathways. Testosterone has opposite effects, increasing cerebral artery tone. Cerebrovascular inflammation is suppressed by estrogen but increased by testosterone and progesterone. Evidence suggests that sex steroids also modulate blood-brain barrier permeability. Estrogen has important protective effects on cerebral endothelial cells by increasing mitochondrial efficiency, decreasing free radical production, promoting cell survival, and stimulating angiogenesis. Although much has been learned regarding hormonal effects on brain blood vessels, most studies involve young, healthy animals. It is becoming apparent that hormonal effects may be modified by aging or disease states such as diabetes. Furthermore, effects of testosterone are complicated because this steroid is also converted to estrogen, systemically and possibly within the vessels themselves. Elucidating the impact of sex steroids on the cerebral vasculature is important for understanding male-female differences in stroke and conditions such as menstrual migraine and preeclampsia-related cerebral edema in pregnancy. Cerebrovascular effects of sex steroids also need to be considered in untangling current controversies regarding consequences of hormone replacement therapies and steroid abuse.

Vascular neuroeffector mechanisms: the next 30 years.

The 10th International Symposium on Vascular Neuroeffector Mechanisms was held on 12-15 July 2002 in Lake Tahoe, California, USA.

17beta-estradiol increases rat cerebrovascular prostacyclin synthesis by elevating cyclooxygenase-1 and prostacyclin synthase.

BACKGROUND AND PURPOSE: It has been reported that estrogens modulate peripheral vascular synthesis of vasodilatory hormones, including prostacyclin. If this occurs in the cerebral circulation, it could have important consequences in the modulation of cerebral hemodynamic function and improvement of stroke outcome. We investigated the hypothesis that in vivo 17beta-estradiol treatment of ovariectomized rats increases cerebrovascular prostacyclin production via elevation of the enzymes responsible for prostacyclin synthesis. METHODS: Cerebral blood vessels from 17beta-estradiol-treated and nontreated ovariectomized rats were isolated and examined for prostacyclin synthesis by enzyme-linked immunosorbent assay or for protein levels of cyclooxygenase-1, prostacyclin-synthase, and cytosolic phospholipase A2 by immunoblot analysis. RESULTS: We report that chronic in vivo 17beta-estradiol treatment significantly enhanced basal prostacyclin synthesis in rat cerebral blood vessels by 2.6-fold over control. 17beta-estradiol treatment also resulted in a 5.1-fold increase of cyclooxygenase-1 protein and a 6.7-fold increase of prostacyclin-synthase protein in the cerebral vasculature. There was no effect of estrogen on levels of cytosolic phospholipase A2. CONCLUSIONS: Our findings suggest that estrogen influences the biosynthesis of prostacyclin, which may be important in the regulation of cerebral blood flow and thrombosis. This finding may shed light on the mechanisms that govern sex-based differences in cerebrovascular disease.

Estrogen increases endothelial nitric oxide synthase via estrogen receptors in rat cerebral blood vessels: effect preserved after concurrent treatment with medroxyprogesterone acetate or progesterone.

BACKGROUND AND PURPOSE: In vivo and in vitro rat models of hormone therapy were used to test the following hypotheses: (1) estrogen acts directly on cerebrovascular estrogen receptors to increase endothelial nitric oxide synthase (eNOS); (2) increased protein correlates with higher NOS activity; and (3) effects of estrogen on eNOS are altered by concurrent treatment with either medroxyprogesterone acetate (MPA) or progesterone. METHODS: Blood vessels were isolated from brains of ovariectomized female rats; some were treated for 1 month with estrogen, estrogen and progesterone, or estrogen and MPA. Isolated cerebral vessels were also treated in vitro with estrogen in the absence and presence of progesterone, MPA, tamoxifen, and the estrogen receptor antagonist ICI 182 780. Levels of eNOS were measured by Western blot, and NOS activity was measured by [14C]arginine-[14C]citrulline conversion. RESULTS: Chronic hormone treatment in vivo resulted in plasma levels of 17beta-estradiol, progesterone, and MPA in the range of values found in humans. Estrogen treatment resulted in higher levels of cerebrovascular NOS activity that paralleled increases in eNOS protein. In vitro estrogen treatment for 18 hours also resulted in a concentration-dependent increase in eNOS protein (EC50 approximately 300 pmol/L) that was completely prevented by estrogen receptor antagonists tamoxifen or ICI 182 780. However, cotreatment with progesterone or MPA, either in vivo or in vitro, did not alter the effect of estrogen on eNOS protein. CONCLUSIONS: Estrogen receptor activation in cerebrovascular tissue results in increased eNOS activity and protein levels. The latter effect persists in the presence of either progesterone or MPA. Thus, increased NO production by eNOS may contribute to the neuroprotective effects of estrogen.

Chronic hypoxia alters the function of NOS nerves in cerebral arteries of near-term fetal and adult sheep.

In addition to adrenergic innervation, cerebral arteries also contain neuronal nitric oxide synthase (nNOS)-expressing nerves that augment adrenergic nerve function. We examined the impact of development and chronic high-altitude hypoxia (3,820 m) on nNOS nerve function in near-term fetal and adult sheep middle cerebral arteries (MCA). Electrical stimulation-evoked release of norepinephrine (NE) was measured with HPLC and electrochemical detection, whereas nitric oxide (NO) release was measured by chemiluminescence. An inhibitor of NO synthase, N(omega)-nitro-l-arginine methyl ester (l-NAME), significantly inhibited stimulation-evoked NE release in MCA from normoxic fetal and adult sheep with no effect in MCA from hypoxic animals. Addition of the NO donor S-nitroso-N-acetyl-dl-penicillamine fully reversed the effect of l-NAME in MCA from normoxic animals with no effect in MCA from hypoxic animals. Electrical stimulation caused a significant increase in NO release in MCA from normoxic animals, an effect that was blocked by the neurotoxin tetrodotoxin, whereas there was no increase in NO release in MCA from hypoxic animals. Relative abundance of nNOS as measured by Western blot analysis was similar in normoxic fetal and adult MCA. However, after hypoxic acclimitization, nNOS levels dramatically declined in both fetal and adult MCA. These data suggest that the function of nNOS nerves declines during chronic high-altitude hypoxia, a functional change that may be related to a decline in nNOS protein levels.

17 Beta-estradiol increases endothelial nitric oxide synthase mRNA copy number in cerebral blood vessels: quantification by real-time polymerase chain reaction.

The enzyme endothelial nitric oxide synthase (eNOS) plays a critical role in the maintenance of vascular tone. The mechanism by which estrogen increases eNOS function remains controversial. We demonstrate here using real-time polymerase chain reaction (PCR) and immunoblot analysis that in vivo estrogen treatment leads to a 100% increase in eNOS messenger RNA (mRNA) copy number and increases eNOS protein levels by 47% in mouse cerebral blood vessels. These data suggest that estrogen can modulate eNOS at the transcriptional level in blood vessels in vivo.

Male-female differences in the relative contribution of endothelial vasodilators released by rat tail artery.

Several different vasodilator substances can be released by vascular endothelium in response to mechanical stimuli and vasoactive agents. The purpose of this study was to determine whether there is a male-female difference in the relative contributions of nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) to endothelium-dependent vasodilation. Perfusion pressure was measured in isolated tail arteries from male and female rats. Vasodilators released by mechanical shear stress were assessed by constricting the artery with methoxamine; acetylcholine was applied to induce receptor-mediated vasodilation. We used an inhibitor of NO synthase, N(G)-monomethyl-L-arginine acetate (L-NMMA), and elevated levels of K(+) (27 mM) to reveal the relative contributions of NO and EDHF, respectively. Indomethacin was present in all experiments to block prostanoid production. The results indicate that NO was the primary vasodilator released by male tail arteries in response to both mechanical stress and acetylcholine (the L-NMMA-sensitive component of the combined L-NMMA/K(+) effect was 83 +/- 8% and 101 +/- 4%, respectively). However female tail arteries appeared to utilize both NO and EDHF for vascular relaxation (e.g., L-NMMA sensitivity: 58 +/- 9%; K+-sensitivity: 42 +/- 9% in mechanical stress experiments). These findings suggest endothelial regulation differs between males and females.

Aging and calcium buffering in adrenergic neurons.

The aging process at the cellular, organ and whole organism levels is in many respects a mystery. A common bias among those who study aging is that cellular homeostasis "generally falls apart". The assumption of a general deterioration in cellular homeostasis does not take into account that many individuals age quite well maintaining even robust physiological and mental functions. One facet of aging studies that has come to the forefront is the impact of age on the control of the ion messenger, calcium. Emerging evidence suggests that despite age-related declines in any one component or multiple components of the calcium buffering systems, compensatory mechanisms may be able to maintain overall calcium homeostasis. This brief review focuses specifically on the ability of peripheral neurons to maintain control of the ion messenger calcium with advancing age. In addition, the idea that the impact of age on calcium homeostasis may be more subtle due to complex and integrated mechanisms that control this ion is discussed.

Genomic and non-genomic regulation of PGC1 isoforms by estrogen to increase cerebral vascular mitochondrial biogenesis and reactive oxygen species protection.

We previously found that estrogen exerts a novel protective effect on mitochondria in brain vasculature. Here we demonstrate in rat cerebral blood vessels that 17ß-estradiol (estrogen), both in vivo and ex vivo, affects key transcriptional coactivators responsible for mitochondrial regulation. Treatment of ovariectomized rats with estrogen in vivo lowered mRNA levels of peroxisome proliferator-activated receptor-γ coactivator-1 alpha (PGC-1α) but increased levels of the other PGC-1 isoforms: PGC-1ß and PGC-1 related coactivator (PRC). In vessels ex vivo, estrogen decreased protein levels of PGC-1α via activation of phosphatidylinositol 3-kinase (PI3K). Estrogen treatment also increased phosphorylation of forkhead transcription factor, FoxO1, a known pathway for PGC-1α downregulation. In contrast to the decrease in PGC-1α, estrogen increased protein levels of nuclear respiratory factor 1, a known PGC target and mediator of mitochondrial biogenesis. The latter effect of estrogen was independent of PI3K, suggesting a separate mechanism consistent with increased expression of PGC-1ß and PRC. We demonstrated increased mitochondrial biogenesis following estrogen treatment in vivo; cerebrovascular levels of mitochondrial transcription factor A and electron transport chain subunits as well as the mitochondrial/nuclear DNA ratio were increased. We examined a downstream target of PGC-1ß, glutamate-cysteine ligase (GCL), the rate-limiting enzyme for glutathione synthesis. In vivo estrogen increased protein levels of both GCL subunits and total glutathione levels. Together these data show estrogen differentially regulates PGC-1 isoforms in brain vasculature, underscoring the importance of these coactivators in adapting mitochondria in specific tissues. By upregulating PGC-1ß and/or PRC, estrogen appears to enhance mitochondrial biogenesis, function and reactive oxygen species protection.

Endogenous ovarian hormones affect mitochondrial efficiency in cerebral endothelium via distinct regulation of PGC-1 isoforms.

Mitochondria support the energy-intensive functions of brain endothelium but also produce damaging-free radicals that lead to disease. Previously, we found that estrogen treatment protects cerebrovascular mitochondria, increasing capacity for ATP production while decreasing reactive oxygen species (ROS). To determine whether these effects occur specifically in endothelium in vivo and also explore underlying transcriptional mechanisms, we studied freshly isolated brain endothelial preparations from intact and ovariectomized female mice. This preparation reflects physiologic influences of circulating hormones, hemodynamic forces, and cell-cell interactions of the neurovascular unit. Loss of ovarian hormones affected endothelial expression of the key mitochondrial regulator family, peroxisome proliferator-activated receptor γ coactivator 1 (PGC-1), but in a unique way. Ovariectomy increased endothelial PGC-1α mRNA but decreased PGC-1ß mRNA. The change in PGC-1ß correlated with decreased mRNA for crucial downstream mitochondrial regulators, nuclear respiratory factor 1 and mitochondrial transcription factor A, as well as for ATP synthase and ROS protection enzymes, glutamate-cysteine ligase and manganese superoxide dismutase. Ovariectomy also decreased mitochondrial biogenesis (mitochondrial/nuclear DNA ratio). These results indicate ovarian hormones normally act through a distinctive regulatory pathway involving PGC-1ß to support cerebral endothelial mitochondrial content and guide mitochondrial function to favor ATP coupling and ROS protection.

17ß-Estradiol prevents cell death and mitochondrial dysfunction by an estrogen receptor-dependent mechanism in astrocytes after oxygen-glucose deprivation/reperfusion.

17ß-Estradiol (E2) has been shown to protect against ischemic brain injury, yet its targets and the mechanisms are unclear. E2 may exert multiple regulatory actions on astrocytes that may greatly contribute to its ability to protect the brain. Mitochondria are recognized as playing central roles in the development of injury during ischemia. Increasing evidence indicates that mitochondrial mechanisms are critically involved in E2-mediated protection. In this study, the effects of E2 and the role of mitochondria were evaluated in primary cultures of astrocytes subjected to an ischemia-like condition of oxygen-glucose deprivation (OGD)/reperfusion. We showed that E2 treatment significantly protects against OGD/reperfusion-induced cell death as determined by cell viability, apoptosis, and lactate dehydrogenase leakage. The protective effects of E2 on astrocytic survival were blocked by an estrogen receptor (ER) antagonist (ICI-182,780) and were mimicked by an ER agonist selective for ERα (PPT), but not by an ER agonist selective for ERß (DPN). OGD/reperfusion provoked mitochondrial dysfunction as manifested by an increase in cellular reactive oxygen species production, loss of mitochondrial membrane potential, and depletion of ATP. E2 pretreatment significantly inhibited OGD/reperfusion-induced mitochondrial dysfunction, and this effect was also blocked by ICI-182,780. Therefore, we conclude that E2 provides direct protection to astrocytes from ischemic injury by an ER-dependent mechanism, highlighting an important role for ERα. Estrogen protects against mitochondrial dysfunction at the early phase of ischemic injury. However, overall implications for protection against brain ischemia and its complex sequelae await further exploration.

Estrogen-receptor-mediated protection of cerebral endothelial cell viability and mitochondrial function after ischemic insult in vitro.

Protective effects of estrogen against experimental stroke and neuronal ischemic insult are well-documented, but it is not known whether estrogen prevents ischemic injury to brain endothelium, a key component of the neurovascular unit. Increasing evidence indicates that estrogen exerts protective effects through mitochondrial mechanisms. We previously found 17beta-estradiol (E2) to improve mitochondrial efficiency and reduce mitochondrial superoxide in brain blood vessels and endothelial cells. Thus we hypothesized E2 will preserve mitochondrial function and protect brain endothelial cells against ischemic damage. To test this, an in vitro ischemic model, oxygen-glucose deprivation (OGD)/reperfusion, was applied to immortalized mouse brain endothelial cells (bEnd.3). OGD/reperfusion-induced cell death was prevented by long-term (24, 48 h), but not short-term (0.5, 12 h), pretreatment with 10 nmol/L E2. Protective effects of E2 on endothelial cell viability were mimicked by an estrogen-receptor (ER) agonist selective for ERalpha (PPT), but not by one selective for ERbeta (DPN). In addition, E2 significantly decreased mitochondrial superoxide and preserved mitochondrial membrane potential and ATP levels in early stages of OGD/reperfusion. All of the E2 effects were blocked by the ER antagonist, ICI-182,780. These findings indicate that E2 can preserve endothelial mitochondrial function and provide protection against ischemic injury through ER-mediated mechanisms.

Dihydrotestosterone stimulates cerebrovascular inflammation through NFkappaB, modulating contractile function.

Our previous studies show that long-term testosterone treatment augments vascular tone under physiological conditions and exacerbates endotoxin-induced inflammation in the cerebral circulation. However, testosterone can be metabolized by aromatase to estrogen, evoking a balance between androgenic and estrogenic effects. Therefore, we investigated the effect of the nonaromatizable androgen receptor agonist, dihydrotestosterone (DHT), on the inflammatory nuclear factor-kappaB (NFkappaB) pathway in cerebral blood vessels. Cerebral arteries were isolated from orchiectomized male rats treated chronically with DHT in vivo. Alternatively, pial arteries were isolated from orchiectomized males and were exposed ex vivo to DHT or vehicle in culture medium. DHT treatment, in vivo or ex vivo, increased nuclear NFkappaB activation in cerebral arteries and increased levels of the proinflammatory products of NFkappaB activation, cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). Effects of DHT on COX-2 and iNOS were attenuated by flutamide. In isolated pressurized middle cerebral arteries from DHT-treated rats, constrictions to the selective COX-2 inhibitor NS398 or the selective iNOS inhibitor L-nil, [L-N6-(Iminoethyl)lysine], were increased, confirming a functional consequence of DHT exposure. In conclusion, activation of the NFkappaB-mediated COX-2/iNOS pathway by the selective androgen receptor agonist, DHT, results in a state of vascular inflammation. This effect may contribute to sex-related differences in cerebrovascular pathophysiology.

Cerebrovascular effects of oestrogen: multiplicity of action.

1. Cerebral vessels express oestrogen receptors (ER) in both the smooth muscle and endothelial cell layers of cerebral blood vessels. Levels of ERalpha are higher in female rats chronically exposed to oestrogen, either endogenous or exogenous. 2. Chronic exposure to oestrogen, either endogenous (normally cycling females) or exogenous (ovariectomized with oestrogen replacement), results in cerebral arteries that are more dilated than arteries from ovariectomized counterparts when studied in vitro. This effect is primarily mediated by an increase in the production of vasodilator factors, including nitric oxide (NO) and prostacylin. In contrast, oestrogen appears to suppress the production of endothelial-derived hyperpolarizing factor. Oestrogen treatment increases cerebrovascular levels of endothelial nitric oxide synthase (eNOS), cyclo-oxygenase (COX)-1 and prostacyclin synthase. In addition, via activation of the phosphatidylinositol 3-kinase/Akt pathway, both acute and chronic oestrogen exposure increases eNOS phosphorylation, increasing NO production. 3. Oestrogen receptors have also been localized to cerebrovascular mitochondria and exposure to oestrogen increases the efficiency of energy production while simultaneously reducing mitochondrial production of reactive oxygen species. Oestrogen increases the production of mitochondrial proteins encoded by both mitochondrial and nuclear DNA, including cytochrome c, subunits I and IV of complex IV and Mn-superoxide dismutase. Oestrogen treatment increases the activity of citrate synthase and complex IV and decreases mitochondrial production of H(2)O(2). 4. Oestrogen also has potent anti-inflammatory effects in the cerebral circulation that may have important implications for the incidence and severity of cerebrovascular disease. Administration of lipopolysaccharide or interleukin-1beta to ovariectomized female rats induces cerebrovascular COX-2 and inducible nitric oxide synthase (iNOS) protein expression and increases prostaglandin E(2) expression. Levels of COX-2 and iNOS expression vary with the stage of the oestrous cycle, and the cerebrovascular inflammatory response is suppressed in ovariectomized animals treated with oestrogen. Interleukin-1beta induction of COX-2 protein is prevented by treatment with a nuclear factor (NF)-kappaB inhibitor, and oestrogen treatment reduces cerebrovascular NF-kappaB activity. 5. Cerebrovascular dysfunction and pathology contribute to the pathogenesis of stroke, brain trauma, oedema and dementias, such as Alzheimer's disease. A better understanding of the action of oestrogen on cerebrovascular function holds promise for the development of new therapeutic entities that could be useful in preventing or treating a wide variety of cerebrovascular diseases.

Estrogen suppresses brain mitochondrial oxidative stress in female and male rats.

Mitochondria are a major source of reactive oxygen species (ROS) and oxidative stress, key contributors to aging and neurodegenerative disorders. We report that gonadal hormones influence brain mitochondrial ROS production in both females and males. Initial experiments showed that estrogen decreases mitochondrial superoxide production in a receptor-mediated manner, as measured by MitoSOX fluorescence in differentiated PC-12 cells. We then assessed in vivo effects of gonadal hormones on brain mitochondrial oxidative stress in female and male rats. Brain mitochondria were isolated to measure a functional indicator of ROS, i.e., activity of the ROS-sensitive mitochondrial enzyme, aconitase. Gonadectomy of both males and females caused a decrease in aconitase activity, suggesting that endogenous gonadal hormones influence mitochondrial ROS production in the brain. In vivo treatment of gonadectomized animals with testosterone or dihydrotestosterone (DHT) had no effect, but estrogen replacement significantly increased aconitase activity in brain mitochondria from both female and male rats. This indicates that estrogen decreases brain mitochondrial ROS production in vivo. Sex hormone treatments did not affect protein levels of brain mitochondrial uncoupling proteins (UCP-2, 4, and 5). However, estrogen did increase the activity, but not the levels, of manganese superoxide dismutase (MnSOD), the mitochondrial enzyme that catalyzes superoxide radical breakdown, in brain mitochondria from both female and male rats. Thus, in contrast to the lack of effect of androgens on mitochondrial ROS, estrogen suppression of mitochondrial oxidative stress may influence neurological disease incidence and progression in both females and males.