Atheroprotective Effects Of Estrogen Research Articles

Abstract 452: Estrogen Inhibits LDL Transcytosis by Coronary Artery Endothelial Cells via GPER and SR-BI

Objectives: Premenopausal women have a significantly lower rate of atherosclerosis than men, an effect attributed to estrogen. The atheroprotective effects of estrogen are independent of circulating lipid levels and may be due to an effect on the vessel wall. Early on in atherosclerosis, circulating LDL is able to cross an intact endothelial monolayer by transcytosis and accumulate in the intima. Little is known about the mechanisms of transcytosis but using novel assays we recently discovered a role for the SR-BI and ALK1 receptors. We hypothesized that estrogen can attenuate LDL transcytosis by coronary artery endothelial cells. Approach and Results: Using a recently described assay based on total internal reflection fluorescence microscopy, we quantified transcytosis of LDL across human coronary artery endothelial cells treated with physiological concentrations of estrogen. Estrogen significantly attenuated LDL transcytosis by endothelial cells from male donors; the effect was ligand-specific as transcytosis of albumin was not affected. Estrogen caused down-regulation of endothelial SR-BI but not ALK1 and over-expression of SR-BI was sufficient to restore LDL transcytosis. Similarly, depletion of SR-BI by siRNA attenuated endothelial LDL transcytosis and prevented any further effect of estrogen. In contrast, treatment with estrogen had no effect on SR-BI expression by liver cells from a male donor. Inhibition of estrogen receptors α and β had no effect on estrogen-mediated attenuation of LDL transcytosis. However, estrogen’s effect was blocked by depletion of the G-protein coupled estrogen receptor (GPER) by siRNA. GPER was found to be enriched in endothelial cells compared to hepatocytes and is known to signal via transactivation of the epidermal growth factor receptor (EGFR); we observed that inhibition of EGFR also prevented the effect of estrogen on LDL transcytosis. Lastly, male mice demonstrated more vascular deposition of LDL than age-matched female mice after acute injection. Conclusions: Physiological concentrations of estrogen significantly inhibit LDL transcytosis by down-regulating endothelial SR-BI; this effect requires GPER. This may contribute to the lower rates of atherosclerosis in premenopausal women.

17β-Estradiol Suppresses the Macrophage Foam Cell Formation Associated with SOCS3

Evidence from clinical trials and animal experiments has shown that estrogen has anti-atherosclerotic effects when administered to young women or experimental animals. The mechanisms involve the modulation of vascular inflammation, growth factor expression, and oxidative stress injured arteries. However, whether estrogen modulates the foam cell formation in plaque remains unknown. Here, we investigated the effects of 17β-estradiol (E2) on cholesterol efflux in vivo and in vitro. ApoE null mice underwent an ovariectomy at 5(th) week of age and then were treated with E2 or vehicle for the following 8 weeks. Compared with the vehicle-treated mice, the serum total cholesterol level, atherosclerotic plaque size, and lipid deposits were decreased and meanwhile ATP-binding cassette transporter A1 (ABCA1) expression in the plaque was increased in mice with E2 treatment. E2 also increased suppressor of cytokine signaling 3 (SOCS3) expression in the atherosclerotic plaques and in RAW264.7 cells. In vitro, E2 treatment reversed janus kinase/signal transducers and activators of transcription (JAK/STAT)-inhibited ABCA1 expression in RAW264.7 cells but had no effect on ABCA1 expression in SOCS3 knockdown cells. SOCS3 overexpression elevated ABCA1 expression through the inhibition of JAK2/STAT3 phosphorylation. Finally, we also found that E2 enhanced the cholesterol efflux to apoA I in RAW264.7 cells. In summary, E2 reduces atherosclerosis in ApoE null mice associated with upregulating ABCA1 expression and modulating the cholesterol efflux, which are dependent on SOCS3 upregulation. These results provide new insight into the athero-protective effects of estrogen.

Association of Estrogen Receptor α Genotypes/ Haplotypes With Carotid Intima-Media Thickness in Taiwanese Women

The estrogen receptor alpha gene (ESR1) is an important mediator of the atheroprotective effect of estrogen on the vasculature system. We examined the potential associations between common single nucleotide polymorphism (SNP) variants of ESR1 and intima-media thickness (IMT) in carotid arteries, a strong predictor of cardiovascular disease (CVD). A total of 760 study participants (343 men and 407 women), who had undergone a Duplex ultrasonographic examination of carotid artery, were investigated. Measurement of IMT was performed on a 10-mm segment of the common carotid artery (CCA). Fourteen sequence-validated SNPs of high frequency of Oriental origin were selected and genotyped by the method of Light-Cycler-480-assisted real-time polymerase chain reaction (PCR) followed by melting curve analysis. Results from multiple linear regression analyses showed significant associations of SNPs rs2228480 (Ex8+229G>A) and rs3798758 (Ex8+1988C>A) with the carotid IMT values in women but not in men. Women with SNP rs2228480 (Ex8+229G>A) A/A genotype had a 0.048 mm (7.1%) increase in IMT values versus the other genotypes combined (P = .030). In women who carried the rs3798758 (Ex8+1988C>A) CA+AA combined genotypes, their carotid IMT measures were 0.020 mm (2.9%) decreased as compared with those in women who carried C/ C genotype (P = .042). In haplotype analysis, women with the T-A haplotype versus C-C haplotype of combined rs3798577 (Ex8+1264T>C) and rs3798758 (Ex8+1988C>A) were also found to be associated with a decreased IMT value at a borderline significance (P = .057). Some common SNPs in the ESR1 could be important in modulating carotid atherosclerosis and thereby CVD susceptibility in Taiwanese women.

Estrogen stimulation of COX-2-derived PGI 2 confers atheroprotection

Although selective cyclooxygenase-2 (COX-2) inhibitors provide relief from pain and inflammation, they also reduce the formation of the atheroprotective prostaglandin I2 (PGI2). They do not reduce the formation of the COX-1-derived thromboxane A2 (TXA2), however, which is both atherogenic and a potent vasoconstrictor. For this reason, the effects of TXA2 might be exacerbated during extended therapy with COX-2 inhibitors, potentially predisposing patients to heart attack and stroke. Recent studies have demonstrated that the atheroprotective effects of estrogen are induced through PGI2 production, through COX-2 activation. This explains how estrogen production in pre-menopausal females is beneficial for the heart and also raises the possibility that COX-2 inhibitors might be particularly hazardous to females.

Estrogen Receptors Act in Atherosclerosis

Men experience a more rapid progression of atherosclerosis, but the basis for this gender difference has not been clear. The prostacyclin PGI 2 prevents many processes associated with the formation of atherosclerotic lesions, and the atheroprotective effect of estrogen in women may be via stimulation of PGI 2 production. Egan et al . (see the news story by Couzin) now show in a mouse model of atherosclerosis that estrogen acts through the estrogen receptor subtype α to generate PGI 2 through cyclooxygenase 2 (COX-2). Female mice lacking a receptor for PGI 2 developed atherosclerosis as rapidly as male mice and had poor response to estrogen therapy. This mechanism may be important in assessing the effects of hormone replacement therapy and selective COX-2 inhibitors. K. M. Egan, J. A. Lawson, S. Fries, B. Koller, D. J. Rader, E. M. Smyth, G. A. FitzGerald, COX-2-derived prostacyclin confers atheroprotection on female mice. Science 306 , 1954-1957 (2004). [Abstract] [Full Text] J. Couzin, Estrogen's ties to COX-2 may explain heart disease gender gap. Science 306 , 1277 (2004). [Summary] [Full Text]

Estrogen and homocysteine.

Cardiovascular diseases are the major causes of illness and death in women. Premenopausal women are relatively protected from coronary artery disease and atherosclerosis as compared to postmenopausal women, and this protection is attributed to the effects of the female sex hormone (estrogen). The vasculature, like the reproductive tissues, bone, liver, and brain, is now recognized as an important site of estrogen's action. Although estrogen's beneficial effects on the cardiovascular system are well described in many studies, the molecular basis of estrogen protective mechanisms are still quite vague. Both genomic mechanisms, mediated primarily through estrogen receptor alpha (ER alpha) and estrogen receptor beta (ER beta), and non-genomic mechanisms, through nitric oxide (NO), of estrogen action are controversial and do not entirely explain the effects of estrogen on vascular preservation during conditions of oxidative stress. Until recently, the atheroprotective effects of estrogen were attributed principally to its effects on serum lipid concentrations and cholesterol levels. However, two recent reports that estrogen therapy has no effect on the progression of coronary atherosclerosis in women with established disease, despite the favorable changes in LDL and cholesterol levels, leads to questions about the lipid/cholesterol mechanism of estrogen-mediated effects on atherosclerosis. Alternatively, the high level of homocysteine, found to correlate with accelerated cardiovascular disease and identified as an independent risk factor for atherosclerosis, was recently described to be diminished by estrogen. Protection against disturbed sulfhydryl metabolism and higher homocysteine level could be the missing link in understanding how exactly estrogen affects vascular cells metabolism and responses to oxidative stress. This review focuses on estrogen/homocysteine interactions and their relevance to the cardiovascular system.

Angioscopic evaluation of the effect of estrogen on ovariectomized Japanese monkeys.

Using angioscopy to study the atheroprotective effect of estrogen, atherosclerotic changes in surgically postmenopausal Japanese monkeys were observed for a long period. Changes in serum lipids and estradiol were also followed. About 3-6 months after the 2% cholesterol diet, serum cholesterol increased up to three times normal value, and atheroma was first found at the orifice of major branches of the abdominal aorta. However, in the other group of 2% cholesterol including an oral estrogen diet, the appearance of atheroma was inhibited until 12 months, although serum cholesterol increased gradually. The chronologic investigation was continued over 3 years. From these results we concluded that the most important atheroprotective effect of estrogen is the direct inhibition of atherosclerosis in the arterial wall, and improvement in lipid metabolism is of secondary importance.

Effect of 17-β estradiol on pre-existing atherosclerotic lesions: role of the endothelium

The atheroprotective effects of estrogen during the process of atherogenesis is well documented, whereas limited information is available about the effect of estrogen on pre-existing atherosclerotic lesions. After bilateral ovariectomy, 24 New Zealand White rabbits were randomized into three groups of eight animals each and subsequently fed a 0.5% cholesterol diet. In group I, the vessels were excised at day 84, whereas in group II, the cholesterol diet was continued for a total of 168 days. In group III, the animals were first fed with a cholesterol diet for 84 days; in the second phase of the experiment, the cholesterol diet was continued for a further 84 days with a combined estrogen treatment (1 mg estradiol valerate per kg body weight per week intramuscularly). At the end of the experiment, the proximal aortic arch, right carotid artery, thoracical aorta and abdominal aorta of each animal were excised and prepared for histological and immunohistological examination. By day 168, morphometrical analysis displayed a significantly lower plaque development under estrogen therapy in the carotid artery (0.08±0.18 mm 2 vs. 0.60±0.39 mm 2), the thoracic aorta (0.56±0.94 mm 2 vs. 3.63±2.06 mm 2), and in the abdominal aorta (0.55±0.70 mm 2 vs. 1.71±1.05 mm 2) in comparison with the corresponding 168 day control group. However, estrogen treatment has failed to reduce further atherosclerotic plaque development in the aortic arch (9.42±1.79 mm 2 vs. 11.64±3.29 mm 2). Immunohistological detection of the ‘anti-human factor VIII related antigen’, i.e. the ‘von Willebrand factor’ (vWF), showed a significantly lower number of luminal cells positive for vWF in the aortic arch in the 84-day cholesterol group, compared with the corresponding controls of normocholesterolemic rabbits (65.9±12.4% vs. 83.1±6.2%; P<0.05). Estradiol was able to inhibit the further progression of atherosclerosis when moderate vessel wall alterations were present, whereas pre-existing severe atherosclerosis was associated with a failure of the anti-atherosclerotic estrogen action. As suggested by the in situ detection of vWF as a morphological marker for endothelial cells, an intact endothelial layer might play an important role in mediating the beneficial effect of estrogen in the process of atherosclerosis.

Estrogen receptor-alpha gene transfer into bovine aortic endothelial cells induces eNOS gene expression and inhibits cell migration.

It has been suggested that estrogen may improve endothelial cell function to delay the onset of atherosclerosis in pre-menopausal females, though its mechanism of action is not fully understood. We examined the hypothesis that human estrogen receptor-alpha (ER alpha) gene transfection improves the endothelial cell function. A replication deficient adenoviral vector was used to transfect the ER alpha gene into bovine aortic endothelial cells (BAEC) and a GFP gene containing vector was used as control. Expression of the eNOS gene was determined by Northern blot analysis and enzyme activity assay; cell migration was assayed using a Transwell apparatus; and tyrosine phosphorylation of FAK was estimated by Western blot analysis. ER alpha gene transfection of endothelial cells produced a 2-3-fold increase in eNOS mRNA and protein levels as well as a significant increase (P < 0.05) in NOS activity as measured by citrulline assay and nitrite accumulation in the media in response to bradykinin stimulation. Treatment of cells with estrogen blocking agent ICI 182780 inhibited eNOS induction in response to ER alpha transfection. ER alpha gene transfection significantly inhibited (P < 0.05) bFGF-induced chemotactic migration of endothelial cells but increased cell attachment to fibronectin, laminin, and type I and IV collagens. ER alpha gene transfer also inhibited bFGF-stimulated tyrosine phosphorylation of FAK. Our results suggest that the atheroprotective effects of estrogen may in part be mediated by ER alpha-induced upregulation of eNOS gene expression and maintenance of endothelial cell function and integrity.

Localized expression of aromatase in human vascular tissues.

The atheroprotective effects of estrogen are well established and the presence of an estrogen receptor in vascular tissues has recently been reported. Therefore, we investigated the localization of the estrogen-producing enzyme aromatase in vascular tissues to assess the possible contribution of endocrine, paracrine, and autocrine modes of action. Aromatase was found in human vascular smooth muscle cells (SMCs) but not in endothelial cells on in situ hybridization. These observations were further supported by quantitative analysis of aromatase mRNA and the activity in 15 human vascular specimens. Only trace levels of expression were detected in the 3 infants examined, whereas 0.0088 to 0.0806 amol/ microg RNA of aromatase mRNA and 12.9 to 122.3 fmol. h-1. mg-1 protein of the activity were detected in 12 of the adult individuals. The switching of tissue-specific exon 1 of the human aromatase gene was also observed in some cases. Aromatase was found to be expressed only in cultured SMCs and not in cultured endothelial cells of human aorta and pulmonary artery and to be regulated through dexamethasone and the signaling pathways of protein kinase A and C. Study results revealed the localized expression of aromatase in vascular SMCs, which indicated a possible direct action of locally produced estrogen in an autocrine or paracrine manner, with possible cross talk between smooth muscle and endothelial cells.

Estrogen inhibits the vascular injury response in estrogen receptor alpha-deficient mice.

The atheroprotective effects of estrogen in women are well recognized, but the underlying mechanisms responsible are not well understood. Blood vessel cells express the classic estrogen receptor, ER alpha (ref. 2-6), and are directly affected by estrogen, which inhibits the development of atherosclerotic and injury-induced vascular lesions. We have generated mice in which the ER alpha gene is disrupted and have used a mouse model of carotid arterial injury to compare the effects of estrogen on wild-type and estrogen receptor-deficient mice. Increases in vascular medial area and smooth muscle cell proliferation were quantified following vascular injury in ovariectomized mice treated with vehicle or with physiologic levels of 17 beta-estradiol. Surprisingly, in both wild-type and estrogen receptor-deficient mice, 17 beta-estradiol markedly inhibited to the same degree all measures of vascular injury. These data demonstrate that estrogen inhibits vascular by a novel mechanism that is independent of the classic estrogen receptor, ER alpha.

P101 Direct atheroprotective effect of estrogen on human smooth muscle cells and endothelial cells

P101 Direct atheroprotective effect of estrogen on human smooth muscle cells and endothelial cells

Different effects of estrogen and progesterone on experimental atherosclerosis in female versus male rabbits. Quantification of cellular proliferation by bromodeoxyuridine.

The aim of the present study was to compare the effect of estrogen and progesterone on the development of experimental atherosclerosis in female versus male rabbits to assess possible sex-specific differences. A total of 32 female and 32 male New Zealand White rabbits were ovariectomized or castrated. In addition to a 0.5% cholesterol diet, the rabbits received estradiol alone (1 mg/kg body wt [BW] per week), progesterone alone (25 mg/kg BW per week), or combined estradiol-progesterone in these dosages during 12 weeks. Ovariectomized female and castrated male rabbits served as control groups without hormone treatment. Before excision of the vessels, bromodeoxyuridine labeling was performed to determine the extent of cellular proliferation in the atherosclerotic lesions. The aortic arch was analyzed immunohistologically and morphometrically. An inhibitory effect of estrogen on intimal plaque size was found in female rabbits compared with the ovariectomized control group (0.7 +/- 0.5 versus 3.7 +/- 2.5 mm2, P < .002; proliferating cells, 3.1 +/- 1.8% versus 8.5 +/- 2.6%, P < .002). In combination with progesterone, however, estrogen was not able to reduce intimal plaque size or cellular proliferation. In contrast, estradiol in castrated male rabbits was not associated with an inhibitory effect on cellular proliferation or intimal thickening compared with controls (estrogen treatment, 7.6 +/- 2.1% proliferating cells and 2.8 +/- 1.0 mm2 neointima; control group, 7.2 +/- 2.1% cellular proliferation and 2.9 +/- 1.2 mm2 intimal thickening). Our data suggest that the atheroprotective effect of estrogen is probably due to a mechanism that is present in female rabbits only.

Differential-display polymerase chain reaction identifies nucleophosmin as an estrogen-regulated gene in human vascular smooth muscle cells

Atheroprotective effects of estrogen (E2) are well documented and are due in part to direct effects of E2 on vascular cells. We recently demonstrated that human vascular smooth muscle cells (VSMCs) express a functional E2 receptor capable of activatine gene expression. This study was designed to identify E2-regulated genes in human VSMCs. VSMC mRNA was screened by differential-display polymerase chain reaction (ddPCR) to identify E2-regulated genes. Quiescent human VSMCs were stimulated with 10% fetal bovine serum in the absence or presence of 10(-8) mol/L E2, and total cellular RNA was harvested. ddPCR was performed in triplicate on each RNA sample and differentially expressed candidate genes were isolated. Differential expression of candidate genes were confirmed by dot blotting and positive bands were identified by sequence analysis. E2-mediated regulation at the protein level was investigated by immunoblotting. ddPCR of RNA harvested from aortic VSMCs identified a 462 bp gene product, EAo8, as a candidate E2- regulated gene. Dot blotting with probes derived from four independent VSMC cultures demonstrated a 5.8 +/- 3.9-fold increase in EAo8 expression in response to E2 treatment (p < 0.05 vs control). Sequence analysis identified EAo8 as nucleophosmin, a nucleolar phosphprotein implicated in the regulation of cell growth and protein synthesis. E2-induced expression of nucleophosmin protein was demonstrated by immunoblotting in both saphenous vein and mammary artery VSMCs. E2 induces nucleophosmin expression in human VSMCs, and ddPCR is a useful approach for studying estrogen-regulated gene expression in VSMCs.

Estrogen inhibits the response-to-injury in a mouse carotid artery model.

The atheroprotective effects of estrogen are well documented, but the mechanisms responsible for these effects are not well understood. To study the role of physiologic (nanomolar) estrogen levels on the arterial response-to-injury, we applied a mouse carotid artery injury model to ovariectomized C57BL/6J mice. Mice were treated with vehicle (-E2, n = 10) or 17 beta-estradiol (+E2, n = 10) for 7 d, subjected to unilateral carotid injury, and 14 d later contralateral (normal = NL) and injured carotids from -E2 and +E2 animals were pressure fixed, harvested, and analyzed by quantitative morphometry. E2 levels in +E2 mice were consistently in the nanomolar range (2.1-2.5 nM) at days 0, 7, and 14. At 14 d, measures of both intimal and medial area were markedly increased in the -E2 group: (-E2 vs NL, P < 0.05 for both), but were unchanged from normal levels in the +E2 group (+E2 vs NL, P = NS and +E2 vs -E2, P < 0.05 for both). Cellular proliferation, as assessed by bromodeoxyuridine (BrdU) labeling, was significantly increased over NL in the -E2 mice, but this increase was markedly attenuated in the estrogen replacement group (total BrdU positive cells/section: NL = 6.4 +/- 4.5; -E2 = 113 +/- 26, +E2 = 40 +/- 3.7; -E2 vs NL, P < 0.05; +E2 vs NL, P = NS; -E2 vs +E2, P < 0.05). These data (a) demonstrate significant suppression of the mouse carotid response-to-injury by physiologic levels of estrogen replacement; (b) support the utility of this model in the study of the biologic effects of estrogen on the vascular-injury response; and (c) suggest a direct effect of estrogen on vascular smooth muscle cell proliferation in injured vessels.