Oestrogen Inhibits VEGF Expression And Angiogenesis In Triple-Negative Breast Cancer By Activating GPER-1.

Triple-negative breast cancer (TNBC) is the most malignant type of breast cancer with ample vascularisation and high vascular endothelial growth factor (VEGF) expression. The sex steroid hormone oestrogen is involved in several cellular activities associated with TNBC regulation. However, the role of oestrogen in VEGF expression and angiogenesis in TNBC remains unclear. In this study, we found that treatment with 17ß-oestradiol (E2) inhibited VEGF mRNA and protein expression in the TNBC cell lines MDA-MB-468 and MDA-MB-436. To further elaborate on the phenomenon of E2-regulated angiogenesis, we showed that conditioned medium from the TNBC cell line MDA-MB-468 treated with E2 inhibits the tube formation ability of human umbilical vein endothelial cells (HUVECs). Additionally, the G-protein-coupled oestrogen receptor-1 (GPER-1)-specific agonist G-1 has a function similar to that of E2. While G-15, the selective antagonist of GPER-1, notably reversed the inhibitory effects of E2 and G-1 on VEGF expression and tube formation, suggesting that GPER-1 is involved in the E2-induced angiogenesis suppression in TNBC cells. Moreover, E2 inhibited in vivo tumour growth and angiogenesis and reduced the expression levels of VEGF, NF-κB/p65, STAT3, and the endothelial marker CD34 in MDA-MB-468 xenograft tumours. Our findings provide important evidence that E2 can inhibit VEGF expression and angiogenesis in TNBC by activating GPER-1, offering additional insight into tumour angiogenesis and targets for drug intervention in TNBC.

[Advances of the Role of Ezrin in Migration and Invasion of Breast Cancer Cells].

Ezrin, also known as cytovillin or vilin 2, is one of the members of ERM (Ezrin/Radixin/Moesin) protein family. Ezrin, which is a tyrosine kinase substrate, functions to bridge membrane proteins and the actin cytoskeleton. Recent studies have demonstrated that Ezrin regulates the proliferation, apoptosis, adhesion, invasion, metastasis and angiogenesis of breast cancer cells. These processes are not only associated with changes in expression level and subcellular localization of Ezrin itself, but also influenced by alteration in microenvironment of primary breast cancer cells. The regulation of Ezrin in mammary carcinoma cells involves interactions among signaling pathways mediated by adhesion molecules (CD44, ICAM, E-cadherin) and the tyrosine kinase growth factors, Epidermal Growth Factor (EGF), and Platelet-derived Growth Factor (PDGF) and their receptors. The determination of the functions and mechanism(s) of action of Ezrin in the migration and invasion of breast cancer cells will provide new information on the basic mechanisms of metastasis of breast cancer cells and has the potential to identify a novel drug target for the prevention and treatment of breast cancer. This article addresses the role of Ezrin in the migration and invasion of breast cancer cells.

Oestrogen exerts anti-inflammation via p38 MAPK/NF-κB cascade in adipocytes.

BACKGROUND: Oestrogen has anti-inflammatory property in obesity. However, the mechanism is still not defined. OBJECTIVE: To investigate the effect of oestrogen on LPS-induced monocyte chemoattractant protein-1 (MCP-1) production in adipocytes. METHODS: Lipopolysaccharides (LPS) was used to imitate inflammatory responses and monocyte chemotactic protein-1 (MCP-1) was selected as an inflammatory marker to observe. 17ß-Estradiol (E2), SB203580 (SB), pyrrolidine dithiocarbamate (PDTC), pertussis toxin (PTX), wortmannin (WM), p65 siRNA and p38 MAPK siRNA were pre-treated respectively or together in LPS-induced MCP-1. Then p38 MAPK and NF-κB cascade were silenced successively to observe the change of each other. Lastly, oestrogen receptor (ER) α agonist, ERß agonist and ER antagonist were utilised. RESULTS: LPS-induced MCP-1 largely impaired by pre-treatment with E2, SB, PDTC or silencing NF-κB subunit. E2 inhibited LPS-induced MCP-1 in a time- and dose-dependent manner, which was related to the suppression of p65 translocation to nucleus. Furthermore, LPS rapidly activated p38 MAPK, while E2 markedly inhibited this activation. It markedly attenuated LPS-stimulated p65 translocation to nucleus and MCP-1 production by transfecting with p38 MAPK siRNA or using p38 MAPK inhibitor. The oestrogen's inhibitory effect was mimicked by the ERα agonist, but not by the ERß agonist. The inhibition of E2 on p38 MAPK phosphorylation was prevented by ER antagonist. CONCLUSIONS: E2 inhibits LPS-stimulated MCP-1 in adipocytes. This effect is related to the inhibition of p38 MAPK/NF-κB cascade, and ERα appears to be the dominant ER subtype in these events.

Caveolin-3 is involved in the protection of resveratrol against high-fat-diet-induced insulin resistance by promoting GLUT4 translocation to the plasma membrane in skeletal muscle of ovariectomized rats.

Insulin resistance is recognized as a common metabolic factor which predicts the future development of both type 2 diabetes and atherosclerotic disease. Resveratrol (RSV), an agonist of estrogen receptor (ER), is known to affect insulin sensitivity, but the mechanism is unclear. Evidence suggests that caveolin-3 (CAV-3), a member of the caveolin family, is involved in insulin-stimulated glucose uptake. Our recent work indicated that estrogen via ER improves glucose uptake by up-regulation of CAV-3 expression. Here, we investigated the role of CAV-3 in the effect of RSV on insulin resistance in skeletal muscle both in vivo and in vitro. The results demonstrated that RSV ameliorated high-fat-diet (HFD)-induced glucose intolerance and insulin resistance in ovariectomized rats. RSV elevated insulin-stimulated glucose uptake in isolated soleus muscle in vivo and in C2C12 myotubes in vitro by enhancing GLUT4 translocation to the plasma membrane rather than increasing GLUT4 protein expression. Through ERα-mediated transcription, RSV increased CAV-3 protein expression, which contributed to GLUT4 translocation. Moreover, after knockdown of CAV-3 gene, the effects of RSV on glucose uptake and the translocation of GLUT4 to the plasma membrane, as well as the association of CAV-3 and GLUT4 in the membrane, were significantly attenuated. Our findings demonstrated that RSV via ERα elevated CAV-3 expression and then enhanced GLUT4 translocation to the plasma membrane to promote glucose uptake in skeletal muscle, exerting its protective effects against HFD-induced insulin resistance. It suggests that this pathway could represent an effective therapeutic target to fight against insulin resistance syndrome induced by HFD.

E2-BSA activates caveolin-1 via PI3K/ERK1/2 and lysosomal degradation pathway and contributes to EPC proliferation.

BACKGROUND: The mechanism that estrogen (E(2)) increases the number of endothelial progenitor cells (EPC) is largely unknown. Here we used E(2)-conjugated bovine serum albumin (E(2)-BSA, membrane impermeable) to investigate whether the membrane estrogen receptor (mER) and its related protein caveolin-1 (CAV-1) are involved in these processes. METHODS AND RESULTS: E(2)-BSA promoted [(3)H]-thymidine incorporation of EPC through increasing CAV-1 expression via mER (ERα, but not ERß or GPR30). Both cholesterol depletion and CAV-1 knockdown with use of CAV-1 siRNA significantly attenuated E(2)-BSA-induced [(3)H]-thymidine incorporation. Western blot showed that E(2)-BSA increased membrane CAV-1 protein expression 12h after treatment, whereas mRNA levels of CAV-1 were augmented until 24h after E(2)-BSA treatment. Furthermore, pre-incubated EPC with ICI 182780 (a specific ER antagonist), LY 294002 (a selective PI(3)K inhibitor) or PD 98059 (a specific ERK1/2 inhibitor) before E(2)-BSA inhibited the late-stage effect of E(2)-BSA (≥24 h) on up-regulation of CAV-1 mRNA and protein expression. Pulse chase results demonstrated that E(2)-BSA inhibited lysosome-mediated degradation of CAV-1 protein at the early stage (≤12 h), and then resulted in the increased CAV-1 protein. CONCLUSION: In the present work we demonstrated that E(2)-BSA promotes EPC proliferation through mER (ERα) in CAV-1-dependent manner: prolonging the stability of CAV-1 protein through quick inhibition of the lysosomal degradation pathway at the early stage (≤12 h) and up-regulating CAV-1 at transcription levels through PI(3)K/ERK1/2 signaling pathway at the late stage (≥24 h). These data indicated that a there is a novel mechanism of E(2)-BSA in the regulation of EPC proliferation through CAV-1.

17 beta-estradiol attenuates pressure overload-induced myocardial hypertrophy through regulating caveolin-3 protein in ovariectomized female rats.

Our findings indicate that in ovariectomized female rats abdominal aortic constriction led to significant increases in left ventricular mass, myocyte diameter and heart weight/body weight (HW/BW) value, and decreases in interventricular septal thickness at diastole (IVSd), left ventricular percent fractional shortening (FS) and ejection fraction (EF). These pathophysiological alterations were largely reversed by administration with 17ß-estradiol for eight weeks. Furthermore, the enhanced expression of extracellular signal-regulated kinases 1/2 and decreased expression of caveolin-3 were found in left ventricle of AAC group. 17ß-estradiol (E(2)) administration increased the expression of caveolin-3 and reduced the level of ERK phosphorylation in these pressure-overloaded rats. Moreover, in cultured neonatal rat cardiomyocytes, E(2) inhibited the hypertrophic response to angiotensin II. This effect was reinforced by the addition of extracellular signal-regulated kinases 1/2 inhibitor PD98059, but was impaired when the cells were pretreated with caveolae disruptor, methyl-ß-cyclodextrin (M-ß-CD). In conclusion, our data indicate that estrogen attenuates the hypertrophic response induced by pressure overload through down-regulation of extracellular signal-regulated kinases 1/2 phosphorylation and up-regulation of caveolin-3 expression.

[Role of caveolin-1 on membrane estrogen receptor mediated proliferation of endothelial progenitor cells].

OBJECTIVE: To investigate the potential role of caveolin-1 (CAV-1) on membrane estrogen receptor (mER) mediated proliferation of endothelial progenitor cells (EPCs). METHODS: Bone marrow (BM)-derived EPCs were cultured. The proliferation of EPCs induced by estradiol (E2)-BSA in the absence or presence of ICI 182, 780 (a pure ER inhibitor), MßCD and CAV-1 siRNA was determined by [³H]-thymidine incorporation. The expression of CAV-1 was detected by Western blot. RESULTS: Proliferation of EPC peaked after 10(-8) mol/L E2-BSA culture for 24 h (87.5% increase vs. control), and this effect could be inhibited by estrogen receptor blocker ICI 182, 780, indicating that mER-initiated membrane signaling pathways was involved in the proliferation effect of estrogen on EPC. Both cholesterol depletion and CAV-1 siRNA significantly attenuated E2-BSA induced [³H]-thymidine incorporation. Western blot result confirmed that cholesterol depletion or CAV-1 siRNA significantly decreased CAV-1 protein expression (-18.6% or -41.2% vs. 10(-8) mol/L E2-BSA alone). CONCLUSION: Our results suggested that estradiol promoted EPC proliferation through activating CAV-1 pathway.

Modulatory role of estradiol in nicotinic antinociception in adult female rats.

AIMS: Differences in the response to nicotinic analgesia in males and females have been suggested by recent studies, and such differences are presumed to be due to the regulatory effects of gonadal hormones. The aim of this study was to investigate nicotinic antinociception and the effect of estradiol (E2) on this response in female rats. MAIN METHODS: Ovariectomized female rats were implanted with subcutaneous silastic tubes containing E2. On day 28 after implantation, epibatidine, a high-potency nicotinic acetylcholine receptor (nAChR) agonist, was administered intrathecally, and antinociception at the spinal level was assessed by the tail-flick test. In addition, immunohistochemical staining for nAChRalpha4 was performed in spinal cord sections. KEY FINDINGS: We found that female rats showed shorter nociceptive latencies than males, but there was no effect of ovarian status. However, OVX significantly increased epibatidine-induced antinociception compared to that in intact females, and this increase was attenuated by E2 treatment. In addition, OVX resulted in increased nAChRalpha4 immunostaining in the dorsal horn compared to that in intact females, and this increase was also attenuated by E2 treatment. SIGNIFICANCE: Results of this study provide new evidence that E2 modulates epibatidine-induced antinociception at the spinal level in female rats.

Non-genomic effects of 17beta-estradiol in activation of the ERK1/ERK2 pathway induces cell proliferation through upregulation of cyclin D1 expression in bovine artery endothelial cells.

OBJECTIVE: Growing evidence indicates that estrogen's non-genomic effects play important roles in cellular functions and backs up the hypothesis of the existence of a membrane estrogen receptor (mER) in a number of cell types, but little is known about the complementary effects between traditional genomic and novel non-genomic effects of estrogen. The aim of the present study was to explore the non-genomic activation of ERK1/2 mitogen-activated protein kinase (MAPK) by 17beta-estradiol (E(2)) through mER and its role in cell proliferation. METHODS: On cultured bovine artery endothelial cells (BAECs) we used the [(3)H]thymidine incorporation assay to evaluate the influence of E(2) on cell proliferation and fluorescence microscopy to show the presence of mER on the cell membrane. Scatchard analysis was performed to identify and characterize the mER on a purified membrane fraction of BAECs. RESULTS: E(2) upregulated cyclin D1 protein expression and enhanced cell proliferation. Inhibition of the MAPK cascade with PD98059 or of G protein with pertussis toxin (PTX) completely abolished the above effects, while the estrogen receptor antagonist tamoxifen attenuated E(2)-dependent upregulation of cyclin D1 and cell proliferation. Accordingly, E(2) rapidly led to ERK1/ERK2 activation, which was prevented by tamoxifen or PTX and was entirely reproduced by membrane-impermeable estradiol-bovine serum albumin conjugate (E(2)coBSA). Immunofluorescent staining with E(2)coBSA-fluorescein isothiocyanate resulted in a punctuate staining pattern of the plasma membrane and Scatchard analysis of the E(2)-binding protein in a purified membrane fraction of BAECs showed that E(2) binds to the membrane fraction with a dissociation constant of 0.2394 nmol/l. CONCLUSION: Our findings showed that E(2) induces cell proliferation through upregulation of cyclin D1 via non-genomic activation of the ERK1/ERK2 pathway mediated by mER and G protein.

Raloxifene plus 17beta-estradiol inhibits proliferation of primary cultured vascular smooth muscle cells and human mammary endothelial cells via the janus kinase/signal transducer and activator of transcription3 cascade.

Long-term use of estrogen replacement therapy increases the risk of breast cancer. Presently, we investigated the effects and mechanisms of Raloxifene, a second generation selective estrogen receptor modulator, plus 17beta-estradiol on the proliferation of primary cultured vascular smooth muscle cells (VSMC) and human mammary endothelial cells (HMEC). Raloxifene plus 17beta-estradiol inhibited angiotensin II-induced VSMC proliferation and rapid phosphorylation of STAT(3); these effects were blocked by AG490, the janus kinase/signal transducer and activator of transcription3 (JAK/STAT(3)) inhibitor. STAT(3) production was not affected. In primary cultured HMEC, immunofluorescence identified the ERbeta subtype, but not the ERalpha subtype, in the nucleus. Raloxifene plus 17beta-estradiol inhibited 17beta-estradiol-induced proliferation of HMEC. Western blot analysis established that Raloxifene attenuated the 17beta-estradiol-induced phosphorylation of STAT(3), and that this effect was blocked by AG490. We conclude that Raloxifene plus 17beta-estradiol inhibits the proliferation of VSMC and HMEC through the JAK/STAT(3) cascade, which in primary cultured HMEC may be implemented through ERbeta.

[Role of NO signal pathway in the inhibitory of 17beta-estradiol on the production of endothelin-1 in vascular smooth muscle cells].

AIM: To investigate the mechanisms of 17beta-estradiol on the production of endothelin-1 in vascular smooth muscle cells. METHODS: After incubation VSMC with various concentrations of 17beta-estradiol (10(-9) - 10(-7) mol/L) or plus L-NAME(10(- 6) mol/L) for different times, the concentration of endothelin-1 was measured. At the same time, the activity of endothelin converting enzyme-1 was analyzed, and the expression of preproET-1mRNA was measured by RT-PCR. RESULTS: In basal conditions, 17beta-estradiol could inhibit the production of endothelin-1 in VSMC, and the action of 17beta-estradiol had nothing to do with the activity of endothelin converting enzyme-1. L-NAME inhibited the effect of 17-estradiol on the production of endothelin-1 in VSMC. RT-PCR results showed that 17-estradiol inhibited the preproET-1 mRNA expression, and whereas L-NAME reversed this action of 17beta-estradiol. CONCLUSION: In basal conditions, 17beta-estradiol decreases the preproET-1 mRNA expression through NO-pathway to inhibit the production of endothelin-1 in cultured VSMC.

Effects of caveolin-1 on the 17beta-estradiol-mediated inhibition of VSMC proliferation induced by vascular injury.

Estrogen has a protective effect on the cardiovascular system. Yet the mechanism of how estrogen inhibits vascular smooth muscle cell (VSMC) proliferation after vascular injury and the role of caveolin-1 in this process are not clear. To understand the protection effect of estrogen and caveolin-1, we employed a vascular balloon-injury model. Sixteen New Zealand White rabbits with or without estrogen were tested. 17beta-estradiol is able to inhibit VSMC proliferation in a range from 10(-10)-10(-5) mol/L, with an optimal concentration of 10(-8) mol/L. Estrogen exerted its effect through suppressing the activity of p42/44 MAPK, which can be blocked by tamoxifen. Moreover, in estrogen pretreated cells as well as in common carotid arteries of the balloon injury model, expression of caveolin-1 is enhanced compared to the estrogen-deficient group, as assessed by both western blotting and RT-PCR and morphological studies. Our results showed that the inhibition effect of estrogen in VSMCs is mediated by p42/44 MAPK. Caveolin-1 plays an important role in this protective process.

Estrogen receptor alpha gene polymorphism associated with type 2 diabetes mellitus and the serum lipid concentration in Chinese women in Guangzhou.

BACKGROUND: Estrogen might play an important role in type 2 diabetes mellitus pathogenesis. A number of polymorphisms have been reported in the estrogen receptor alpha (ERalpha) gene (also named ESR1), including the XbaI and PvuII restriction enzyme polymorphisms of ESR1, which may be involved in disease pathogenesis. The aim of this study was to determine whether ERX gene polymorphisms are associated with type 2 diabetes mellitus and serum lipid level. METHODS: Two hundred and ninety-nine patients with type 2 diabetes mellitus were compared with three hundred and forty-one health controls of Guangzhou in China, both were male and postmenopausal female residents at 51 - 70 years. ESR1 genotyping was performed using polymerase chain reaction (PCR) and PvuII and XbaI restriction fragment length polymorphism (PCR-RFLP) analysis. RESULTS: ESR1 allelic frequencies of P, p and X, x alleles were 0.408, 0.592; 0.360, 0.640 in the type 2 diabetes mellitus group and 0.318, 0.682; 0.328, 0.672 in the control group, respectively. In case-control study, there was significant difference in PvuII, but not XbaI, allele frequency between the type 2 diabetes mellitus and control groups (P = 0.001 and P = 0.122). When the group was separated into men and women, the difference was significant in women (P < 0.001) but not in men (P = 0.854) with the PvuII genotype, and the effect of PvuII variant on the development of type 2 diabetes mellitus was improved with aging. In addition, PvuII genotype was associated with blood glucose [fasting blood glucose (FBG), postprandial blood glucose (PBG)] and serum lipid [total cholesterol (TC) and low density lipoprotein (LDL)-c] concentration in healthy women. CONCLUSIONS: PvuII polymorphism of ESR1 increases susceptibility to type 2 diabetes mellitus in Chinese Guangzhou women. ESR1 variants may also impact serum lipid metabolism, which might provide a mechanism connecting ESR1 to type 2 diabetes.

Effects of selective estrogen receptor modulator raloxifene plus 17-beta estradiol in aorta and mammary gland of female experimental atherosclerosis rabbits and possible involvement of ERK signal transduction pathway.

The present study investigated the effects of raloxifene, a second generation selective estrogen receptor modulator (SERM), plus 17-betaE2 on aortic atherosclerosis and mammary gland hyperplasia in ovariectomized, cholesterol-fed rabbits. Following 10 weeks of raloxifene, 17-betaE2, or raloxifene plus 17-betaE2 administration, serum total cholesterol, triglyceride, low density lipoprotein were significantly decreased in the drug groups compared to the placebo group. Consistent with serum lipid results, the total lesion area for each aorta of the drug groups decreased significantly as compared to the placebo group. HE staining of aorta paraffin section showed that in the drug groups the endothelial monolayer was almost continuous. While in mammary gland, HE staining of paraffin sections indicated the hyperplasia of epithelial cells (in 17-betaE2 group) was obviously inhibited in raloxifene plus 17-betaE2 group. In cultured vascular smooth muscle cell (VSMC), the results of MTT and [3H]TdR incorporation showed that the drug groups could inhibit AngII-induced proliferation of VSMC. Western blotting proved that raloxifene plus 17-betaE2 inhibited the expression of phosphorylated ERK protein, similar to 17-betaE2 but different from raloxifene. This effect was inhibited by PD98059 (inhibitor of MAPK) or ICI182780 (ER antagonist). In conclusion, this study suggests that SERM raloxifene plus 17-betaE2 improves the lipid metabolism and relieves the aorta changes of female experimental atherosclerosis rabbits, which are partly implemented by the inhibition of VSMC growth through ERK cascade. The hyperplasia of mammary gland epithelial cells could be significantly inhibited by raloxifene plus 17-betaE2.

[Possible involvement of caveolin-1 in the inhibition of endothelin-1 induced proliferation of vascular smooth muscle cells by 17beta-estradiol].

The aim of the present study was to investigate the role of caveolin-1 in the inhibition of endothelin-1 induced proliferation of vascular smooth muscle cells (VSMCs) by 17beta-estradiol. In the cultured rat thoracic aortic VSMCs, proliferation of VSMCs was determined by using [(3)H]-thymidine incorporation and the expression of caveolin-1 protein was measured by immunofluorescence assays and Western blotting. The measurement demonstate VSMCs exposed to various concentrations of endothelin-1 (1-100 nmol/L) for 24 h induced an increase in [(3)H]-thymidine incorporation. Pretreament with various concentrations of 17beta-estradiol (0.1-10 nmol/L) for 24 h inhibited the proliferation effect of endothelin-1. Immunofluorescence assays showed that after 24 h treatment of VSMCs with endothelin-1 (100 nmol/L), the expression of caveolin-1 in VSMCs was significantly increased, whereas pretreament with 17beta-estradiol (10 nmol/L) for 24 h inhibited the effect. Western blotting results further proved that endothelin-1 inhibited and 17beta-estradiol increased the expression of caveolin-1 in VSMCs. These results demonstrate that 17beta-estradiol inhibits the VSMCs proliferation induced by endothelin-1, and that the effect of estradiol is probably mediated by caveolin-1.

[Role of inducible nitric oxide synthase in the vascular smooth muscle cells cycle arrest induced by 17 beta-estradiol].

Clinical epidemiologic data and animal experimental studies regard estrogen as being protective against the development of cardiovascular diseases. The mechanisms by which estrogen affects the development of vascular diseases are not clear. Recent studies demonstrated that the cardiovascular protective effects of estrogen are closely related to nitric oxide (NO) pathway. Our previous study proved that estrogen inhibited the proliferation and oncogene expression of vascular smooth muscle cells (VSMCs) induced by endothlin 1 (ET-1) and serum,this effect was mediated by NO release. In the present study, we investigated the role of inducible nitric oxide synthase (iNOS) in the VSMCs cycle arrest induced by 17 beta-estradiol (E(2)). The effects of E(2) on iNOS activity and protein expression in cultured rat VSMCs and the influence of NOS inhibitor N(G)-nitro-L-arginine methylester (L-NAME) on the inhibitory effect of E(2) on cell cycle were investigated. NOS assay kit was used to measure the activity of iNOS and protein expression of iNOS was determined by Western-blot. Cell cycle analysis was accessed by flow cytometry. The results obtained showed that E(2) increased iNOS activity of VSMCs but not in a dose-dependent manner. E(2) 10 nmol/L increased the iNOS activity of VSMCs distinctly at two time points: 30 min and 12 h. These effects were significantly inhibited by estrogen receptor (ER) antagonist Tamoxifen (0.1 micromol/L) and NOS inhibitor L-NAME (1 micromol/L). E(2) increased iNOS protein expression of VSMCs in a dose-dependent manner. The effect of E(2) on iNOS protein expression of VSMCs started at 3 h, distinctly increased at 12 h and then decreased. Tamoxifen significantly inhibited the E(2)-induced iNOS protein expression of VSMCs. ET-1 increased cell percentage of S phase and G(2)+S/G(1). This effect was inhibited by E(2). L-NAME significantly attenuated the inhibitory effect of E(2) on cell cycle of VSMCs. The results suggest that E(2) induced G(1) arrest of VSMCs, which was associated with an increase in iNOS activity and protein expression of VSMCs. These effects were at least mediated by estrogen receptor partly.

Mechanisms of 17beta-estradiol on the production of ET-1 in ovariectomized rats.

In order to clarify the mechanism underlying the possible preventive effect of estrogen on atherogenesis, we investigated the role of 17beta-estradiol (E2) in the regulation of endothelin-1 (ET-1) production in ovariectomized rats, which may contribute to atherogenesis. Female Spragure-Dawly rats were randomly divided into three groups: sham-operated group (sham), ovariectomized group (OVX) and 17beta-estradiol replacement group (OVX + E2, 20 microg(-1).kg.d(-1),s.c.). 4 weeks after operation, the plasma concentration of ET-1, clearance of ET-1, functional ECE activity and preproET-1 mRNA expression in aorta were measured. Concentration of plasma ET-1 change from 107.8 +/- 18.3 pg/ml (sham) and 135.5 +/- 27.6 pg/ml (OVX + E2) to 190.7 +/- 25.5 pg/ml (OVX ) (n = 8, p < 0.05). There was no significant difference in the clearance of 125IET-1 among three groups (p > 0.05). Functional ECE activity was increased in OVX group in comparison to that in sham group (p < 0.05). The OVX increased the preproET-1 mRNA expression in sham, whereas treatment with estrogen reversed these changes (p < 0.05). The present study have shown that estrogen down-regulates plasma ET-1 levels by inhibiting the preproET-1 mRNA expression and functional ECE activity. Clearance of ET-1 was not affected. Inhibition of ET-1 production mediated by modulating ECE activity may be one of the novel mechanisms of the protective of estrogens on the cardiovascular system.

[Effect of ERK on 17beta-estradiol-induced inhibition of VSMC proliferation in rats after vascular injury].

The aim of the present study was to investigate the effect of ERK on 17beta-estradiol (E(2)) inhibition of vascular smooth muscle cell (VSMC) proliferation in rats after vascular injury. Common carotid artery balloon-injury (Inj) model was established in ovariectomized rats (OVX). Female SD rats were randomly divided into 4 groups: OVX, E(2)+OVX, OVX+Inj, and E(2)+OVX+Inj groups. The thickness of the vessels, the plasma content of NO, and the expression of ERK, phosphorylated ERK as well as eNOS protein were measured. The results showed that compared with OVX, the vessel wall was significantly thickened and the plasma content of NO was significantly decreased in OVX+Inj group. E(2) significantly decreased the vessel thickness but increased the plasma NO content after balloon injury. E(2) inhibited the expression of ERK, phosphorylated ERK and induced the eNOS expression. There is a positive correlation between plasma NO content and eNOS protein expression, while there is a negative correlation between plasma NO content and the thickness of vessel. The plasma NO content and the expression of ERK protein were negatively correlated. These results suggest that E(2) increases the vascular eNOS protein expression and NO release, leading to the inhibition of VSMC proliferation after balloon injury by inhibiting the ERK and phosphorylated ERK protein expression.