Nitric oxide (NO), derived from activity of the endothelial isoform of NO synthase (eNOS), is a regulator of antiatherogenic processes including the maintenance of vascular tone, inhibition of monocyte adhesion to the endothelial surface, inhibition of platelet aggregation, and inhibition of vascular smooth muscle cell proliferation.1 Estrogen exerts antiatherogenic effects on the vasculature in part through the production of NO.2–4 In cultured endothelial cells, estrogen through ligation of estrogen receptor alpha (ERα) increases expression of eNOS mRNA through genomic transcription of eNOS gene.5 In addition, estrogen also regulates eNOS activity through posttranslational modification of the enzyme in caveolae involving regulatory proteins such as caveolin-1, which inhibits eNOS activity and conversely, heat shock protein 90 (Hsp90), which stimulates catalytic function.6,7 Calmodulin also serves as an allosteric activator for eNOS.6,8 However, it is unclear to what extent results from in vitro experiments can be applied to endothelial cells in vivo and whether different types of estrogenic compounds commonly used in clinical practice affect eNOS and eNOS regulatory proteins in the same way.
Therefore, this study was designed to compare expression of ERs, eNOS, and regulatory proteins for eNOS in endothelial cells from gonadally intact and ovariectomized pigs treated with clinically relevant estrogenic compounds: 17β-estradiol, the prominent ovarian hormone; conjugated equine estrogen (CEE), a mixture of estrogen metabolites; or raloxifene, a synthetic estrogen receptor modulator.
MATERIALS AND METHODS
Animals and Experimental Design
The Institutional Animal Care and Use Committee of Mayo Clinic approved this study. Ovaries were removed laparoscopically from sexually mature female pigs (4 crossbreeds: Yorkshire, Hampshire, Duroc, and Landrace, 7 months of age, 80–120 kg, n=24), which were anesthetized with a mixture of xylazine (2 mg/kg), glycopyrrolate (0.44 mg/kg), and telazol (5 mg/kg) given intramuscularly.9 Blood samples were collected from femoral arteries of anesthetized pigs immediately before ovariectomy (intact) and 4 weeks after ovariectomy (4wk-OVX). At 4 weeks postovariectomy, pigs were randomly assigned to 1 of 4 treatment groups: untreated (8wk-OVX, n=6), oral 17β-estradiol (E2, 2 mg/day, n=6), oral conjugated equine estrogen (CEE, 0.625 mg/day, n=6), or oral raloxifene (60 mg/day, n=6) for 4 weeks. These drugs are approved for treatment of menopausal symptoms and osteoporosis in women; dosages represented those used clinically at the time the study began. Since the publication of the results of the Women's Health Initiative,10 conjugated equine estrogen is prescribed at either 0.425 or 0.325 mg/day. Animals were fed measured portions of Lean Grow 93 diet (Land O'Lakes Farmland Feed LLC, Fort Dodge, IA) twice per day; water was available ad libitum. All of the medications were mixed into the morning food, which was consumed in the presence of the veterinary or laboratory technician.
After 4 weeks of treatment, or in total 8 weeks postovariectomy (for untreated pigs, 8wk-OVX, or for treated pigs, 4wk-OVX+4wk hormone treatment), pigs were anesthetized by intramuscular injection of xylazine (2 mg/kg), glycopyrrolate (0.44 mg/kg), telazol (5 mg/kg), and ketamine (4.5 mg/kg). Within 10 min of anesthetic administration, the carotid artery was exposed and cannulated. Blood was collected for measurement of NO. The cannula was removed and the pig was exsanguinated via the carotid arteries. The thoracic aorta was removed. Aortic endothelial cells were scraped and stored for detection of eNOS mRNA, ERα, ERβ, eNOS, and related proteins. Aortic endothelial cells collected similarly from sexually mature female pigs (intact, 6 months old, n=6). The carotid arteries from these animals were used in another study.11 Platelets and coronary arteries from ovariectomized and hormone-treated animals were used in other studies.9,12
Western Blot Analysis
Descending aorta from gonadally intact, ovariectomized, and estrogen-treated pigs were removed and opened longitudinally. Cells were scraped, using a spatula, from half of the length of the aorta and were placed into tubes containing lysis buffer (1% sodium dodecyl sulfate, 1 mmol/L sodium orthovanadate, 10 mmol/L Tris HC, pH 7.4) with protease inhibitors and stored at −70°C until analysis. Aortic endothelial cell lysate preparation for Western blotting was made as described previously.11,13 Western blotting of eNOS, Hsp90, calmodulin, caveolin-1, and estrogen receptors (ERα and ERβ) was determined using the standard method as described previously.9,11,13–15
Antibodies were purchased as follows: anti-monoclonal ERα (clone AER311 directed against the 500–595 amino acid of the C-terminal of the human receptor), anti-calmodulin, and anti-phosphotyrosine (clone 4G10, mouse monoclonal IgG2bk) antibodies were obtained from Upstate Cell Signaling Solutions (Lake Placid, NY); anti-ERβ and anti-β-actin monoclonal antibodies were obtained from Sigma Chemical (St Louis, MO); monoclonal anti-eNOS (anti-NOS type III) and anti-caveolin-1 antibodies were obtained from Transduction Laboratories (Lexington, KY); anti-Hsp90 monoclonal antibody was obtained from Stressgen Biotechnologies (Victoria, Canada).
Assay for NO
Plasma was separated from blood that was collected into tubes containing ethylenediamine-tetraacetic acid. Plasma concentrations of total oxidized products of nitric oxide (NOx) were determined using the Griess reaction with the Total Nitric Oxide Assay kit (R&D Systems, Minneapolis, MN). Plasma NOx is expressed as micromoles per liter.
Real-Time Polymerase Chain Reaction for eNOS mRNA
Cells scraped from the remaining half of the aorta (those not used for Western blotting) were placed into tubes containing TRIzol reagent and stored at −70°C until analysis. Total RNA was extracted from aortic endothelial cells by TRIzol reagent. DNA contamination in isolated RNA was eliminated with DNase treatment. Endothelial NOS mRNA expression was determined by quantitative polymerase chain reaction (PCR). Reverse transcriptase reaction and PCR for eNOS mRNA determination was performed in accordance with guidelines from Applied Biosystems (Foster City, CA) and described by our group using the following primers:9,11,13
Forward primer 5′-CAAAGTGACCATTGTGGACCAT-3′
Reverse primer 5′-TGCTCGTTCTCCAGGTGCTT-3′
Probe sequence 5′-FAM-CCGCCACGGCCTCCTTCATG-TAMPRA-3′
Quantification of mRNA for eNOS was expressed as a ratio of eNOS mRNA to 18S rRNA.
All data are presented as mean±SEM. All proteins detected by Western blotting were normalized to β-actin. Each membrane contained samples from each treatment group. Pixel values for each specific protein/receptor were obtained using UN-SCAN-IT positive segment analysis. These values were corrected (normalized) with corresponding membrane pixel values of control protein β-actin. Results for specific proteins/β-actin were averaged for pigs in each treatment group. Tyrosine phosphorylation was calculated as pixel values of phosphorylated eNOS/β-actin corrected pixel values of nonphosphorylated eNOS protein for each animal. The number of different animals from which samples were obtained is designated by “n”. Data were analyzed by 1-way analysis of variance (ANOVA) followed by Fisher progressively lowered stress threshold test to identify differences among groups. Statistical significance was accepted as P<0.05.
Body weight increased similarly in all treatment groups. Concentrations of plasma 17β-estradiol were below the detection limit of the assay in all groups of treated pigs. However, efficacy of treatment was evidenced by increases in secretory cells of the uterine epithelium glands and vascularity of uterine myometrium.9
NOx in Plasma
Mean plasma concentrations of NOx decreased significantly after ovariectomy (4wk-OVX and 8wk-OVX compared with intact; P<0.05). With estrogenic treatments, mean NOx was not statistically different from either intact or 8wk-OVX animals (Fig. 1A). When values from individual animals were compared at 8 weeks compared with 4wk-OVX, NOx did not increase in any animal from the 8wk-OVX group. After 4 weeks of treatment with either E2 or CEE, plasma NOx increased in 4 of 5 and 4 of 6 animals, respectively; whereas with raloxifene treatment, NOx increased in 2 of 6 animals (Fig. 1B).
Real-Time PCR for eNOS mRNA, Western Blotting for eNOS Protein, and Phosphorylation of eNOS
Expression of mRNA for eNOS showed large variability with ovariectomy and treatment and thus did not reach statistical significance among groups (Fig. 2A). However, eNOS identified as a single band of protein with an estimated molecular weight of 140 kDa increased significantly in endothelial cells with ovariectomy. There was no significant difference in eNOS expression between the 8wk-OVX and E2 groups, but there were significant decreases in eNOS expression with CEE and raloxifene treatments (Fig. 2B). Tyrosine phosphorylation of eNOS did not change with ovariectomy but increased significantly with all treatments. Tyrosine phosphorylation was greater with raloxifene treatment than with either E2 or CEE (Fig. 2C).
Expression of eNOS-Related Proteins
Hsp90 increased significantly following ovariectomy (Fig. 3A). Only treatment with CEE and raloxifene significantly reduced expression of Hsp90 (Fig. 3A). Expression of calmodulin was not significantly affected by ovariectomy but increased with all hormone treatments (Fig. 3B). In contrast to Hsp90, caveolin-1 expression decreased with ovariectomy (Fig. 3C). CEE and raloxifene treatments increased expression of caveolin-1 compared with 8wk-OVX animals (Fig. 3C). Raloxifene treatment significantly increased expression of caveolin-1 above that observed in animals treated with E2 or CEE.
Expression of Estrogen Receptors
Expression of ERα but not ERβ increased significantly in endothelial cells with ovariectomy (Fig. 4A). Only raloxifene treatment significantly decreased expression of ERα compared with 8wk-OVX. Expression of ERβ was significantly greater in endothelial cells from animals treated with CEE compared with E2 (Fig. 4B). The intensity ratio of ERα/ERβ significantly increased with OVX and E2 compared with intact animals (Fig. 5). With CEE and raloxifene treatment, the ratio of ERα/ERβ was not different from that of intact animals (Fig. 5).
The major finding of this study is that 3 different estrogenic treatments given to ovariectomized pigs do not have the same effect on expression of estrogen receptors and regulatory proteins associated with posttranslational modulation of eNOS activity in aortic endothelial cells; thus, plasma NO is not consistently elevated by each treatment. These results are important for 2 reasons. First, these results report the first direct comparison of 3 different but clinically relevant estrogen compounds on proteins associated with regulation of eNOS and NO production in vivo. Second, changes in endogenous NO are consistent with those observed in women with menopause or surgical ovariectomy and with estrogen treatment,16,17 thus indicating that pigs are useful experimental models for studying hormonal effects on the vasculature.
Changes in plasma NOx reflect activity and quantity of eNOS.2–4 Loss of ovarian hormones tended to increase mRNA for eNOS, suggesting that either NO or the ovarian hormones exert a negative feedback on gene transcription, an observation that is consistent with what is observed in endothelial cells of male pigs transitioning to sexual maturity.13 The reciprocal relationship between plasma NO and eNOS mRNA in ovariectomized pigs is not in agreement with other studies that showed decreases in mRNA with ovariectomy and increases with exogenous estrogen treatment.18–23 Differences among studies may represent differences in the duration of ovariectomy and the analysis of tissue homogenates, which contain many cell types compared with a single cell type, or differences in anatomical origin of the tissue or species.20,24 Variability in mRNA for eNOS will also reflect the ability of E2 to stabilize mRNA.25
In addition to changes in the amount of eNOS, activity of the enzyme is regulated posttranslationally by processes such as protein phosphorylation8 and association with intracellular and membrane-bound proteins like caveolin-1.6 Tyrosine phosphorylation was associated with decreases in NOS activity in cultured bovine aortic endothelial cells.26 In the present study, raloxifene treatment in vivo increased tyrosine phosphorylation of eNOS to a greater level than the other 2 estrogen treatments. This, coupled with an increase expression caveolin-1 with raloxifene treatment, may explain why plasma NOx was increased in only 2 of 6 animals treated with raloxifene. However, phosphorylation of Tyr-83 resulted in a 3-fold increase in NO production in transfected COS-7 cells.27 Specific studies are needed to investigate how various estrogenic compounds regulate eNOS activity through phosphorylation pathways.
Activity of eNOS also is regulated by Hsp90 and calmodulin.7 Therefore, posttranslational regulation of eNOS requires the interaction of at least 3 (and perhaps other) proteins in vivo to regulate endothelial production of NO. Although stimulation of ERs may participate in regulation of eNOS, Hsp90, caveolin-1, and calmodulin, the selective estrogen receptor modulator, raloxifene, did not affect changes in these proteins in the same way as E2 or CEE treatments. These differences most likely reflect differences in binding affinity of the natural compounds compared with the synthetic compound for estrogen receptors and the biological activity of the natural metabolites of E2 and CEE,28,29 thus providing selective effects to biological targets. For example, raloxifene is antagonistic to breast and uterus in women, does not reduce hot flashes as does E2 and CEE, increases risk for venous thrombosis, and does not reduce formation of coronary arterial plaque in monkeys.29–32
Although both ERα and ERβ are present in endothelial cells, factors affecting their expression are not clearly understood. ERα is considered the dominant receptor vascular endothelium.3,33 In this in vivo study, expression of ERα increased in aortic endothelial cells with ovariectomy. Raloxifene was more effective than either E2 or CEE in returning ERα expression to that observed in intact animals. In contrast to ERα, neither ovariectomy nor estrogenic treatments modified ERβ expression significantly compared with intact animals. Studies of cultured endothelial cells,34 human uterus,35 and vaginal tissue36 also suggest differential regulation of ER isoforms by estrogen.
Estrogen receptors exist as functional homodimers in cell membranes.37,38 However, both homo- and heterodimers may be important in affecting estrogen-ligand receptor genomic effects.39 Therefore, part of the variability in changes in mRNA for eNOS may be a result of ERα and ERβ dimerization. Much remains to be learned about reactivity between the ERs and their genomic and nongenomic signaling mechanism for eNOS and other enzymes and receptors.
The present study has several limitations. First, only 1 dose of each estrogenic treatment was studied. Although these doses were comparable to those used clinically to treat menopausal women, pigs may not metabolize the drugs the same as women. Also, variability in responses may have been reduced if these treatments were extended beyond 4 weeks. Finally, functional consequences of changes in NO and eNOS protein or its phosphorylation were not evaluated (eg, differences in response to a vascular injury or binding of eNOS to the various regulatory proteins), thus forming the basis for future studies. Both E2 and CEE reduce the incidence of cardiovascular disease in women taking these hormones for symptoms of menopause.40,41 Raloxifene had a neutral effect on adverse arterial cardiovascular events in older, high-risk women of the Multiple Outcomes of Raloxifene Evaluation (MORE) study.42,43 Additional information about the effects of raloxifene on the arterial system of older women at risk for adverse arterial events await results of the international clinical trial Raloxifene Use for the Heart (RUTH).44 Results of the present study and others9,45–47 identifying differences in mechanisms between the natural estrogens and raloxifene may help to interpret differences in cardiovascular outcomes of clinical studies of hormone treatments.
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Keywords:© 2006 Lippincott Williams & Wilkins, Inc.
caveolin-1; conjugated equine estrogen; 17β-estradiol; estrogen receptors; raloxifene