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J Appl Physiol 91: 2400-2406, 2001;
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Vol. 91, Issue 5, 2400-2406, November 2001

HIGHLIGHTED TOPICS
Genome and Hormones: Gender Differences in Physiology
Selected Contribution: Estrogen receptor-alpha gene transfer inhibits proliferation and NF-kappa B activation in VSM cells from female rats

Ram V. Sharma, Milind V. Gurjar, and Ramesh C. Bhalla

Department of Anatomy and Cell Biology, and The Cardiovascular Center, The University of Iowa College of Medicine, Iowa City, Iowa 52242


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Epidemiological studies have demonstrated that hormone replacement therapy with estrogen (E2) or E2 plus progesterone in postmenopausal women decreases the age-associated risk of cardiovascular disease by 30-50%. Treatment of vascular smooth muscle (VSM) cells with physiological concentrations of E2 has been shown to inhibit growth factor-stimulated cell proliferation. In this study, we tested the hypothesis that E2 inhibits the age-associated increase in VSM cell proliferation by inhibiting nuclear factor (NF)-kappa B pathway. We investigated the effects of E2 treatment and adenovirus-mediated estrogen receptor (ER)-alpha gene transfer on cell proliferation and NF-kappa B activation using VSM cells cultured from 3-mo-old and 24-mo-old Fischer 344 female rats. Our results demonstrate that VSM cell proliferation was significantly increased (P < 0.05) in aged compared with young adult female rats. Treatment of VSM cells with physiological concentrations of E2 inhibited VSM cell proliferation, and this inhibition was significantly greater (P < 0.05) in cells from aged female rats compared with young adults. The inhibitory effects of E2 on cell proliferation in aged female rats were significantly potentiated by overexpression of the human ER-alpha gene into VSM cells. Constitutive and interleukin (IL)-1beta -stimulated NF-kappa B activation was significantly greater (P < 0.05) in VSM cells from aged compared with young female rats. E2 treatment of VSM cells from aged female rats inhibited both constitutive and IL-1beta -stimulated NF-kappa B activation. ER-alpha gene transfer into VSM cells from aged female rats further augmented the inhibitory effects of E2. In conclusion, our data demonstrate that constitutive and IL-1beta -stimulated NF-kappa B activation is increased in VSM cells from aged female rats due to loss of E2 and this can be restored back to normal levels by ER-alpha gene transfer and E2 treatment. In addition, increased NF-kappa B signaling may be responsible for increased incidence of cardiovascular disease in postmenopausal females.

vascular smooth muscle cells; nuclear factor-kappa B; Fischer 344 aged female rats


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

AGING IS ASSOCIATED WITH A progressive increase in the incidence of cardiovascular disease (CVD); in women, a steep increase in CVD commences at the time of menopause (3). Moreover, in premenopausal women with impaired ovarian function, a thickened and sclerotic arterial tunica intima is significantly more common than in regularly menstruating women (34). The results of hormone replacement therapy studies in postmenopausal women have indicated that estrogen (E2) as well as E2 plus progesterone therapy reduces the incidence of myocardial infarction by 30-50% (20, 22, 26, 29-31). In addition, results from animal models have shown that E2 replacement therapy inhibits neointima formation and restenosis after vascular injury (14, 26, 32). Results from our laboratory and several others have shown that treatment of cultured vascular smooth muscle (VSM) cells with E2 inhibits their proliferation, and these responses appear to be sexually dimorphic (5, 12, 27). All of these data support the hypothesis that the majority of the vasculoprotective effects of E2 are due to its direct effect on the vascular cells (2, 26).

Most of the effects of E2 on target cells are mediated via binding to estrogen receptors (ERs) that act as ligand-activated transcription factors (11). Two types of ERs have been cloned (ER-alpha and ER-beta ), and they are present in most tissues, including vascular cells (reviewed in Ref. 26). E2 binding to ER-alpha and ER-beta leads to their activation, and activated receptors mediate specific gene expression and cell function by binding to cis-acting regulatory sequences termed ER response element (11). Although expression of both ER-alpha and ER-beta has been shown to increase in cardiac allografts and after vascular injury (reviewed in Ref. 26), only ER-alpha expression has been shown to decrease in atherosclerotic arteries from postmenopausal women (25). Similarly, methylation-dependent inactivation of ER-alpha is increased in vascular cells from human atherosclerotic lesion (33). Moreover, a recent study has demonstrated that ER-alpha activation, but not ER-beta , inhibits expression of insulin-like growth factor-1 and insulin-like growth factor-1 receptor genes in VSM cells (38). Results obtained in our laboratory have demonstrated that adenovirus-mediated expression of the ER-alpha gene in endothelial cells increases induction of E2-sensitive endothelial nitric oxide synthase (NOS) gene (41). Taken together, these data suggest that ER-alpha may play a dominant role in regulating VSM cell function.

Although vasculoprotective effects of E2 are well established, the mechanism(s) of ER-alpha -mediated vasculoprotection is not well understood. In addition to regulating gene transcription via ER response elements, ER-alpha has also been shown to modulate gene transcription by negatively interfering with other transcription factor pathways in a DNA binding-independent manner (15, 38). Activated ER-alpha has been shown to repress cytokine-induced activation of a number of inflammatory genes by negatively interfering with nuclear factor (NF)-kappa B transcriptional activity, suggesting that functional antagonism between ligand-activated ER-alpha and NF-kappa B may play an important role in E2-mediated protection against CVD (15, 18, 40).

Several lines of evidence suggest that transcription factors of the NF-kappa B/Rel family may play a causative role in vasculoproliferative diseases like atherosclerosis and restenosis (11a). First, constitutively activated NF-kappa B is found in the nuclei of VSM cells in atherosclerotic lesions and NF-kappa B activation increases in an injury-induced model of restenosis (8-10). Second, activation of NF-kappa B is required for VSM cell proliferation in response to several growth factors and hormones (1, 4, 8). Third, inhibition of NF-kappa B signaling using antisense oligonucleotides to the p65 subunit, overexpression of Ikappa Balpha , or dominant-negative inhibitors of Ikappa B kinases have been demonstrated to inhibit VSM cell proliferation, increase apoptosis, and inhibit restenosis after vascular injury (1, 13, 21, 37). Fourth, a variety of genes involved in inflammation and proliferation that are induced in atherosclerotic lesions and in response to vascular injury are regulated by NF-kappa B transactivation (9, 11a). Some of the gene products, such as tumor necrosis factor-alpha and interleukin (IL)-1beta , are potent activators of NF-kappa B in VSM cells and may participate in producing continuous inflammatory conditions in atherosclerotic lesions (9, 10). All these studies suggest that the activation of NF-kappa B plays an integral role in the development and progression of CVD.

Therefore, we have tested the hypothesis that NF-kappa B activation is increased in VSM cells from aged female rats, resulting in increased proliferation, and that E2 and ER-alpha inhibit VSM cell proliferation in aged female rats by inhibiting NF-kappa B activation. Our results show that constitutive and IL-1beta -stimulated NF-kappa B activity is markedly increased in VSM cells from aged female rats compared with young adults. The ER-alpha gene transfer in VSM cells from aged female rats inhibits constitutive and cytokine-stimulated activation of NF-kappa B.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Chemicals and materials were obtained from the following sources: [gamma -32P]ATP (3,000 Ci/mmol) from Amersham; electrophoretic mobility shift assay (EMSA) kit for measuring NF-kappa B activation from Promega; human recombinant IL-1beta from R&D Systems (Minneapolis, MN); and water-soluble 17beta -estradiol, nitro-L-arginine, as well as other chemicals not listed, were of the highest grade available from Sigma. Cell culture reagents were purchased from the Cell Culture and Hybridoma Center of the University of Iowa. Full-length human ER-alpha (hER-alpha ) cDNA was a generous gift from Dr. Geoffrey L. Greene, University of Chicago (16).

Cell culture. In this study, young adult (3-mo-old) and aged (24-mo-old) female Fischer 344 rats (National Institutes of Health Aging Institute) were used to isolate and culture aortic smooth muscle cells. Thoracic aorta from four rats were pooled for isolating and culturing each batch of cells according to the procedure established in our laboratory (17, 39). For each experiment, we have used cells from two different batches between passages 2 and 4. The purity of cultures was confirmed by immunocytochemical localization of smooth muscle-specific alpha -actin using monoclonal antibodies raised against the NH2-terminal decapeptide of a smooth muscle alpha -actin (5). In all experiments, phenol red-free DMEM and charcoal-treated estrogen-free (<2 pg/ml) fetal bovine serum (FBS) were used.

E2 treatment protocol. E2 treatment of cultured cells was done in the absence of phenol red, which is a weak E2-receptor agonist, as described earlier (5). The cultured VSM cells were first treated with a low concentration of E2 (50 pg/ml) for 24-48 h. This concentration was similar to typical nonpregnant levels of E2 in plasma of noncycling female rats (26). Cells were then treated with 250 pg/ml of E2, a concentration similar to that found in cycling and pregnant rats (26), for 4-6 days. The rationale for this protocol was to allow significant time for the genomic effects of E2 to appear in the cultured cells.

Adenovirus vector-mediated gene transfer. The replication-deficient recombinant adenovirus vector AdCMVhERalpha was used to transfer hER-alpha gene into VSM cells in vitro (41). AdCMVlacZ was used as control vector and served as negative control along with nontransfected cells. AdCMVGFP containing the fluorescent marker for green fluorescent protein (GFP) was used to determine transfection efficiency in parallel cultures. The DNA constructs of replication-deficient adenovirus comprise almost a full-length copy of the adenovirus genome in which the ER-alpha , lacZ, or gfp expression cassette is incorporated at the site of E1 region deletions. In this cassette, a RSV (Rous sarcoma virus) promoter to drive transcription of ER-alpha gene precedes ER-alpha cDNA. A polyadenylation sequence of SV40 is cloned downstream of endothelial NOS. Purified and concentrated adenoviral vectors (serotype 5, produced in 293 cells) [containing ~2 × 1010 plaque-forming unit (pfu)/ml] were obtained from the Gene Transfer Vector Core at the University of Iowa College of Medicine.

VSM cells were transfected with hER-alpha or a control vector as described earlier (17, 39). Briefly, VSM cells in passages 2-4 were plated in cell culture dishes and semi-confluent cells were infected with 100 MOI of viral vector in DMEM supplemented with 0.1% BSA, 100 U/ml penicillin, and 100 µg/ml streptomycin (phenol red-free and serum-free defined medium) for 2 h. One MOI is defined as 1 pfu of virus per cell. After a 2-h incubation, virus-containing culture medium was removed and replaced with fresh virus-free and serum-free culture medium supplemented with 0.4% FBS for 48 h. E2 (250 pg/ml) treatment of ER-alpha -transfected cells was started during the serum-deprivation period. The extent of ER-alpha gene expression was quantified by Northern and Western blotting 48 h after gene transfer as described earlier (42).

Cell proliferation. VSM cell proliferation was measured by counting cell numbers as described previously (5). For these experiments, semiconfluent cells were treated with 50 pg/ml E2 for 48 h. After this treatment, cells were left untreated or transfected with hER-alpha gene or a control vector; 48 h after gene transfer, the cells were plated into six-well dishes. At the time of plating, 10 pfu/cell of hER-alpha or control vector was added so that replicating cells were transfected with the hER-alpha gene. Immediately after culture, cells were treated with or without E2 (250 pg/ml) in phenol red-free medium. Fresh E2-containing medium with or without 5% FBS was supplemented every day, and cells were counted on indicated days.

EMSA. The NF-kappa B activity was measured by EMSA using consensus NF-kappa B binding 32P-labeled oligonucleotides according to published procedures (28, 36). VSM cells from young and aged female rats with or without E2 treatment and/or ER-alpha gene transfer were either left untreated or stimulated with human recombinant IL-1beta (1 ng/ml) for 90 min. Cells were washed with ice-cold PBS, scraped, and collected into chilled microcentrifuge tubes. Nuclear extracts were prepared as described earlier (36). Aliquots of nuclear extracts were assayed for protein concentration using Bio-Rad reagent and stored at -80°C. NF-kappa B-binding oligonucleotides (5'-AGTTGAGGGGAGTTTCCCAGG-3', Promega) were radiolabeled with [gamma -32P]ATP and T4 polynucleotide kinase and purified by gel filtration through a column of polyacrylamide beads (Bio-Rad). Nuclear extracts (3-5 µg) were incubated in a total volume of 20 µl containing 32P-oligonucleotide (50,000 counts/min), 2 µg of poly(dI · dC), 10 µg of BSA, 10 mM Tris · HCl, pH 7.5, 50 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, and 5% glycerol. Reaction tubes were incubated at room temperature for 20 min. Controls included heat-inactivated nuclear extracts and excess unlabeled oligonucleotides. Binding complexes were resolved on 4% nondenaturing polyacrylamide gels via electrophoresis in 0.5 × Tris-borate-EDTA buffer. Gels were dried and then quantified and analyzed using PhosphorImager and ImageQuant analysis software (Molecular Dynamics, Sunnyvale, CA).

Data analysis. Statistical analysis was carried out by Student's t-test, and difference was considered significant at P < 0.05. The results are presented as means ± SE (with n representing the number of separate experiments). Data were analyzed to estimate the effect of age, E2 treatment, and ER-alpha gene transfer on NF-kappa B activation and whether ER-alpha gene transfer differentially inhibit NF-kappa B activation in VSM cells from young and aged rats.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

VSM cell proliferation is increased in aged female rats. Our results show that cultured VSM cells from aged rats proliferate at a faster rate compared with 3-mo-old female rats in response to serum stimulation (Fig. 1). Interestingly, cell number at confluence was two- to threefold higher in cells cultured from aged female rats compared with young adults (Fig. 1B). These results demonstrate that VSM cells from aged female rats have altered properties with respect to proliferative or apoptotic response. However, it remains to be demonstrated whether an increase in cell number in aged animals is due to an increased rate of cell proliferation or a decreased rate of apoptosis.


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  		Fig. 1.   Vascular smooth muscle (VSM) cell proliferation is increased in cells cultured from aged female rats compared with young adult female rats. Cells were cultured from thoracic aorta of 3-mo-old and 24-mo-old Fischer 344 female rats, and proliferation was measured in passages 2 to 4 in 24-well dishes. A: 20,000 cells were plated in each well of a 24-well dish. At indicated times, the medium was changed to serum-free (-FBS) or with 10% fetal bovine serum (+FBS). B: cell number at confluence is expressed as cells per cm2. *Significant increase (P < 0.05) vs. 3-mo-old female rats (n = 6).

E2 treatment inhibits proliferation of VSM cells in aged female rats. VSM cells in females are cyclically exposed to E2 during the follicular phase (50-100 pg/ml) and at ovulation (250-600 pg/ml) (reviewed in Ref. 26). This may be important in maintaining functional ERs in VSM cells. Therefore, we have recently developed protocols for chronic treatment of coronary artery VSM cells with physiological concentrations of E2 (50-250 pg/ml or <1 nM E2) (5). VSM cells were first treated with 50 pg/ml of E2 for 2 days followed by treatment with 250 pg/ml E2. Using these treatment protocols, we observed that E2 (250 pg/ml) inhibited serum-stimulated VSM cell proliferation from both young and aged female rats; however, the extent of inhibition was significantly greater (P < 0.05) in cells from aged female rats compared with young adult rats (Fig. 2).


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  		Fig. 2.   Estrogen (E2) treatment and human estrogen receptor (hER)-alpha gene transfer inhibit proliferation of VSM cells cultured from 24-mo-old Fisher 344 rats. VSM cells from young adult and aged female rats were cultured in 24-well dishes in phenol red-free medium containing 10% FBS for 24 h. Cells were then treated with 50 pg/ml E2 in phenol red-free medium containing 0.4% FBS for 48 h. E2 concentration was then increased to 250 pg/ml in 5% FBS, and increases in cell number were determined at confluence. Every alternate day, fresh phenol red-free DMEM containing 5% FBS and 250 pg/ml E2 was added. Cells were cultured and treated with E2 before or after hER-alpha transfection using adenoviral vector as described in MATERIALS AND METHODS. One MOI is defined as 1 plaque-forming unit of virus per cell. Cell proliferation was measured by counting cell number on day 3 after treatment with 250 pg/ml E2 in 5% FBS as described in Fig 1. *Significant inhibition (P < 0.05) vs. untreated cells (n = 4). aSignificant difference vs. 3-mo-old cells (n = 4).

ER-alpha gene transfer augments E2-mediated inhibition of VSM cell proliferation in aged female rats. To further demonstrate that the inhibitory effect of E2 on VSM cell proliferation is mediated via ER-alpha activation, we first transfected cells with hER-alpha using adenovirus vector. Fluorescent microscopy observations demonstrated that 80-90% of VSM cells infected with 100 MOI of AD5/RSVGFP expressed high levels of GFP protein (data not shown). Thus, in all other experiments, we have used 100 MOI of either control vectors (Ad5/RSVGFP or Ad5/RSVlacZ) or ER-alpha vector (Ad5/RSVERalpha ) to transfect VSM cells. Cells were treated with 250 pg/ml E2 immediately after gene transfer, and proliferation was measured 3 days after E2 treatment. Transfection of ER-alpha gene into VSM cells increased the inhibitory effects of E2 in both young and aged animals (Fig. 2).

Constitutive and cytokine-stimulated NF-kappa B activation is increased in VSM cells from aged female rats. Recent evidence suggests that NF-kappa B signaling plays an important role in VSM cell proliferation and apoptosis (11a). Furthermore, ER-alpha may antagonize NF-kappa B signaling in VSM cells to inhibit cell apoptosis and increase VSM cell proliferation (40). Therefore, we tested the hypothesis that NF-kappa B activation is increased in VSM cells from aged female rats. VSM cells from aged female rats exhibit significantly higher (P < 0.05) levels of constitutive activity of NF-kappa B DNA binding activity compared with cells from young female rats (Fig. 3). In addition, IL-1beta -stimulated activity is also significantly higher (P < 0.05) in cells from older female rats compared with young female rats. An increase in constitutive as well as IL-1beta -stimulated NF-kappa B activation in nuclear extracts from aged female rats support our hypothesis that NF-kappa B may be playing a significant role in regulating expression of genes of potential importance in inflammation, cell proliferation, and apoptosis.


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  		Fig. 3.   Nuclear factor (NF)-kappa B activity is increased in aged female rat compared with young adult female rat VSM cells. Confluent cells in 100-mm Petri dishes were serum starved for 48 h before stimulation with interleukin (IL)-1beta (1 ng/ml). Nuclear extracts were collected, and NF-kappa B activity was estimated by electrophoretic mobility shift assay (EMSA) using consensus NF-kappa B binding 32P-labeled oligonucleotides according to kit manufacturer's instructions (Promega, Madison, WI). Gels were dried and quantified using PhosphorImager and ImageQuant analysis software (Molecular Dynamics, Sunnyvale, CA). *Significant (P < 0.05) difference between 3-mo-old and 24-mo-old female rat cells. #Significant (P < 0.05) increase in activity by IL-1beta (n = 3).

ER-alpha gene transfer inhibits basal and cytokine-stimulated NF-kappa B activation in VSM cells from aged female rats. To determine whether increased NF-kappa B activity in VSM cells from aged female rats is due to loss of E2, we chronically treated confluent VSM cells for 3 days with physiological concentrations of E2 (250 pg/ml) and estimated NF-kappa B activity by EMSA. E2 treatment of cultured cells from aged female rats partially inhibited both constitutive and IL-1beta -stimulated NF-kappa B DNA binding activity in VSM cell nuclear extracts from aged female rats (Fig. 4). Transfection of ER-alpha gene before E2 treatment further increased the E2 inhibitory effects on constitutive and cytokine-stimulated NF-kappa B activity (Fig. 4). These results suggest that E2 may play an important role in regulating NF-kappa B activation in VSM cells and that loss of E2 regulation of NF-kappa B after menopause may enhance expression of inflammatory and growth-promoting genes implicated in atherogenesis that increases with age.


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  		Fig. 4.   E2 treatment and hER-alpha gene transfer inhibit NF-kappa B activity in aged female rat VSM cells. Confluent cells were infected with 100 MOI of hER-alpha vector or a control green fluorescent protein (GFP) vector for 2 h. Cells were then serum starved for 72 h and simultaneously treated with E2 (250 ng/ml E2) before stimulation with IL-1beta (1 ng/ml; indicated by +) for 90 min. Nuclear extracts were collected, and NF-kappa B activity was estimated by EMSA, as described in Fig. 3. A: representative EMSA blot. B: quantitative data from 3 similar experiments. aSignificant differences (P < 0.05) between untreated GFP vector-transfected cells vs. IL-1beta -stimulated cells (n = 3). *Significant (P < 0.05) decrease in activity by E2 treatment of hER-transfected cells from aged female rats (n = 3).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present study demonstrates that 1) VSM cell proliferation is increased in aged female rats, 2) treatment of VSM cells with physiological concentration of E2 inhibits VSM cell proliferation to a greater extent in aged female rats compared with young adult rats, 3) ER-alpha gene transfection in VSM cells increases E2-mediated inhibition of VSM cell proliferation, and 4) constitutive and IL-1beta -stimulated NF-kappa B activity is increased in VSM cells from aged female rats compared with young adult rats and that ER-alpha gene transfer and E2 treatment significantly inhibit both constitutive and cytokine-stimulated NF-kappa B activation. These results suggest that increased NF-kappa B activation in VSM cells from aged female rats could be in part responsible for increased cell proliferation or decreased apoptosis. E2 treatment and ER-alpha gene transfer in VSM cells can inhibit the excessive NF-kappa B signaling and VSM cell proliferation observed in aged female rats. Similar to our findings, a recent study has demonstrated that E2 treatment inhibits NOS II induction in the rat uterus (43). Because the promoter region of NOS II contains putative NF-kappa B binding sites (42), it is possible that inhibition of NF-kappa B activation by E2 in rat uterus may be, at least in part, responsible for E2-mediated inhibition of NOS II induction.

It has been demonstrated that, with aging, VSM cells dedifferentiate and migrate from the arterial medial layer to the intima where they proliferate (reviewed in Ref. 6). VSM cells derived from aged animals replicate more actively than corresponding cells from newborn or young adult rats and require lower concentrations of growth factors for proliferation (6, 7, 24). Our findings support and extend these results by demonstrating that VSM cells cultured from 24-mo-old Fisher 344 female rats proliferate at a faster rate and reach higher cell density at confluence. It is possible that increased VSM cell proliferation in older females is due to a decrease in E2 levels and may represent the primary cause of development of CVD after menopause. In support, our results show that chronic treatment of VSM cells from aged female rats with E2 significantly inhibited VSM cell proliferation. Overexpression of functional hER-alpha receptor potentiated the inhibitory effects of E2 in aged female rats and almost abolished the differences in proliferation of VSM cells between young and aged female rats. Together, these studies suggest that of E2 may inhibit the progression of atherosclerotic lesions and CVD in postmenopausal women by inhibiting VSM cell proliferation.

The mechanisms of E2-mediated inhibition of VSM cell proliferation are not fully understood. We have observed that constitutive and IL-1beta -stimulated NF-kappa B activity is markedly increased in VSM cells from aged female rats compared with young adult female rats. We have also demonstrated that E2 treatment inhibits NF-kappa B activation in VSM cells from aged female rats and that the inhibitory effects of E2 on NF-kappa B activation are significantly potentiated by ER-alpha gene transfer. These findings support the hypothesis that ER-alpha receptor function is decreased in VSM cells from aged female rats and that enforced expression of ER-alpha inhibits VSM cell proliferation, at least in part, by inhibiting activation of NF-kappa B signaling. The role of the NF-kappa B pathway in increased VSM cell proliferation in aged female rats is further supported by the findings that NF-kappa B activation is increased in VSM cells at the site of vascular injury and in atherosclerotic lesions (8-10). Our results, demonstrating ER-alpha -mediated inhibition of NF-kappa B activation and VSM cell proliferation in aged female rats, underscore the importance of classical ER-alpha in regulating inflammatory and growth-promoting genes implicated in atherogenesis. These data support and extend earlier findings that suggested that a decrease in the expression of ER-alpha in atherosclerotic vessels may play an important role in the development of CVD in postmenopausal women (25).

The mechanism(s) of increased NF-kappa B activation in VSM cells from aged females and inhibition of NF-kappa B activation by E2 is not known. We postulate that ligand-activated ER-alpha may inhibit NF-kappa B signaling by competing for common transcription coactivators with NF-kappa B, thus inhibiting NF-kappa B-mediated gene transactivation. Several recent studies have demonstrated that reciprocal inhibition between ER-alpha and NF-kappa B is due to competition for CREB binding protein (CBP/p300) transcriptional coactivator proteins (18, 19, 23, 40). Thus it is possible that in aged female rats, due to a decrease in plasma E2 levels and/or decreased expression of functional ER-alpha number (25, 33), unopposed activation of NF-kappa B by proinflammatory stressors such as reactive oxygen species and cytokines would exaggerate inflammation. Experiments will be needed to demonstrate whether decreased competition for CBP/p300 by ER-alpha in VSM cells from aged female rats leads to increased, unopposed NF-kappa B activation.


    ACKNOWLEDGEMENTS

This work was supported by Grants AG-16504 and HL-14388 from the National Institutes of Health and a Grant-in-Aid from the American Heart Association (Heartland Affiliate).


    FOOTNOTES

Address for reprint requests and other correspondence: R. C. Bhalla, Dept. of Anatomy and Cell Biology, The Univ. of Iowa College of Medicine, Iowa City, IA 52242 (E-mail: ramesh-bhalla{at}uiowa.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 18 May 2001; accepted in final form 19 July 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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J APPL PHYSIOL 91(5):2400-2406
8750-7587/01 $5.00 Copyright © 2001 the American Physiological Society


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