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The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 8 3922-3931
Copyright © 2004 by The Endocrine Society

Catecholamines Block the Antimitogenic Effect of Estradiol on Human Coronary Artery Smooth Muscle Cells

Raghvendra K. Dubey, Edwin K. Jackson, Delbert G. Gillespie, Lefteris C. Zacharia and Bruno Imthurn

Department of Obstetrics and Gynecology, Clinic for Endocrinology, University Hospital Zurich (R.K.D., B.I.), 8091-CH Zurich, Switzerland; and Center for Clinical Pharmacology (R.K.D., E.K.J., D.G.G., L.C.Z., B.I.), Departments of Medicine (R.K.D., E.K.J., D.G.G., L.C.Z., B.I.) and Pharmacology (E.K.J., L.C.Z.), University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania 15213

Address all correspondence and requests for reprints to: Dr. Raghvendra K. Dubey, Department of Obstetrics and Gynecology, Clinic for Endocrinology (NORD-1, D217), University Hospital Zurich, 8091-CH Zurich, Switzerland. E-mail: raghvendra.dubey{at}usz.ch.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Sequential conversion of estradiol to catecholestradiols and methoxyestradiols by cytochrome-P450 (CYP450) and catechol-O-methyltransferase (COMT), respectively, contributes to the antimitogenic effects of estradiol on vascular smooth muscle cell (SMC) growth via estrogen receptor-independent mechanisms. Because catecholamines are also substrates for COMT, we hypothesize that catecholamines may abrogate the vasoprotective effects of estradiol by competing for COMT and inhibiting methoxyestradiol formation. To test this hypothesis, we investigated the antimitogenic/inhibitory effects of estradiol on human coronary artery SMC growth (cell number, DNA synthesis, collagen synthesis, and SMC migration) and ERK1/2 phosphorylation in the presence and absence of catecholamines. Norepinephrine, epinephrine, isoproterenol, and OR486 (COMT inhibitor) abrogated the inhibitory effects of estradiol on SMC growth and ERK1/2 phosphorylation. The interaction of catecholamines with estradiol was not affected by phentolamine or propanolol, {alpha}- and ß-adrenoceptor antagonists, respectively. The antimitogenic effects of 2-hydroxy-estradiol, but not 2-methoxyestradiol, were abrogated by epinephrine, isoproterenol, and OR486. Catecholamines inhibited the conversion of both estradiol and 2-hydroxy-estradiol to 2-methoxyestradiol, and SMCs expressed CYP1A1 and CYP1B1. Our findings suggest that catecholamines within the coronary arteries may abrogate the antivasoocclusive effects of estradiol by blocking the conversion of catecholestradiols to methoxyestradiols. The interaction between catecholamines and estradiol metabolism may importantly define the cardiovascular effects of estradiol therapy in postmenopausal women.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
EVIDENCE FROM MULTIPLE epidemiological and observational studies suggest that estradiol protects the cardiovascular system and is responsible for the lower incidence of coronary artery disease observed in premenopausal women compared with age-matched men (1). However, findings of randomized hormone replacement therapy trials for the primary and secondary prevention of cardiovascular disease do not support this idea (2, 3). The reasons for the negative findings remain unclear and emphasize the need for a greater understanding of the mechanisms by which estradiol influences the vasculature.

Although estradiol is known to positively influence the vasculature via multiple mechanisms (4), its inhibitory effects on smooth muscle cell (SMC) growth play a key role in protecting blood vessels against vasoocclusive disorders. Based on the conventional mechanisms of steroid action, the growth inhibitory effects of estradiol are thought to be mediated via estrogen receptor (ER){alpha} and/or ERß expressed by SMCs (5). However, recent findings that administration of exogenous estradiol inhibits injury-induced SMC proliferation in lesions of mice lacking ER{alpha} (6), ERß (7), or both ER{alpha} and ERß (double knockout) (8) suggest that the antimitogenic effects of estradiol on SMC growth may be ER independent and involve yet another mechanism.

Support for the participation of an ER-independent mechanism in mediating the antimitogenic effects of estradiol also comes from the observations that exogenous estradiol inhibits injury-induced neointima formation in gonadectomized, but not intact, male rats, even though vascular cells from both models express ERs (9). Pharmacological evidence for a role of ERs in mediating the antimitogenic effects of estradiol in SMCs is controversial. ICI182780, an ER antagonist, was effective in blocking the injury-induced neointima formation in one study (10), but not another (11). Due to similarity in its structure with estradiol, ICI182780 not only binds to the ER, but also blocks the metabolism of estradiol to 2-hydroxyestradiol (a potent inhibitor of SMC growth) by competing for cytochrome P450 (CYP450) enzymes (12). In in vitro studies with SMCs, we have demonstrated that ICI182780 blocks the antimitogenic effects of estradiol only at concentrations that inhibit the metabolism of estradiol to hydroxyestradiol (12, 13). Taken together, the above findings provide strong evidence that the antiproliferative actions of estradiol may be ER independent; however, the exact mechanism involved remains undiscovered.

Endogenous estradiol is sequentially metabolized to catecholestradiols (e.g. 2-hydroxyestradiol) by CYP450, and catecholestradiols are metabolized to methoxyestradiols (e.g. 2-methoxyestrdaiol) by catechol-O-methyltransferase (COMT). Catecholestradiols and methoxyestradiols have, respectively, little or no binding affinity for ERs, yet are potent inhibitors of cancer cell growth (14, 15). Because the antigrowth effects of estradiol are in part ER independent, we hypothesized that methoxyestradiols mediate the antiproliferative actions of estradiol on SMC growth. Subsequently, using pharmacological agents to block or induce the conversion of estradiol to catecholestradiols and methoxyestradiols and using COMT knockout mice, we provided strong evidence that methoxyestradiols mediate the antimitogenic effects of estradiol in SMCs (12, 13, 16). Moreover, we found similar effects in cardiac fibroblasts (17) and glomerular mesangial cells (18), cell types that are relevant for the cardiovascular system.

Apart from metabolizing estradiol to methoxyestradiols, COMT is a key enzyme responsible for catabolizing catecholamines (19). Therefore, it is conceivable that increased levels of catecholamines may abrogate the vasoprotective effects of estradiol by competing for COMT and inhibiting the conversion of catecholestradiols to methoxyestradiols. This hypothesis is supported by the observations: 1) that postmenopausal compared with premenopausal women exhibit greater stress-induced increases in catecholamine levels and sympathetic activity (20, 21); 2) that, in general, patients with cardiovascular disease (atherosclerosis and hypertension) have higher levels of catecholamines (22, 23, 24, 25); 3) that increased sympathetic activity accelerates the process of vasoocclusive disorders in animal models (26, 27); and 4) that increased synthesis of catecholamines under pathological conditions induces vasoocclusive disorders (28, 29).

As outlined in Fig. 1Go, the goals of the present study were to test the hypothesis that catecholamines can reduce the antigrowth effects of estradiol on SMCs via a mechanism that involves the competitive inhibition of methoxyestradiol formation by COMT. To test this hypothesis, we determined whether catecholamines abrogate the antimitogenic effects of estradiol and whether the abrogatory effects of catecholamines were due to inhibitory effects on the metabolism of estradiol to methoxyestradiols or to direct growth stimulatory effects of catecholamines via {alpha}- or ß-adrenoceptors.



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  		FIG. 1. A schematic representation of the hypothesis that the antimitogenic effects of estradiol are mediated via the sequential metabolism of ß-estradiol to hydroxyestradiol and methoxyestradiol by cytochrome P450 (CYP450) and COMT, respectively; and that catecholamines generated in response to pathological stimuli can interfere with the antiproliferative/antivasoocclusive actions of estradiol by competing for COMT and inhibiting methoxyestradiol formation. TH, Tyrosine hydroxylase, the rate-limiting enzyme for catecholamine (norepinephrine) synthesis. The arrow with the broken line indicates inhibitory effects.

 

   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials

All tissue culture reagents and culture ware were purchased from Life Technologies, Inc. (Grand Island, NY). Fetal calf serum (FCS) was obtained from HyClone (Logan, UT). 17ß-Estradiol, isoproterenol, norepinephrine, and epinephrine were purchased from Sigma-Aldrich Corp. (St. Louis, MO). 2-Hydroxyestradiol and 2-methoxyestradiol were procured from Steraloids (Newport, RI). The ER antagonist, ICI182780, and the COMT inhibitor, OR486, were obtained from Tocris (Langford, UK). [3H]Thymidine (specific activity, 11.8 Ci/mmol) was purchased from ICN Biomedicals (Costa Mesa, CA). L-[3H]Proline (specific activity, 23 Ci/mmol) was obtained from Amersham Pharmacia Biotech (Piscataway, NJ). Antibodies for ERK1/2 were obtained from Calbiochem (La Jolla, CA).

Cell culture

Well characterized female human coronary artery SMCs were obtained from Cascade Biologics (Portland, OR) and cultured as described by us previously (4). SMC purity was recharacterized by immunofluorescence staining with smooth muscle-specific, antismooth muscle {alpha}-actin monoclonal antibodies and by morphological criteria specific for SMC as described in detail previously (5). SMCs in the fourth passage were used for all studies.

Growth studies

[3H]Thymidine incorporation (index of DNA synthesis) and cell number (cell proliferation) studies were conducted to investigate the effects of various test agents on cell growth. SMCs were plated at a density of 5 x 103 cells/well in 24-well tissue culture dishes and allowed to grow to subconfluence in DMEM/Ham’s F-12 (phenol red-free) medium containing 10% FCS (steroid free and delipidated) under standard tissue culture conditions. The cells were then growth arrested by feeding them DMEM (phenol red free) containing 0.4% albumin for 48 h. For DNA synthesis, growth was initiated by treating growth-arrested cells for 20 h with DMEM containing 2.5% FCS and containing or lacking the test agent(s). To evaluate the roles of ERs and adrenergic receptors ({alpha} and ß), cells were pretreated for 30 min with ICI182780, phentolamine, or propranolol before treatment with the test agents. After 20 h of incubation, the treatments were repeated with freshly prepared solutions, but supplemented with [3H]thymidine (1 µCi/ml) for an additional 4 h. The experiments were terminated by washing the cells twice with Dulbecco’s PBS and twice with ice-cold trichloroacetic acid (10%). The precipitate was solubilized in 500 µl 0.3 N NaOH and 0.1% sodium dodecyl sulfate after incubation at 50 C for 2 h. Aliquots from four wells for each treatment with 10 ml scintillation fluid were counted in a liquid scintillation counter. For cell number experiments SMCs were allowed to attach overnight, were growth-arrested for 48 h, and then were treated every 24 h for 4 d; on d 5, cells were dislodged and counted on a Coulter counter (Beckman Coulter, Fullerton, CA).

[3H]Proline incorporation studies were performed to investigate the effects of various test agents on collagen synthesis. Confluent monolayers of SMCs were made quiescent by feeding DMEM containing 0.4% albumin for 48 h. SMCs growth arrested for 48 h were treated with DMEM supplemented with 2.5% FCS plus L-[3H]proline (1 µCi/ml) and containing or lacking the test agents. To evaluate the roles of ERs and adrenergic receptors ({alpha} and ß), cells were pretreated for 30 min with ICI182780, phentolamine, or propranolol before treatment with the test agents. The experiments were terminated by washing the cells twice with PBS and twice with ice-cold trichloroacetic acid (10%). The precipitate was solubilized as described above, and aliquots from four wells for each treatment were counted in a liquid scintillation counter. Each experiment was conducted in triplicate and with three separate cultures of SMCs. To make sure that the inhibitory effects of the experimental agents on collagen synthesis were not due to changes in cell number, the experiments were conducted in confluent monolayers of cells in which changes in cell number were precluded. Additionally, cell counting was performed in cells treated in parallel with the cells used for the collagen synthesis studies, and data were normalized to cell number.

SMC migration

Modified Boyden’s chambers (NeuroProbe, Inc., Cabin John, MD) were used to evaluate the effects of various treatments on platelet-derived growth factor-BB (PDGF-BB)-induced SMC migration, as previously described (5).

ERK1/2 phosphorylation

The effects of the test agents on ERK1/2 phosphorylation were also assessed because ERK1/2 is an important mediator of cell growth. SMCs grown to subconfluence in 35-mm2 culture dishes were treated for 24 h with or without various test agents in the presence of FCS (2.5%). After the treatments, the cells were washed with ice-cold PBS and solubilized by adding lysis buffer. Equal amounts of proteins (20 µg/lane) were denatured by boiling at 95 C for 5 min and resolved in 10% sodium dodecyl sulfate-polyacrylamide gel. After separation, the proteins were transferred to a nitrocellulose membrane, the membranes were probed with ERK1/2 antibodies, and the bands were visualized after staining with peroxidase-conjugated secondary antibody and the enhance chemiluminescence system and using X-OMAT LS films (Eastman Kodak Co., Rochester, NY) for exposure.

Metabolism studies

To assess the effects of catecholamines on the conversion of 2-hydroxyestradiol to 2-methoxyestradiol, confluent SMCs were incubated with 2-hydroxyestradiol for 1 h in the presence or absence of catecholamines (isoproterenol, epinephrine, or norepinephrine). Next, internal standard (16{alpha}-hydroxyestradiol) was added, samples were extracted with methylene chloride, extracts were dried under vacuum, residues were reconstituted in the mobile phase, and samples were analyzed by HPLC with ultraviolet detection using gradient elution, as previously described (13). Due to decreased assay sensitivity, the metabolism of estradiol to 2-methoxyestradiol by SMCs was assessed in the presence of microsomes to facilitate the formation 2-hydroxyestradiol (substrate for 2-methoxyestradiol) from estradiol. Briefly, human microsomes (1 mg/ml) were incubated for 2 h with 75 µmol/liter estradiol, and the supernatants containing the metabolites were collected. Subsequently, confluent monolayers of SMCs were treated for 1 h in the presence or absence of catecholamines (isoproterenol, epinephrine, or norepinephrine) with supernatants collected from human microsomes incubated with estradiol (as described above). After the treatment, the samples were extracted, and 2-methoxyestradiol levels were analyzed by HPLC (13).

CYP1A1 and CYP1B1 expression studies

To investigate whether the SMCs express CYP1A1 and CYP1B1, cell lysates from SMCs treated for 36 h with the CYP450 inducer 3-methylcholantherene (10 µmol/liter) were analyzed by Western blots and probed with antibodies against CYP1A1 (rabbit antihuman polyclonal antibodies; Chemicon International, Inc., Temecula, CA) and CYP1B1 (rabbit antihuman polyclonal antibodies; Gentest Corp., Woburn, MA).

Statistics

All experiments were conducted in triplicate or quadruplicate and repeated three to four times using separate cultures. Statistical analysis was performed using ANOVA. For data presented as a percentage of the control, statistics was performed on the original data using ANOVA. When evaluating a treatment-dependent effect and /or concentration-dependent effect within a group, data were analyzed by one-factor ANOVA, followed by Fisher’s least significant difference test for multiple comparisons. Two-way ANOVA followed by Bonferroni’s t test or Dunnett’s multiple comparison test were employed to compare differences between groups. All treatment-related effects within a group at a specific time point were compared by unpaired t test. P < 0.05 was considered statistically significant. Results are presented as the mean ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Treatment with 2.5% FCS stimulated [3H]thymidine incorporation (DNA synthesis) and [3H]proline incorporation (collagen synthesis) by approximately 8- and 7-fold (P < 0.001 vs. 0.25% albumin), respectively. Treatment with 1–100 nmol/liter estradiol concentration-dependently inhibited FCS-induced [3H]thymidine incorporation (Fig. 2Go), [3H]proline incorporation (Fig. 3Go), and cell number (cell proliferation; Fig. 3Go). Treatment with 100 nmol/liter estradiol inhibited FCS-induced [3H]thymidine incorporation (Fig. 2Go), cell number (Fig. 3AGo), and [3H]proline incorporation (Fig. 3BGo) by approximately 40–50%. Epinephrine, norepinephrine, and isoproterenol abrogated the effects of 100 nmol/liter estradiol on FCS-induced [3H]thymidine incorporation in a concentration-dependent manner (Fig. 2AGo). Treatment with norepinephrine and epinephrine, but not isoproterenol, had a slight, but significant, stimulatory effect on FCS-induced [3H]thymidine incorporation. At concentrations of 1 and 10 µmol/liter, norepinephrine and epinephrine enhanced serum-induced [3H]thymidine incorporation by 5.3 ± 2.4% and 6.2 ± 3%, respectively, and 6.2 ± 1.7% and 7.4 ± 0.8%, respectively. Isoproterenol at concentrations of 0.1, 1, and 10 µmol/liter reversed the inhibitory effect of estradiol on FCS-induced [3H]thymidine incorporation from 42% to 24 ± 2.6%, 10.5 ± 1.9%, and approximately 1 ± 0.04% (Fig. 2AGo), respectively; epinephrine at concentrations of 0.1, 1, and 10 µmol/liter reversed the inhibitory effect from 42% to 25 ± 3%, 8 ± 2.1%, and approximately 1 ± 0.06% (Fig. 2AGo), respectively; norepinephrine at concentrations of 0.1, 1, and 10 µmol/liter reversed the inhibitory effect from 46% to 37 ± 2.3%, 26.3 ± 1.7%, and 3.7 ± 1.1% (Fig. 2AGo), respectively. OR486, a selective inhibitor of COMT, at a concentration of 1 µmol/liter completely reversed estradiol-mediated inhibition of FCS-induced [3H]thymidine incorporation (Fig. 2AGo).



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  		FIG. 2. A, Attenuation by isoproterenol (ISO), norepinephrine (NE), epinephrine (EPI), and OR486 (0.1–10 µmol/liter) of the inhibitory effects of estradiol (ß-Est; 100 nmol/liter) on 2.5% FCS-induced DNA synthesis ([3H]thymidine incorporation). B, Concentration-dependent inhibitory effects of estradiol (ßE; 1–100 nmol/liter) on 2.5% FCS-induced DNA synthesis ([3H]thymidine incorporation) in the presence and absence of 10 µmol/liter ISO, EPI, NE, and OR486 (OR). For data presented as a percentage of the control, the statistical analysis was conducted on original (raw) data. §, P < 0.05 vs. control cells treated with FCS alone; *, P < 0.05 vs. cells treated with estradiol (significant reversal of the inhibitory effects).

 


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  		FIG. 3. A, The inhibitory effects of estradiol (ßE; 1–100 nmol/liter) on 2.5% FCS-induced cell number (A) and collagen synthesis ([3H]proline incorporation; B) in the presence and absence of 10 µmol/liter isoproterenol (ISO), epinephrine (EPI), norepinephrine (NE), and OR486 (OR). For data presented as a percentage of the control, statistical analysis was conducted on original (raw) data. *, P < 0.05 vs. control cells treated with FCS alone; §, P < 0.05 vs. cells treated with estradiol (significant reversal of the inhibitory effects).

 
FCS increased cell number in growth-arrested SMCs by approximately 7-fold (data not shown). Estradiol at 10 nmol/liter inhibited FCS-induced increases in cell number by 30%, and the catecholamines reversed this inhibitory effect (Fig. 3AGo). At a concentration of 10 µmol/liter, isoproterenol, epinephrine, and norepinephrine reversed the inhibitory effects of estradiol from 30 ± 2% to 6 ± 1.2%, 8 ± 0.6%, and 10 ± 1.4%, respectively. OR486 at a concentration of 10 µmol/liter reversed the inhibitory effect from 30 ± 2% to 4 ± 0.8%. Treatment with epinephrine and norepinephrine, but not isoproterenol, induced cell proliferation marginally (5–7%). Treatment with OR486 alone had no effect on basal or serum-induced SMC proliferation.

Similar to the effects on FCS-induced [3H]thymidine incorporation, the catecholamines abrogated the inhibitory effects of estradiol on collagen synthesis ([3H]proline incorporation; Fig. 3BGo). At a concentration of 10 µmol/liter, isoproterenol, epinephrine, and norepinephrine dramatically reversed the inhibitory effects of 100 nmol/liter estradiol on [3H]proline incorporation from 36% to 5 ± 0.7%, 3 ± 0.06%, and 8 ± 0.7%, respectively (Fig. 3BGo). Treatment with norepinephrine and epinephrine, but not isoproterenol, stimulated FCS-induced [3H]proline incorporation (collagen synthesis) by 6–8%. At a concentration of 10 µmol/liter, OR486 completely reversed the inhibitory effect of estradiol (1–100 nmol/liter) on [3H]proline incorporation (Fig. 3BGo).

Treatment of SMCs with PDGF-BB induced SMC migration, and this effect was concentration-dependently inhibited by estradiol (Fig. 4AGo). The inhibitory effects of estradiol on PDGF-BB-induced SMC migration were significantly abrogated in the presence of 10 µmol/liter isoproterenol, epinephrine, norepinephrine, or OR486 (Fig. 4BGo). Treatment of SMCs with catecholamines alone marginally enhanced the stimulatory effect of PDGF-BB on SMC migration. Isoproterenol, epinephrine, and norepinephrine, but not OR486, marginally enhanced the stimulatory effect of PDGF-BB by 3 ± 0.7%, 5 ± 1%, and 6.6 ± 0.4%, respectively. Similar to the effects on SMC proliferation, treatment of SMCs with OR486 alone had no effect on PDGF-BB-induced SMC migration.



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  		FIG. 4. A, Line graph showing the concentration-dependent inhibitory effects of estradiol (ßE; 1–100 nmol/liter) on 25 ng/ml PDGF-BB-induced SMC migration. B, The bar graph depicts the abrogation by 10 µmol/liter of isoproterenol (ISO), epinephrine (EPI), norepinephrine (NE), and OR486 (OR) of the inhibitory effects of estradiol (ß-E; 100 nmol/liter) on PDGF-BB-induced SMC migration. Data on the y-axis show the number of cells migrating at a fixed high power field (HPF). §, P < 0.05 vs. control cells treated with PDGF-BB alone; *, P < 0.05 vs. cells treated with estradiol (significant reversal of the inhibitory effects).

 
Catecholamines mediate their growth effects via {alpha}- and ß-adrenoceptors (30). Norepinephrine and epinephrine activate {alpha}-adrenoceptors (both subtypes 1 and 2) and ß-adrenoceptors (subtype 1 for norepinephrine and both subtypes 1 and 2 for epinephrine), whereas isoproterenol activates primarily ß-adrenoceptors (both subtypes 1 and 2), with lesser effects on {alpha}-adrenoceptors (30). In the present study the stimulatory effects of epinephrine on DNA synthesis, cell proliferation, and collagen synthesis were blocked by phentolamine, an {alpha}-adrenoceptor antagonist (Fig. 5Go, A–C). Similar to epinephrine, norepinephrine induced mitogenesis in SMCs, and these effects were also blocked by phentolamine (data not shown). These findings suggest that {alpha}-adrenoceptors mediate the mitogenic effects of epinephrine and norepinephrine. In contrast to epinephrine and norepinephrine, treatment of SMCs with isoproterenol did not induce mitogenesis; moreover, the effects were not altered by propranolol, a ß-adrenoceptor antagonist (Fig. 5Go, D–F). Together the above findings suggest that the mitogenic effects of catecholamines are mediated via {alpha}-adrenoceptors, but not ß-adrenoceptors.



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  		FIG. 5. Left panels, Modulatory effects of epinephrine (EPI; 1 µmol/liter) on the inhibitory effect of estradiol (ßE; 0.1 µmol/liter) on 2.5% FCS-induced DNA synthesis ([3H]thymidine incorporation; A), cell number (cell proliferation; B), and collagen synthesis ([3H]proline incorporation; C) in the presence and absence of the {alpha}-adrenoceptor blocker, phentolamine (PH; 3 µmol/liter). §, P < 0.05 vs. control (FCS); *, P < 0.05 vs. cells treated with estradiol (significant reversal of the inhibitory effects); {dagger}, P < 0.05 vs. cells treated with epinephrine alone (significant reversal of the mitogenic effects of EPI). Right panels, Modulatory effects of isoproterenol (ISO; 1 µmol/liter) on the inhibitory effect of estradiol (ßE; 0.1 µmol/liter) on 2.5% FCS-induced DNA synthesis ([3H]thymidine incorporation; D), cell number (cell proliferation; E), and collagen synthesis ([3H]proline incorporation; F) in the presence and absence of the ß-adrenoceptor blocker, propranolol (PR; 3 µmol/liter). §, P < 0.05 vs. control (FCS); *, P < 0.05 vs. cells treated with estradiol (significant reversal of the inhibitory effects).

 
To investigate whether the abrogatory effects of catecholamines were mediated via adrenoceptors, we in-vestigated their ability to block the antimitogenic effects of estradiol in the presence or absence of the {alpha}- and ß-adrenoceptor antagonists, phentolamine and propranolol, respectively. As shown in Fig. 5Go, A–C, 1 µmol/liter epinephrine blocked the inhibitory effects of 100 nmol/liter estradiol on FCS-induced [3H]thymidine incorporation (Fig. 5AGo), cell proliferation (Fig. 5BGo), and [3H]proline incorporation (Fig. 5CGo), and the abrogatory effects of epinephrine were not diminished by 3 µmol/liter phentolamine. Similarly, the abrogatory effects of isoproterenol on estradiol-mediated inhibition of FCS-induced [3H]thymidine incorporation, [3H]proline incorporation, and cell proliferation were not prevented by propranolol (Fig. 5Go, D–F).

To determine whether catecholamines block the antimitogenic effects of estradiol by preventing the conversion of catecholestradiols to methoxyestradiols, we investigated the growth inhibitory effects of 2-hydroxyestradiol and 2-methoxyestradiol in the presence and absence of epinephrine and isoproterenol. As shown in Fig. 6Go, the inhibitory effects of 2-hydroxyestradiol on FCS-induced [3H]thymidine incorporation (Fig. 6AGo), cell proliferation (Fig. 6BGo), and [3H]proline incorporation (Fig. 6CGo) were abrogated by epinephrine, norepinephrine, and isoproterenol and also by the COMT inhibitor, OR486. Moreover, the abrogatory effects of epinephrine and isoproterenol were not reversed by phentolamine or propranolol, suggesting that the effects were independent of adrenoceptors. Similar to 2-hydroxyestradiol, 2-methoxyestradiol inhibited FCS-induced SMC growth and [3H]proline incorporation (Fig. 6CGo). However, in contrast to estradiol and 2-hydroxyestradiol, the inhibitory effects of 2-methoxyestradiol were not blocked by catecholamines or OR486 (Fig. 6Go), suggesting that catecholamines abrogate the effects of estradiol by blocking the conversion of catecholestradiols to methoxyestradiols.



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  		FIG. 6. Modulatory effects of epinephrine (EPI; 1 µmol/liter), isoproterenol (ISO; 1 µmol/liter), norepinephrine (NE), and OR486 (OR) on the inhibitory effect of 2-hydroxyestradiol (OE; 0.1 µmol/liter; left panels) and 2-methoxyestradiol (ME; 0.1 µmol/liter; right panels) on 2.5% FCS-induced DNA synthesis (A), cell proliferation (B), and collagen synthesis (C). §, P < 0.05 vs. control; *, P < 0.05 vs. cells treated with 2-hydroxyestradiol or 2-methoxyestradiol alone (significant reversal of the inhibitory effects).

 
The inhibitory effect of estradiol on ERK1/2 is in part responsible for mediating its growth inhibitory effects. Treatment of SMCs with 10 nmol/liter estradiol for 24 h inhibited 2.5% FCS-induced expression of phosphorylated ERK1/2. The inhibitory effect of estradiol was abrogated by 1 µmol/liter isoproterenol, epinephrine, norepinephrine, and OR486 (Fig. 7Go). Treatment of SMCs with catecholamines alone did not significantly enhance the 2.5% FCS-induced expression of phosphorylated ERK1/2 (data not shown).



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  		FIG. 7. Representative Western blot depicting the modulatory effects of epinephrine (EPI; 1 µmol/liter), isoproterenol (ISO; 1 µmol/liter), norepinephrine (NE), and OR486 (OR) on the inhibitory effect of ß-estradiol (ßE; 0.1 µmol/liter) on 2.5% FCS-induced expression of phosphorylated ERK1/2 (ERK-P; 44/42 kDa). The inhibitory effect of estradiol on ERK1/2 phosphorylation was blocked by EPI, ISO, NE, and OR.

 
To determine whether catecholamines can block the conversion of estradiol to 2-methoxyestradiol, we first evaluated the effects of isoproterenol, epinephrine, and norepinephrine on the conversion of 2-hydroxyestradiol to 2-methoxyestradiol. SMCs efficiently metabolized 2-hydroxyestradiol to 2-methoxyestradiol, an effect that was concentration-dependently inhibited by the COMT inhibitor, OR486 (Fig. 8AGo). Similarly, isoproterenol, epinephrine, and norepinephrine inhibited methylation of 2-hydroxyestradiol (Fig. 8AGo). At a concentration of 10 µmol/liter, norepinephrine, epinephrine, and isoproterenol inhibited the methylation of 2-hydroxyestradiol (0.25 µmol/liter) by 16 ± 1%, 21 ± 2%, and 36 ± 1.8%, respectively (P < 0.05). OR486 at a concentration of 1 µmol/liter inhibited 2-hydroxyestradiol methylation by 76 ± 4%. Compared with norepinephrine and epinephrine, isoproterenol more effectively inhibited 2-hydroxyestradiol metabolism.



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  		FIG. 8. A, Line graph showing the concentration-dependent inhibitory effect of COMT inhibitor OR486 (0–2.5 µmol/liter) on the conversion of 2-hydroxyestradiol (0.25 µmol/liter) by SMCs (top panel). The bar graph shows the metabolism of 2-hydroxyestradiol (0.25 µmol/liter) to 2-methoxyestradiol (2-MeOE) by cultured SMCs and the inhibitory effect of isoproterenol (ISO; 10 µmol/liter), epinephrine (EPI; 10 µmol/liter), and norepinephrine (NE; 10 µmol/liter) on the conversion of 2-hydroxyestradiol to 2-methoxyestradiol. *, P < 0.05 vs. 2-MeOE formation in absence of inhibitors. B, The top panel shows concentration-dependent inhibitory effect of isoproterenol on 2-methoxyestradiol formation in SMCs incubated with microsomal extracts (0.25 mg/ml) that were incubated for 2 h with estradiol (75 µmol/liter). In control SMCs treated with the microsomal extracts, 2-methoxyestradiol production was 17 ± 1.4 pmol/min·million cells (represents 100%). The lower panel shows the inhibitory effect of 50 µmol/liter isoproterenol (ISO), epinephrine (EPI), and norepinephrine (NE) on 2-methoxyestradiol formation in SMCs incubated with microsomal extracts. *, P < 0.05 vs. 2-MeOE formation in the absence of inhibitors. C, Western blots showing the expression of cytochrome P450 isozymes, CYP1A1 and CYP1B1, in human coronary artery SMCs pretreated for 36 h with 10 µmol/liter 3-methylcholantherene. Lanes 1–3 depict cell lysates from three separate SMC preparations. D, Line graph comparing the concentration-dependent inhibitory effects of 2-methoxyestradiol (2ME) and 4-methoxyestradiol (4ME) on FCS-induced DNA synthesis ([3H]thymidine incorporation) in SMCs. *, P < 0.05 vs. cells treated with FCS alone; §, P < 0.05 vs. 2ME. Values represent the mean ± SEM from at least three independent experiments. Each experiment was conducted at least in triplicate.

 
In SMCs treated for 1 h with supernatants of microsomes incubated with estradiol, significant amounts (17 ± 1.4 pmol/min·million cells) of 2-methoxyestradiol were formed. The formation of 2-methoxyestradiol from estradiol was inhibited by isoproterenol in a concentration-dependent manner (Fig. 8BGo, top panel). Moreover, the formation of 2-methoxyestradiol was significantly inhibited by catecholamines (Fig. 8BGo, bottom panel). At a concentration of 50 µmol/liter isoproterenol, epinephrine and norepinephrine inhibited 2-methoxyestradiol formation by 53 ± 4%, 39 ± 1.6%, and 32.5 ± 2.8%, respectively (Fig. 8BGo, bottom panel). Estradiol is metabolized to catecholestradiols by CYP450. In this regard, the CYP450 isozymes, CYP1A1 and CYP1B1, are responsible for converting estradiol to 2- and 4-hydroxyestradiols (4) (precursors for 2- and 4-methoxyestradiol), respectively. Importantly, we detected by Western blot analysis the presence of the CYP450 isozymes, CYP1A1 and CYP1B1, in SMCs (Fig. 8CGo). As shown in Fig. 8DGo, both 2- and 4-methoxyestradiol inhibited FCS-induced DNA synthesis in a concentration-dependent manner; moreover, 2-methoxyestradiol was more potent than 4-methoxyestradiol in inhibiting SMC growth (Fig. 8DGo).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The protective effects of estradiol on the cardiovascular system remain controversial. However, it is well established that estradiol can influence both vascular biology and physiology by directly interacting with cells in the vessel wall (4). Mounting evidence suggests that in addition to the conventional ER-dependent mechanism, estradiol can induce its biological effects via nonclassical ER-independent and nongenomic mechanisms (4). To understand the adverse as well as the beneficial effects of estradiol on the cardiovascular system, it is important to elucidate the various mechanisms by which estradiol can influence vascular biology and physiology.

Abnormal growth of SMCs plays a major role in the vascular remodeling process associated with vasoocclusive disorders, including atherosclerosis and coronary artery disease (31). In the present study physiological and pharmacological concentrations of estradiol inhibited SMC growth, and these effects were attenuated by catecholamines. The abrogatory effects of catecholamines were not reduced by {alpha}- and ß-adrenoceptor blockers, and catecholamines blocked the inhibitory effects of 2-hydroxyestradiol, but not 2-methoxyestradiol, on SMC growth. The abrogatory effects of catecholamines were mimicked by the selective COMT inhibitor, OR486. SMCs efficiently metabolized 2-hydroxyestradiol to 2-methoxyestradiol and expressed CYP450 enzymes responsible for converting estradiol to 2-hydroxyestardiol. Moreover, both catecholamines and OR486 inhibited the conversion of estradiol and 2-hydroxyestradiol to 2-methoxyestradiol by SMCs. Taken together, our findings provide evidence that catecholamines block the antimitogenic effects of estradiol in SMCs by inhibiting methoxyestradiol formation. Our results imply that the vasoprotective effects of estradiol in any individual may depend in part on the local levels of catecholamines in the vessel wall.

Adrenergic receptors mediate the effects of catechol-amines on cell growth (30); hence, it is possible that cate-cholamines abrogate the antimitogenic effects of estradiol and 2-hydroxyestradiol by interacting with the adrenergic receptors rather than by inhibiting COMT. In this context, in vascular SMCs, catecholamines induce SMC growth via {alpha}1-adrenoceptors and inhibit SMC growth via ß2-adrenoceptors (30). In the present study SMC growth was induced by the {alpha}-adrenoceptor agonist, epinephrine, but not by the ß-adrenoceptor agonist, isoproterenol. Moreover, the mitogenic effects of catecholamine were blocked by the {alpha}-adrenoceptor antagonist, phentolamine, but not by the ß-adrenoceptor antagonist, propranolol, suggesting that the mitogenic effects in SMCs are mediated by {alpha}-adrenoceptors. Our finding that the abrogatory effects of catecholamines are not blocked by phentolamine or propranolol provides strong evidence that catecholamines abrogate the effects of estradiol by inhibiting COMT and not by activating adrenergic receptors. A direct effect of catecholamines on SMC growth is also ruled out by the observation that in presence of FCS, catecholamines only marginally affect SMC proliferation in the absence of estradiol and 2-hydroxyestradiol. In this regard, the highest concentrations (10 µmol/liter) of epinephrine and norepinephrine enhanced FCS-induced SMC proliferation by approximately 6–7%, and isoproterenol inhibited proliferation by approximately 6%.

We have previously shown that the antimitogenic effects of estradiol on SMCs are blocked by CYP450 inhibitors and enhanced by CYP450 inducers (13). Because CYP450 isozymes are responsible for metabolizing estradiol to catecholestradiols, a precursor of methoxyestradiols, we postulated that the sequential conversion of estradiol to catecholestradiols and methoxyestradiols is responsible for mediating the antigrowth effects of estradiol. Our finding that SMCs expressed CYP1A1 and CYP1B1 and that catecholamines block the conversion of estradiol to 2-methoxyestradiol provides strong evidence that in SMCs estradiol can be locally converted to catecholestradiols and methoxyestradiols. Our contention that local conversion of estradiol to methoxyestradiols is responsible for its ER-independent antimitogenic effects on SMCs is also supported by our recent findings that the antimitogenic effects of estradiol are abrogated in SMCs from COMT knockout mice (16). We have reached similar conclusions in glomerular mesangial cells (18) and cardiac fibroblasts (17), cells relevant to the cardiovascular system.

In the present study, a concentration (10 µmol/liter) of catecholamines that inhibited 2-methoxyestradiol formation by 16–36% was able to completely reverse the inhibitory effect of estradiol on SMC growth. A potential explanation for the difference in the inhibitory effects of catecholamines on metabolism vs. growth might be the difference in ex-perimental conditions (cell number, cell density, time of treatment, and concentration of estradiol). In this context, it is important to note that the inhibitory effects of catecholamines on the metabolism of estradiol and the antigrowth effects of estradiol are time dependent. Importantly, in the metabolism studies the cells were incubated for 1 h, whereas in the growth studies the cells were treated for 24 h or 4 d.

The observations of the present study suggest that catecholamines block the antimitogenic effects of estradiol by blocking the formation of methoxyestradiols; however, additional studies are required to elucidate the exact contribution of 2- and 4-methoxyestradiol in mediating the anti-mitogenic effects of estradiol. It is well established that 2-methoxyestradiol is the major endogenous metabolite of estradiol, whereas 4-methoxyestradiol is a minor metabolite (32). Moreover, CYP1B1 metabolizes estradiol largely to 4-hydroxyestradiol and to a lesser extent to 2-hydroxyestradiol. In contrast, CYP1A1 metabolizes estradiol largely to 2-hydroxyestradiol, whereas 4-hydroxyestradiol is a minor product (4, 14). Because SMCs express both CYP1A1 and CYP1B1, the continuous formation of both 2- and 4-hydroxyestradiols may play an important role in mediating the antimitogenic effect of estradiol on SMCs. This idea is further supported by our finding that both 2-methoxyestradiol and 4-methoxyestradiol inhibit FCS-induced SMC growth.

In vivo metabolism of estradiol to 2-hydroxyestradiol accounts for 50% of the estradiol metabolites formed (4, 14), and the levels of catecholestradiols range from 0.12–0.3 µmol/liter in peripheral blood. Substantial amounts of 2-hydroxyestradiol are thus available to be converted to 2-methoxyestradiol. Due to the rapid conversion of 2-hydroxyestradiol to 2-methoxyestradiol, accurate data on the levels of 2-hydroxyestradiol are not available. Nonetheless, the serum level of 2-methoxyestradiol in pregnant women is 30 nmol/liter, and rough estimates suggest that 2-methoxyestradiol levels may be several-fold higher than the levels of estradiol (4, 14). SMCs and vascular endothelial cells are well endowed with COMT, ensuring pharmacologically active, steady state levels of methoxyestradiols within the vessel wall.

Under normal conditions, circulating levels of norepinephrine and epinephrine range from 1–2 nmol/liter (33); however, after sympathetic nerve stimulation the circulating levels of norepinephrine and epinephrine increase to as high as 12 nmol/liter (34). Moreover, concentrations of norepinephrine in average neuroeffector junctions are almost 4 times greater than circulating levels (35), i.e. approximately 50 nmol/liter. Indeed, both the width of the junction and the efficiency of uptake and metabolism of norepinephrine define the final levels of norepinephrine at a individual neuroeffector junction, which in some junctions can be higher than 50 nmol/liter. In the present study, concentrations of catecholamines as low as 100 nmol/liter significantly attenuated the growth inhibitory effect of 100 nmol/liter estradiol and 2-hydroxyestradiol. As the interaction between catecholamines and estradiol-derived 2-hydroxyestradiol at the level of COMT is competitive, even lower levels of catecholamines would be expected to attenuate the antimitogenic effects of lower levels of estradiol/2-hydroxyestradiol. Moreover, the sequential conversion of estradiol to 2-hydroxyestradiol and 2-methoxyestradiol locally within the SMCs would be more susceptible to inhibition by catecholamines due to the rate-limiting step of 2-hydroxyestradiol formation. In this context we have recently shown that the conversion of 2-hydroxyestradiol to 2-methoxyestradiol in isolated organs is significantly inhibited after nerve stimulation (36). Taken together, these considerations imply that increased synthesis of catecholamines under pathological conditions could effectively attenuate the inhibitory effects of estradiol on SMC growth.

Increases in sympathetic activity, circulating catecholamine levels, and secretion of norepinephrine (26, 27, 28, 29) have been observed in patients with cardiovascular disease, including essential hypertension, atherosclerosis, and coronary artery disease (37, 38, 39). Also, stress-induced sympathetic activity as well as circulating catecholamine levels and catecholamine spill-over are increased by approximately 2-fold in peri- and postmenopausal women compared with premenopausal women (40, 41, 42, 43). This implies that at the neuroeffector junctions the levels of norepinephrine in some people are much higher than normal. The higher levels of norepinephrine could potentially occupy COMT to such an extent as to inhibit or limit the metabolism of other catechols. This could also inhibit or limit the conversion of catecholestrogens and consequently abrogate the vascular protective action of estradiol.

Increased sympathetic activity is positively associated with intimal thickening (26, 28), suggesting that catecholamines can induce vascular remodeling processes associated with vasoocclusive disorders. In animal models in which the sympathetic activity is pharmacologically inhibited (38, 44) or minimized by denervation (24, 26), the progression of cardiovascular disease (hypertension and atherosclerosis) is substantially reduced compared with that in untreated animals. Moreover, the growth-inducing effects of catecholamines are blocked when adrenergic receptors are blocked (30). This indirect evidence indicates the adverse effects of catecholamines on the cardiovascular system. Our findings suggest that increased catecholamines may have an adverse effect on the progression of vasoocclusive disorders by competing for COMT and thereby reducing the methylation of estradiol-derived 2-hydroxyestradiol. Interestingly, we have recently shown that compared with other arterial beds, SMCs from coronary arteries are approximately 2-fold more efficient in converting catecholestradiols to methoxyestradiols (36), suggesting that efficient conversion of estradiol to methoxyestradiol may play a critical role in specifically protecting women against coronary artery disease. Thus, the antivasoocclusive actions of estradiol in postmenopausal women may be defined by the local levels of catecholamines, and increased catecholamine levels may be responsible for the lack of protective effects observed in some studies.

Similar to vascular SMCs, estradiol and 2-hydroxyestradiol inhibit the growth of cardiac fibroblasts (17). Because catecholamines are known to play a key role in the cardiac remodeling process associated with ventricular hypertrophy in myocardial infarction and heart failure (39), it is possible that the interactions between catecholestradiols and catecholamines may participate in the pathophysiology of cardiac disorders.

COMT is not only responsible for converting estradiol to methoxyestradiols; it is also a key enzyme that catabolizes catecholamines (19). Via a competitive interaction for COMT, catecholamines can inhibit the conversion of estradiol to methoxyestradiol and abrogate the antimitogenic effects of estradiol (45, 46). In contrast, because of competition for COMT, via generation of catecholestradiols, it is conceivable that estradiol could elevate catecholamine levels. However, because catecholestradiols directly inhibit tyrosine hydroxylase (47), a rate-limiting enzyme for catecholamine synthesis, it is unlikely that catecholestradiols would increase catecholamine levels. Indeed, administration of estradiol to postmenopausal women has been shown to lower circulating catecholamine levels, catecholamine spill-over, and sympathetic activity (40, 41, 42, 43).

In conclusion, our findings provide evidence that SMCs metabolize estradiol-derived catecholestradiols to methoxyestradiols, and COMT-mediated conversion of catecholestradiols to methoxyestradiols is essential for the inhibitory effects of estradiol on SMC growth. Our findings suggest that interactions between catecholamines and endogenous catecholestradiols may play an important role in defining the overall protective effects of estradiol in the coronary artery. These findings imply that estradiol metabolism may be an important determinant of the cardiovascular protective effects of estradiol. Thus, interindividual differences, either genetic or acquired, in estradiol metabolism may define a given female’s risk of cardiovascular disease and influence the cardiovascular benefit she receives from estradiol replacement therapy in the postmenopausal state.


   Footnotes
 
This work was supported by Swiss National Science Foundation Grant 32–64040.00 and NIH Grant HL-69846.

Abbreviations: COMT, Catechol-O-methyltransferase; ER, estrogen receptor; FCS, fetal calf serum; PDGF-BB, platelet-derived growth factor-BB; SMC, smooth muscle cell.

Received January 26, 2004.

Accepted April 23, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Stampfer MJ, Colditz GA 1991 Estrogen replacement therapy and coronary heart disease: a quantitative assessment of the epidemiologic evidence. Prev Med 20:47–63[CrossRef][Medline]
  2. Rossouw JE, Anderson GL, Prentice RL, LaCroix AZ, Kooperberg C, Stefanick ML, Jackson RD, Beresford SA, Howard BV, Johnson KC, Kotchen JM, Ockene J, Writing Group for the Women’s Health Initiative Investigators 2002 Risks and benefits of estrogen plus progestin in healthy postmenopausal women. JAMA 288:321–333[Abstract/Free Full Text]
  3. Hulley S, Grady D, Bush T, Furberg C, Herrington D, Riggs B, Vittinghoff E 1998 Randomized trial of estrogen plus progestin for secondary prevention of coronary artery heart disease in postmenopausal women: Heart and Estrogen/Progestin Replacement Study (HERS) research group. JAMA 280:605–613[Abstract/Free Full Text]
  4. Dubey RK, Jackson EK 2001 Estrogen-induced cardiorenal protection: potential cellular, biochemical, and molecular mechanisms. Am J Physiol 280:F365–F388
  5. Dubey RK, Gillespie DG, Zacharia LC, Imthurn B, Jackson EK, Keller PJ 2000 Clinically used estrogens differentially inhibit human aortic smooth muscle cell growth and MAP kinase activity. Arterioscler Thromb Vasc Biol 20:964–972[Abstract/Free Full Text]
  6. Iafrati MD, Karas RH, Aronovitz M, Kim S, Sullivan TR, Lubahn DB, O’Donnel TF, Korach KS, Mendelsohn ME 1997 Estrogen inhibits the vascular injury response in estrogen receptor {alpha}-deficient mice. Nat Med 3:545–548[CrossRef][Medline]
  7. Karas RH, Hodgin JB, Kwoun M, Krege JH, Aronovitz M, Mackey W, Gustafsson JA, Korach KS, Smithies O, Mendelsohn ME 1999 Estrogen inhibits the vascular injury response in estrogen receptor ß-deficient female mice. Proc Natl Acad Sci USA 96:15133–15136[Abstract/Free Full Text]
  8. Karas RH, Schulten H, Pare G, Aronovitz MJ, Ohlsson C, Gustafsson JA, Mendelsohn ME 2001 Effects of estrogen on the vascular injury response in estrogen receptor {alpha},ß (double knockout) mice. Circ Res 89:534–539[Abstract/Free Full Text]
  9. Oparil S, Levine RL, Chen SJ, Durand J, Chen YF 1997 Sexually dimorphic response of the balloon-injured rat carotid artery to hormone treatment. Circulation 95:1301–1307[Abstract/Free Full Text]
  10. Bakir S, Mori T, Durand J, Chen YF, Thompson JA, Oparil S 2000 Estrogen-induced vasoprotection is estrogen receptor dependent: evidence from the balloon-injured rat carotid artery model. Circulation 23;101:2342–2344
  11. Finking G, Lenz C, Schochat T, Hanke H 2002 Reduction of post injury neointima formation due to 17ß-estradiol and phytoestrogen treatment is not influenced by the pure synthetic estrogen receptor antagonist ICI 182,780 in vitro. BMC Cardiovasc Disord 2:13 (E-pub)[CrossRef][Medline]
  12. Dubey RK, Gillespie DG, Zacharia LC, Rosselli M, Korzekwa KR, Fingerle J, Jackson EK 2000 Methoxyestradiols mediate the antimitogenic effects of estradiol on vascular smooth muscle cells via estrogen receptor-independent mechanisms. Biochem Biophys Res Commun 278:27–33[CrossRef][Medline]
  13. Dubey RK, Gillespie DG, Zacharia LC, Barchiesi F, Imthurn B, Jackson EK 2003 CYP450- and COMT-derived estradiol metabolites inhibit activity of human coronary artery SMCs. Hypertension 41:807–813[Abstract/Free Full Text]
  14. Zhu BT, Conney AH 1998 Functional role of estrogen metabolism in target cells: review and perspectives. Carcinogenesis 19:1–27[Abstract/Free Full Text]
  15. Dubey RK, Jackson EK 2001 Cardiovascular protective effects of 17ß-estradiol metabolites. J Appl Physiol 91:1868–1884[Abstract/Free Full Text]
  16. Zacharia LC, Gogos JA, Karayiorgou M, Jackson EK, Gillespie DG, Barchiesi F, Dubey RK 2003 Methoxyestradiols mediate the antimitogenic effects of 17ß-estradiol. Direct evidence from catechol-O-methyltransferase knockout mice. Circulation 108:2974–2978[Abstract/Free Full Text]
  17. Dubey RK, Gillespie DG, Zacharia LC, Rosselli M, Imthurn B, Jackson EK 2002 Methoxyestradiols mediate the antimitogenic effects of locally applied estradiol on cardiac fibroblast growth. Hypertension 39:412–417[Abstract/Free Full Text]
  18. Dubey RK, Gillespie DG, Keller PJ, Imthurn B, Zacharia LC, Jackson EK 2002 Role of methoxyestradiols in the growth inhibitory effects of estradiol on human glomerular mesangial cells. Hypertension 39:418–424[Abstract/Free Full Text]
  19. Mànnisto PT, Kaakkola S 1995 Catechol-O-methyltransferase (COMT): biochemistry, molecular biology, pharmacology, and clinical efficacy of new selective COMT inhibitors. Pharmacol Rev 51:593–628
  20. Saab PG, Matthews KA, Stoney CM, McDonald RH 1989 Premenopausal and postmenopausal women differ in their cardiovascular and neuroendocrine responses to behavioral stressors. Psychophysiology 26:270–280[Medline]
  21. Villecco AS, de Aloysio D, Radi D, Sprovieri G, Bargossi AM, Grossi CG, Salgarello M, Cavrini G 1997 Plasma catecholamines in pre- and in postmenopausal women with mild to moderate essential hypertension. J Hum Hypertens 11:157–162[CrossRef][Medline]
  22. Goldstein DS 1983 Plasma catecholamines and essential hypertension: an analytical review. Hypertension 5:86–99[Abstract/Free Full Text]
  23. Cohn JN, Johnson GR, Shabetai R, Loeb H, Tristani F, Rector T, Smith R, Fletcher R 1993 Ejection fraction, peak exercise oxygen consumption, cardiothoracic ratio, ventricular arrhythmias, and plasma norepinephrine as determinants of prognosis in heart failure. The V-HeFT VA Cooperative Studies Group. Circulation 87(Suppl 6):VI5–VI6
  24. Kaplan JR, Pettersson K, Manuck SB, Olsson G 1991 Role of sympathoadrenal medullary activation in the initiation and progression of atherosclerosis. Circulation 84(Suppl 6):VI23–VI32
  25. Adamopoulos S, Piepoli M, McCance A, Bernardi L, Rocadaelli A, Ormerod O, Forfar C, Sleight P, Coats AJ 1992 Comparison of different methods for assessing sympathovagal balance in chronic congestive heart failure secondary to coronary artery disease. Am J Cardiol 70:1576–1582[CrossRef][Medline]
  26. Hart MN, Heistad DD, Brody MJ 1980 Effect of chronic hypertension and sympathetic denervation on wall/lumen ratio of cerebral vessels. Hypertension 2:419–423[Abstract/Free Full Text]
  27. Pauletto P, Scannapieco G, Pessina AC 1991 Sympathetic drive and vascular damage in hypertension and atherosclerosis. Hypertension 17(Suppl-4):III75–III81
  28. Dinenno FA, Jones PP, Seals DR, Tanaka H 2000 Age-associated arterial wall thickening is related to elevations in sympathetic activity in healthy humans. Am J Physiol 278:H1205–H1210
  29. Bauch HJ, Grunwald J, Vischer P, Gerlach U, Hauss WH 1987 A possible role of catecholamines in atherogenesis and subsequent complications of atherosclerosis. Exp Pathol 31:193–204[Medline]
  30. Nakaki T, Nakayama M, Yamamoto S, Kato R 1990 {alpha}1-Adrenergic stimulation and ß2-adrenergic inhibition of DNA synthesis in vascular smooth muscle cells. Mol Pharmacol 37:30–36[Abstract]
  31. Dubey RK, Jackson EK, Rupprecht HD, Sterzel RB 1997 Factors controlling growth and matrix production in vascular smooth muscle and glomerular mesangial cells. Curr Opin Nephrol Hypertens 6:88–105[CrossRef][Medline]
  32. Lee AJ, Kosh JW, Conney AH, Zhu BT 2001 Characterization of the NADPH-dependent metabolism of 17ß-estradiol to multiple metabolites by human liver microsomes and selectively expressed human cytochrome P450 3A4 and 3A5. J Pharmacol Exp Ther 298:420–432[Abstract/Free Full Text]
  33. Izzo JL 1999 The sympathetic nervous system in human hypertension. In: Izzo JL, Black HR, eds. Hypertension primer, 2nd ed. Lippincott Williams & Wilkins; 109–112
  34. Manger WM 1999 Pathophysiology of pheochromocytoma. In: Izzo JL, Black HR, eds. Hypertension primer, 2nd ed. Lippincott Williams & Wilkins; 141–142
  35. Ludwig J, Gerlich M, Halbrugge T, Graefe KH 1990 The synaptic noradrenaline concentration in humans as estimated from simultaneous measurements of plasma noradrenaline and dihydroxyphenylglycol (DOPEG). J Neural Transm Suppl 32:441–445[Medline]
  36. Zacharia LC, Dubey RK, Mi Z, Jackson EK 2003 Methylation of 2-hydroxyestradiol in isolated organs. Hypertension 42:82–87[Abstract/Free Full Text]
  37. Ozawa Y, Yoshizumi M, Inui D, Tsuchiya K, Houchi H, Tamaki T, Minakuchi K 1999 Plasma levels of free and sulfoconjugated catecholamines in patients with atherosclerosis. Biol Pharm Bull 22:657–659[Medline]
  38. Ablad B, Bjorkman JA, Gustafsson D, Hansson G, Ostlund-Lindqvist AM, Pettersson K 1988 The role of sympathetic activity in atherogenesis: effects of ß blockade. Am Heart J 116:322–327[CrossRef][Medline]
  39. Esler M, Kaye D 1998 Increased sympathetic nervous system activity and its therapeutic reduction in arterial hypertension, portal hypertension and heart failure. J Auton Nerv Syst 72:210–219[CrossRef][Medline]
  40. Sudhir K, Elser MD, Jennings GL, Komesaroff PA 1997 Estrogen supplementation decreases norepinephrine-induced vasoconstriction and total body norepinephrine spillover in perimenopausal women. Hypertension 30:1538–1543[Abstract/Free Full Text]
  41. Komesaroff PA, Esler MD, Sudhir K 1999 Estrogen supplementation attenuates glucocorticoid and catecholamine responses to mental stress in perimenopausal women. J Clin Endocrinol Metab 84:606–610[Abstract/Free Full Text]
  42. Ceresini G, Freddi M, Morganti S, Rebecchi I, Modena AB, Rinaldi M, Manca C, Amaducci A, Del Rio G, Valenti G 2000 The effects of transdermal estradiol on the response to mental stress in postmenopausal women: arandomized trial. Am J Med 109:463–468[CrossRef][Medline]
  43. Menozzi R, Cagnacci A, Zanni AL, Bondi M, Volpe A, Del Rio G 2000 Sympathoadrenal response of postmenopausal women prior and durino prolonged administration of estradiol. Maturitas 34:275–281[CrossRef][Medline]
  44. Fingerle J, Sanders KH, Fotev Z 1991 {alpha}1-Receptor antagonists urapidil and prazosin inhibit neointima formation in rat carotid artery induced by balloon catheter injury. Basic Res Cardiol 86(Suppl 1):75–81
  45. Zacharia LC, Jackson EK, Gillespie DG, Dubey RK 2001 Catecholamines abrogate the anti-mitogenic effects of 2-hydroxyestradiol on human aortic vascular smooth muscle cells. Arterioscler Thromb Vasc Biol 21:1745–1750[Abstract/Free Full Text]
  46. Dubey RK, Zacharia LC, Gillespie DG, Imthurn B, Jackson EK 2003 Catecholamines block the antimitogenic effect of estradiol on human glomerular mesangial cells. Hypertension 42:349–355[Abstract/Free Full Text]
  47. Lloyd T, Weisz J 1978 Direct inhibition of tyrosine hydroxylase activity by catechol estrogens. J Biol Chem 253:4841–4843[Abstract/Free Full Text]




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