(Hypertension. 1998;31:522.)
© 1998 American Heart Association, Inc.
Scientific Contributions |
17ß-Estradiol, Its Metabolites, and Progesterone Inhibit Cardiac Fibroblast Growth
From Center for Clinical Pharmacology, Departments of Medicine (R.K.D., E.K.J.) and Pharmacology (E.K.J.), University of Pittsburgh Medical Center, Pittsburgh, PA, USA, and Clinic for Endocrinology, Department of Obstetrics and Gynecology, University Hospital Zurich, Zurich, Switzerland (R.K.D., P.J.K.).
Correspondence to Dr Raghvendra K. Dubey, Center Pharmacology. Department of Medicine, 623 Scaife Hall, 200 Lothrop Street, University of Pittsburgh Medical Center, Pittsburgh, PA 15213-2582, USA. E-mail dubey{at}med1.dept-med.pitt.edu
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Abstract |
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Postmenopausal women (PMW) have increased incidence of cardiovascular disease, and estrogen substitution therapy has been shown to have cardioprotective effects. Since abnormal growth of cardiac fibroblasts (CFs) is associated with hypertension and myocardial infarction and estrogen inhibits vascular smooth muscle cell (SMC) growth, it is feasible that estrogen may attenuate cardiac remodeling by inhibiting CF growth, and this possibility was investigated by using cultured CFs. 17ß-Estradiol and progesterone, but not 17
-estradiol, estrone, or estriol, inhibited 2.5% FCS-induced proliferation (DNA synthesis and cell number) and collagen synthesis (3H-proline incorporation) in a concentration-dependent manner and to a similar extent in male and female CFs. Compared to 17ß-estradiol, its metabolites 2-hydroxyestradiol and 2-methoxyestradiol were more potent in inhibiting FCS-induced DNA synthesis, collagen synthesis, and cell proliferation. The inhibitory effects of 17ß-estradiol and its metabolites were enhanced in presence of progesterone and 4-hydroxytamoxifen (high-affinity estrogen receptor ligand). Moreover, like estrogens, the dietary phytoestrogens biochanin A and daidzein inhibited FCS-induced growth of CFs. In conclusion, 17ß-estradiol, its metabolites, and progesterone inhibit CF growth in a gender independent fashion. Moreover, hormone replacement therapy using 17ß-estradiol and progesterone may protect PMW against cardiovascular disease by inhibiting CF growth and cardiac remodeling; whereas estrogens that do not inhibit CF growth may be less effective in protecting PMW against cardiovascular disease. Finally, our studies provide evidence that phytoestrogens inhibit CF growth and may be clinically useful as a substitute for feminizing estrogens in preventing cardiovascular disease in both women and men.
Key Words: estrogen(s) • phytoestrogens • 17ß-estradiol • cardiac fibroblast • postmenopausal women • hormone replacement therapy • cardiovascular disease • proliferation • hypertrophy
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Introduction |
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Several lines of evidence suggest that estrogens are cardioprotective. First, women in the reproductive age group are protected against cardiovascular disease in comparison with men.1 Second, onset of menopause in women is accompanied with ovarian dysfunction, decrease in circulating estrogen levels, and increased incidence of cardiovascular disease.1 Third, estrogen replacement therapy markedly reduces the risk of cardiovascular disease in postmenopausal women.1,2
Although the cardioprotective effects of estrogen in post-menopausal women (PMW) are well established, the mechanisms by which estrogen induces its cardioprotective effects are not fully understood. Since coronary artery disease is the most frequent cause of death among women,2 most research has focused on evaluating the effects of estrogen on vascular cells. In this regard, several important findings are the following: (1) Functional receptors for estrogen exist on both vascular endothelial and smooth muscle cells (SMCs);3 (2) estrogen has direct effects on vascular cells;3 (3) estrogen improves endothelial function and prevents endothelial damage;4 and (4) estrogen inhibits mitogen-induced proliferation of SMCs, migration of SMCs from the media to the intima, and deposition of extracellular matrix proteins (ECM) such as collagen.3,5 Since endothelial damage/dysfunction and increased SMC proliferation, migration, and ECM synthesis contributes to the vasoocclusive disorders associated with coronary artery disease,6 estrogen may induce its protective effects by inhibiting the vascular remodeling process. Indeed, numerous in vivo studies in various female animal models have now shown that neointima formation in atherosclerosis and after balloon catheterinduced injury is increased in the absence of estrogen and inhibited in the presence of estrogen.3,7,8
Like SMCs within the vasculature, abnormal growth of cardiac fibroblasts (CFs) also are importantly involved in the pathophysiology of cardiovascular diseases including cardiac remodeling induced by hypertension, myocardial infarction, and myocardial reperfusion injury following ischemia.9 CFs that comprise 60% of the total heart cells contribute to pathological structural changes in the heart by undergoing proliferation, depositing ECM proteins, and replacing myocytes with fibrotic scar tissue.9 Thus, CF-induced cardiac remodeling may participate in diastolic and systolic dysfunction leading to congestive heart failure.
A number of autocrine and paracrine factors can stimulate as well as inhibit CF growth. In a normal heart, quiescence is maintained by a balance between circulating and cardiac-derived growth inhibitors and growth promoters that may interact and govern CF growth.9 Disruption of the balanced generation of growth promoters and growth inhibitors under pathological conditions could trigger a cascade of events leading to increased proliferation of CFs, enhanced deposition of ECM by CFs, and enlargement and stiffening of the heart. Therefore, endogenous factors that are generated in substantial amounts and that inhibit CF growth may play a major cardioprotective role. Because CFs, like SMCs, have functional estrogen receptors10 and estrogens inhibit SMC growth,3,5,7,8 we hypothesize that estrogens may also prevent cardiac remodeling by directly inhibiting the growth of CFs and maintaining homeostasis.
Accordingly, the aims of the present study were to determine whether (1) estrogen inhibits growth of CFs; (2) the inhibitory effects of estrogen are receptor mediated; (3) progesterone, which is used in combination with estrogen for hormone replacement therapy, modulates the effects of estrogen on CF growth; (4) various estrogens that are used clinically have similar effects on CF growth; (5) the major metabolites of 17ß-estradiol influence CF growth; (6) the effects of estrogen on CF growth are gender dependent; and (7) like estrogens, dietary phytoestrogens influence CF growth.
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Materials
Dulbecco’s modified Eagle’s medium (DMEM), DMEM/F12 medium, Hanks’ balanced salt solution, penicillin, streptomycin, 0.25% trypsin-EDTA solution, collagenase, and all tissue culture ware were purchased from GIBCO Laboratories. Fetal calf serum (FCS) was obtained from HyClone Laboratories, Inc. 17ß-estradiol, estrone, estriol, 17
-estradiol, 2-hydroxyestradiol, 2 methoxyestradiol, progesterone, testosterone, biochanin A, and daidzein were purchased from Sigma Chemical Co. 4-Hydroxytamoxifen was purchased from Research Biochemicals International. 3H-thymidine (specific activity 11.8 Ci/mmol) was purchased from ICN Biomedicals. L-[3H]-Proline (specific activity 23Ci/mmol) was purchased from NEN. All other chemicals used were of tissue culture or best grade available.
Cardiac Fibroblast Cell Culture
Age-matched male (n=12) and female (n=10) Sprague-Dawley rats weighing 150 to 200 g were obtained from Charles River and were fed standard rat chow and tap water ad libitum. CFs were cultured from the left ventricles by our previously described method, using the enzymatic digestion and selective plating technique.9 The cells were cultured in steroid-free FCS, and since phenol red has weak estrogenic effects, phenol red-free medium was used in all the studies. Purity of the CFs was assessed by morphologic characterization and by positive and negative immunostaining with antibodies against von Willebrand factor VIII, sarcomeric actin (striated muscle, monoclonal), desmin, and vimentin and as we described earlier.9 Our findings suggest that the purity of cultured CFs was greater than 98%. CFs in second and third passages were used in all studies.
DNA Synthesis
3H-thymidine incorporation studies were done to investigate the effects of agents on FCS-induced DNA synthesis. Ventricular CFs from female or male rats were plated at a density of 1x104 cells/well in 24-well tissue culture dishes and allowed to grow to subconfluence in DMEM/F12 containing 10% FCS under standard tissue culture conditions. The cells were then growth arrested by feeding DMEM containing 0.4% bovine serum albumin (BSA; Sigma) for 48 hours. Growth was intiated by treating growth-arrested cells for 20 hours with DMEM supplemented with 2.5% FCS and containing or lacking the following: 17ß-estradiol, estrone, estriol, 17-estradiol, 2-hydroxyestradiol, 2-methoxyestradiol, progesterone, biochanin A, daidzein, 4-hydroxytamoxifen, 17ß-estradiol plus progesterone or 4-hydroxytamoxifen, 2-hyroxyestradiol plus progesterone or 4-hydroxytamoxifen, and 2-methoxyestradiol plus progesterone or 4-hydroxytamoxifen. After 20 hours of incubation, the treatments were repeated with freshly prepared solutions but supplemented with 3H-thymidine (1 µCi/mL) for an additional 4 hours. The experiments were terminated by washing the cells twice with Dulbecco’s phosphate-buffered saline (PBS) and twice with ice-cold trichloroacetic acid (10%). The precipitate was solubilized in 500 µL of 0.3N NaOH and 0.1% SDS after incubation at 50°C for 2 hours. Aliquots from four wells for each treatment with 10 mL of scintillation fluid were counted in a liquid scintillation counter.
Cell Proliferation (Cell Number)
Trypsinized CFs in the third passage were suspended in complete culture media containing 10% FCS and plated in a 24-well culture dish at a density of 1x104 cells/well. After incubation for 18 hours, the cells were fed complete culture media containing 0.25% FCS for 48 hours to growth arrest the cells. To study the effects of hormones on FCS-induced cytokinesis, we treated growth-arrested CFs every 24 hours for 4 days with complete culture media containing 2.5% FCS and supplemented with or without 17ß-estradiol, estrone, estriol, 17-estradiol, 2-hydroxyestradiol, 2-methoxyestradiol, and progesterone. The treatments were terminated on day 5, and cells were dislodged with trypsin-EDTA, diluted in isoton-II, and counted with a hemocytometer-calibrated Coulter counter. Aliquots from four wells were counted for each group, and four to six independent experiments were performed for each treatment.
Collagen Synthesis
3H-proline incorporation studies were done to investigate the effects of hormones on FCS-induced collagen synthesis. Ventricular CFs cultured from male or female rats were plated in 24-well tissue culture dishes and allowed to grow to confluency in DMEM/F12 containing 10% FCS under standard tissue culture conditions. The cells were made quiescent by feeding DMEM containing 0.4% BSA for 48 hours. Collagen synthesis was initiated by treating growth arrested CFs for 48 hours with culture medium supplemented with 2.5% FCS and 3H-L-proline (1 µCi/mL) and containing or lacking the various treatments as described above (see "DNA Synthesis"). The experiments were terminated by washing the cells twice with PBS and twice with ice-cold trichloroacetic acid (10%). The precipitate was solubilized in 500 µL of 0.3N NaOH and 0.1% SDS. Aliquots from four wells for each treatment with 10 mL of scintillation fluid were counted in a liquid scintillation counter. Each experiment was repeated three to four times. 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 that were treated in parallel to the cells used for the collagen synthesis studies, and the data were normalized to cell number.
Statistics
All growth experiments were performed in triplicate or quadruplicate with four to six separate cultures. Data for the DNA synthesis, cell number, and collagen synthesis are presented as mean±SEM. Statitical analysis was performed by using ANOVA, paired or unpaired Student’s t test, or Fisher’s least significant difference test as appropriate. A value of P<.05 was considered statistically significant.
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Results |
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Effects of 17ß-Estradiol, Its Metabolites, and Progesterone on FCS-Induced Growth of Male and Female Rat Ventricular Cardiac Fibroblasts
Treatment with 2.5% FCS stimulated DNA synthesis by eightfold to elevenfold (P<.001 versus 0.4% BSA) and collagen synthesis by sevenfold to ninefold (P<.05 versus 0.4% BSA). In experiments conducted in parallel with male and female CFs, 2.5% FCS induced DNA synthesis and collagen synthesis to a similar extent (P>.05, male versus female). In male and female CFs, both 17ß-estradiol and progesterone inhibited FCS-induced 3[H]thymidine and 3H-proline incorporation to a similar extent and in a concentration-dependent manner (Fig 1A and 1B). The lowest concentrations of 17ß-estradiol and progesterone that significantly inhibited FCS-induced DNA and collagen synthesis in male and female CFs was 1 nmol/L for estradiol and 10 nmol/L for progesterone (Fig 1). A 50% decrease in FCS-induced DNA and collagen synthesis in male and female CFs was observed at approximately 1 µmol/L of 17ß-estradiol and progesterone (Fig 1A and 1B).
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P<.05 versus 17ß-estradiol, 2-hydroxyestradiol, or 2-methoxyestradiol.17ß-Estradiol is metabolized to 2-hydroxyestradiol and 2-methoxyestradiol,11 and these metabolites have been shown to be more potent then 17ß-estradiol in inhibiting SMC growth.11 However, the effects and relative potencies of these metabolites on CF growth are not known and were investigated. Compared to 17ß-estradiol, its metabolites 2-hydroxyestradiol and 2-methoxyestradiol were more potent in inhibiting FCS-induced DNA synthesis (Fig 2A) and collagen synthesis (Fig 2B) in both male CFs and female CFs and in the following order of potency: 2-methoxyestradiol > 2-hydroxyestradiol > 17ß-estradiol. A 50% decrease in DNA synthesis in female CFs by 17ß-estradiol, 2-hydroxyestradiol, and 2-methoxyestradiol was observed at 2 µmol/L, 0.3 µmol/L, and 0.03 µmol/L, respectively.
FCS induced proliferation (cell number) of growth-arrested CFs by eightfold to tenfold (data not shown). 17ß-Estradiol and progesterone inhibited FCS-induced increases in cell number in a concentration-dependent manner (Fig 3A). Low concentrations (0.001 µmol/L) of both progesterone and 17ß-estradiol significantly inhibited FCS-induced increases in cell number by 26±2% and 16±3%, respectively. Trypan blue exclusion tests indicated no loss in viability of cells treated with 0.001 to 10 µmol/L of 17ß-estradiol. In cells treated with progesterone, cell toxicity was observed at the maximal concentration of progesterone used (10 µmol/L); however, at lower concentrations (0.001 to 1 µmol/L), no loss in cell viability was evident (data not shown).
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Like 17ß-estradiol, both 2-methoxyestradiol and 2-hydroxyestradiol also inhibited FCS-induced proliferation of female CFs in a concentration-dependent manner (Fig 3B). The lowest concentrations (0.001 µmol/L) of 2-methoxyestradiol and 2-hydroxyestradiol inhibited FCS-induced increases in cell number by 59±4% and 32±3%, respectively. Compared to DNA synthesis, the inhibitory effects of 2-methoxyestradiol and 2-hydroxyestradiol on cell proliferation were much greater (compare Fig 1 to Fig 3). Trypan blue exclusion tests indicated no loss in viability of cells treated with 0.001 to 10 µmol/L of 2-methoxyestradiol or 2-hydroxyestradiol (data not shown).
Effects of Various Estrogens (Estrone, Estriol, 17-Estradiol, and 17ß-Estradiol) and Phytoestrogens (Biochanin A and Daidzein) on FCS-Induced Growth of Female Rat Cardiac Fibroblasts
A wide spectrum of estrogens are used clinically for hormone replacement therapy in PMW.1,2 We investigated whether various clinically used estrogens are equipotent in inhibiting FCS-induced growth of female CFs. Compared to 17ß-estradiol, 17-estradiol (inactive isomer of 17ß-estradiol), estrone, and estriol did not inhibit FCS-induced increases in DNA and collagen synthesis (Fig 4A). 17ß-Estradiol inhibited DNA synthesis by 50% and collagen synthesis by 37% at a concentration of 
1 µmol/L. At this concentration, estrone, estriol, and 17-estradiol inhibited thymidine incorporation by approximately 3%, 6%, and 8%, respectively (P>.05 versus 2.5% FCS) and proline incorporation by 1% to 3% (P>.05 versus 2.5%FCS).
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Phytoestrogens have been shown to induce cardioprotective effects.12 Whether phytoestrogens inhibit growth of CFs is not known and was investigated. Biochanin A and daidzein inhibited FCS-induced DNA and collagen synthesis in female CFs in a concentration-dependent manner (Fig 4B). The lowest concentrations of biochanin A and daidzein that significantly inhibited FCS-induced DNA synthesis were 0.1 µmol/L and 1 µmol/L, respectively. Moreover, as in female CFs, both biochanin A and daidzein also inhibited FCS-induced DNA and collagen synthesis in CFs cultured from male rats (data not shown). Trypan blue exclusion tests indicated no loss in viability of cells treated with 0.001 to 10 µmol/L of biochanin A and daidzein (data not shown).
Modulatory Effects of Progesterone and 4-Hydroxytamoxifen on 17ß-Estradiol, 2-Hydroxyestradiol, and 2-Methoxyestradiol-Induced Inhibition of Cardiac Fibroblast Growth
The inhibitory effects of 17ß-estradiol (0.1 µmol/L), 2-methoxyestradiol (0.001 µmol/L), and 2-hydroxyestradiol (0.01 µmol/L) on DNA synthesis were significantly enhanced in presence of progesterone (1 µmol/L; Fig 5A, top panel). Moreover, progesterone also increased the inhibitory effects of 17ß-estradiol and its metabolites on collagen synthesis (Fig 5A, bottom panel). Like the modulatory effects of progesterone, the inhibitory effects of 17ß-estradiol (0.1 µmol/L), 2-methoxyestradiol (.001 µmol/L), and 2-hydroxyestradiol (.01 µmol/L) or DNA synthesis were not reversed but rather were enhanced in the presence of 4-hydroxytamoxifen (1 µmol/L; Fig 5B, top panel). Moreover, 4-hydroxytamoxifen did not block the inhibitory effects of 17ß-estradiol, 2-methoxyestradiol, and 2-hydroxyestradiol on collagen synthesis but rather enhanced them (Fig 5B, bottom panel).
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Discussion |
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Cardiac remodeling associated with hypertension, myocardial infarction, and reperfusion injury following ischemia9 is a complex process that involves accumulation of ECM proteins and proliferation of CFs. In a normal heart, CF quiescence and homeostasis are maintained by the simultaneous and balanced release of growth-promoting and growth-inhibiting factors.9 Because menopause is accompanied by ovarian insufficiency/ dysfunction and a decrease in estrogen levels,1,2 it is feasible that decreased estrogen levels tilt the balance toward CF growth, resulting in increased growth of CFs and contributing to the increased risk of cardiac remodeling, hypertrophy, and dysfunction in PMW. Hence, detailed knowledge of the role of ovarian hormones that regulate CF growth and maintain CF homeostasis is of considerable clinical and therapeutic importance. In this regard, even though functional estrogen receptors have been identified in CFs,10 the role of estrogen(s) in regulating CF growth has not been well investigated.
Structural changes in the heart probably involve multiple autocrine/paracrine/endocrine factors.9 FCS contains a battery of growth factors including platelet-derived growth factor, epidermal growth factor, FGF, AngII, endothelin, and norepinephrine, which may contribute to the remodeling process.9 Therefore, we thought it important to evaluate the effects of 17ß-estradiol on FCS-induced growth of CFs so as to elucidate the growth-regulatory effects of 17ß-estradiol under more physiological conditions. The finding that 17ß-estradiol, its metabolites, and progesterone inhibit FCS-induced CF growth provides the first evidence that these ovarian hormones are important modulators of CF growth.
Physiological concentrations (1 nmol/L) of 17ß-estradiol inhibit cell proliferation by 25%. Moreover, 1 nmol/L of 2-hydroxyestradiol and 2-methoxyestradiol, the major metabolites of 17ß-estradiol,11 inhibit cell proliferation by 32% and 59%, respectively. This suggests that physiological concentrations of 17ß-estradiol inhibit CF growth and that the inhibitory effects of 17ß-estradiol in vivo may be considerably higher, owing to the presence of 17ß-estradiol metabolites. In addition, our findings suggest that the cardioprotective effects of 17ß-estradiol may vary and be dependent on the metabolic capability of the individual. Notably, estrogen replacement therapy is not beneficial in all PMW.1,2 We have recently shown that estrogen replacement therapy in PMW differentially increases nitric oxide synthesis,13 and similar effects have been shown for the changes in LDL levels.1,13 Moreover, recent studies have shown that 17ß-estradiol must be metabolized to prevent LDL oxidation.14 On the basis of these findings, it is possible that the decreased cardioprotective effects of estrogen that are observed in some PMW may be due to the lack of metabolism of 17ß-estradiol to 2-methoxyestradiol and 2-hydroxyestradiol. Indeed, differences in metabolism of 17ß-estradiol to 2-hydroxyestradiol have recently been shown to be associated with the carcinogenic effects of 17ß-estradiol in women.15 However, detailed clinical studies will be needed to address this possibility.
In agreement with our studies, 17ß-estradiol, 2-hydroxyestradiol, and 2-methoxyestradiol have been shown to inhibit proliferation of SMCs cultured from rabbit aortas.11 However, in contrast to our findings, an earlier study showed that 17ß-estradiol and estrone, but not 2-methoxyestradiol, induced DNA synthesis in CFs cultured from neonatal rats in the absence of a growth stimulus.10 The reasons for these disparate results are unclear; however, the effects of estrogens on neonatal CFs may be different from the effects on adult CFs, and the effects of estrogens on DNA synthesis may depend on whether a growth stimulus is present. Additional studies are required to resolve the discrepancy between these two studies.
Our finding that the inhibitory effects of 17ß-estradiol and its metabolites on FCS-induced growth of male and female CFs are similar suggests that the direct effects of these hormones on CF growth in vitro are not sexually dimorphic. Several studies provide evidence that the dimorphic effects of 17ß-estradiol are not due to the intrinsic property of the vascular SMCs, but rather to androgenic factors produced by gonads in males.3,7,16 The most compelling evidence supporting this conclusion comes from the recent studies demonstrating that exogenous 17ß-estradiol prevents balloon injury–induced neointima formation in gonadectomized rats but not in intact male rats.3,7,16 It is feasible that like SMCs, male hormones may regulate the effects of 17ß-estradiol on CFs in vivo; however, this possibility needs to be investigated.
In the present study, the inhibitory effects of 17ß-estradiol and its metabolites were enhanced, rather than inhibited, by progesterone. To reduce the risk of endometrial cancer, combined administration of a progestin with an estrogen is currently the preferred method of hormone replacement therapy in nonhysterectomized PMW.1,2 Our finding suggests that treatment with 17ß-estradiol plus progesterone may be more protective against cardiovascular disease in PMW. Co-administration of progestin(s) with estrogen has been shown to both increase and decrease the cardioprotective effects of estrogen.1,16 Progesterone has been shown to enhance the protective effects of 17ß-estradiol on neointima formation.8 In contrast to progesterone, synthetic progestins such as medroxyprogesterone acetate (MPA), cyproterone acetate (CPA), and norethisterone acetate (NETA), which are used clinically, have been shown to reduce the various cardioprotective effects of 17ß-estradiol.13,16 Moreover, MPA has been shown to abrogate the inhibitory effects of 17ß-estradiol on neointima formation.16 Progesterone is the naturally occurring progestin without any androgenic effects.16 In contrast, NETA is a testosterone-derived progestin with both gestagenic and androgenic properties, and MPA and CPA are derivatives of 17-hydroxy-progesterone.13,17 Hence, it is feasible that progestins that are used clinically have differential effects that are governed by their chemical properties. Therefore, it is important to evaluate the effects of various clinically used progestin(s) on 17ß-estradiol-induced cardioprotective effects.
The inhibitory effects of 17ß-estradiol and its metabolites on DNA synthesis were enhanced in the presence 4-hydroxytamoxifen, a high-affinity estrogen receptor antagonist.18 This suggests that the effects of 17ß-estradiol are not estrogen receptor-mediated. In this regard, it has recently been dem- onstrated that 17ß-estradiol inhibits balloon injury-induced neointima formation to an equal extent in wild-type and estrogen receptor-deficient mice.19 Indeed, estrogen receptor antagonists such as tamoxifen and 4-hydroxytamoxifen are also known potent cardioprotective agents.18 Together, these findings demonstrate that 17ß-estradiol inhibits proliferation of vascular SMCs and CFs by a novel mechanism that is estrogen receptor independent.
The observation that 17ß-estradiol, but not 17-estradiol, estrone, and estriol, is effective in inhibiting CF growth suggests that the inhibitory effects of estrogens may vary considerably. Moreover, it also raises the question of whether all estrogens that are used clinically have cardioprotective effects. Even though various estrogens such as 17ß-estradiol, estradiol valerate, estrone (used in conjugated equine estrogen), and estriol are used clinically for hormone replacement therapy, most in vitro and in vivo studies have used 17ß-estradiol to investigate the possible mechanisms by which estrogen induces cardioprotective effects. However, the effects of 17ß-estradiol may not reflect the cardioprotective effects of all other clinically used estrogens. This is evident from our finding that estrone and estriol are unable to inhibit FCS-induced growth of CFs. We have also observed similar differential effects of these estrogens on rat vascular SMCs (unpublished observations). Taken together, these findings raise the concern that if various estrogens do indeed have differential effects on the cardiovascular system, then the choice of estrogen in hormone replacement therapy will have to be reevaluated.
Our finding that the inhibitory effects of 17ß-estradiol are similar in CFs from both males and females suggests that the inhibitory effects are not gender dependent. Although 17ß-estradiol may be protective in males, its use may have feminizing effects. This has led to the search for nonfeminizing estrogen substitutes that may protect both men and women against cardiovascular disease. Since, compared to 17ß-estradiol, estrone and estriol have significantly lower affinity for estrogen receptors and are estrogens with weak feminizing effects, their use in men to protect against cardiovascular disease has been proposed. However, our finding that estrone and estriol are unable to inhibit CF and SMC (unpublished observation) growth suggests that they may not induce cardioprotective effects. Indeed, in premenopausal women with functional ovaries, the levels of 17ß-estradiol are higher than those of estrone;20 however, in postmenopausal women, the levels of 17ß-estradiol are significantly lower than those of estrone.20 Since estrone is synthesized in peripheral fat tissue, its levels are not dramatically reduced in menopause,20 and men also have substantial levels of estrone.20 Our findings suggest that the cardiovascular complications in PMW may be due largely to the reduction in 17ß-estradiol, and the use of estrone or estriol may not induce cardioprotection.
Our finding that the natural phytoestrogens biochanin A and daidzein inhibited FCS-induced growth of male and female CFs suggests that inhibitory effects of phytoestrogens are not sexually dimorphic. More important, these data suggest that dietary phytoestrogens may be cardioprotective. In this regard, earlier studies have shown that phytoestrogens improve cardiovascular risk factors and prevent neointima formation without affecting the reproductive system.12 Since, unlike 17ß-estradiol, phytoestrogens do not have feminizing effects,12 phytoestrogens may provide a safe estrogen substitute to protect against cardiovascular disease in both women and men.
Estrogens prevent cardiovascular disease in PMW via multiple mechanisms. Endothelium-dependent mechanisms by which estrogen may induce its beneficial effects involve protecting the vascular endothelium by reducing apoptosis and inducing endothelial cell recovery and growth,4 improving endothelial-mediated degradation of LDL cholesterol,3 suppressing collagen and elastin synthesis,3 and restoring endothelium-dependent vasodilator mechanisms after injury.3,4 17ß-Estradiol also favorably influences the lipid profile by reducing LDL levels and increasing HDL levels and preventing the oxidation of LDL to oxidized LDL, a potent mediator of vascular cell damage.1,3 Estrogens also induce the release of both vasodilatory and growth-inhibitory molecules such as nitric oxide13 and prostaglandins3 from vascular endothelial cells. Furthermore, estrogens reduce the adhesion of activated monocytes to the endothelium by inhibiting the expression of adhesion molecules, such as E-selectin, VCAM-1, and ICAM-1.21 Estrogens also inhibit proliferation, migration, and extracellular matrix synthesis in vascular SMCs and thereby reduce neointima formation.3,5,7 Together, these findings provide convincing evidence that estrogens protect against vasoocclusive disorders in women by interacting with vascular cells. Our finding that 17ß-estradiol inhibits CF growth provides evidence of yet another mechanism by which estrogen may prevent cardiovascular disease in PMW.
In conclusion, we provide evidence that 17ß-estradiol, its metabolites, and progesterone inhibit FCS-induced growth of CFs. Moreover, our findings suggest that combined treatment of 17ß-estradiol with progesterone may be more protective than 17ß-estradiol alone in preventing cardiovascular disease in PMW. Also, our study provides evidence that the protective effects of clinically used estrogens may vary considerably and estrogens that do not inhibit cell proliferation may be less effective in protecting PMW against cardiovascular disease. Moreover, our studies provide evidence that phytoestrogens inhibit CF growth and may be clinically useful as a substitute for feminizing estrogens in protecting against cardiovascular disease in both women and men.
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Acknowledgments |
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This work was supported by grants from National Institutes of Health (HL-55314, HL-35909).
Received September 17, 1997; first decision October 6, 1997; accepted October 22, 1997.
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