17-Epiestriol, an Estrogen Metabolite, Is More Potent Than Estradiol in Inhibiting Vascular Cell Adhesion Molecule 1 (VCAM-1) mRNA Expression

17-ॆ estradiol (17-ॆ E2) attenuates the expression of vascular cell adhesion molecule 1 (VCAM-1)in vivo at physiological levels (pg/ml), whereas supraphysiological concentrations of 17-ॆ E2 (ng/ml) are required in vitro. We assessed whether a metabolite of estrogen, which could only be generated in vivo, might be a more potent inhibitor of VCAM-1 expression and thereby explain this discrepancy. We report here that 17-epiestriol, an estrogen metabolite and a selective estrogen receptor (ER) ॆ agonist, is ∼400× more potent than 17-ॆ E2 in suppressing tumor necrosis factor (TNF) α-induced VCAM-1 mRNA as well as protein expression in human umbilical vein endothelial cells. Genistein, an ERॆ agonist, at low concentrations (1 and 10 nm) also suppressed TNFα-induced VCAM-1 mRNA expression. These actions of 17-epiestriol and genistein were significantly attenuated in the presence of the estrogen receptor antagonist ICI-182780. Other estrogenic compounds such as ethinyl estradiol and estrone did not have any effect on TNFα-induced VCAM-1 expression at the concentrations tested. We further show that, 1) 17-epiestriol induces the expression of endothelial nitric-oxide synthase mRNA and protein, 2) 17-epiestriol prevents TNFα-induced migration of NFκB into the nucleus, 3) NG-nitro-l-arginine methyl ester, an inhibitor of NO synthesis, abolishes 17-epiestriol-mediated inhibition of TNFα-induced VCAM-1 expression and migration of NFκB from the cytoplasm to the nucleus. Our results indicate that 17-epiestriol is more potent than 17-ॆ E2 in suppressing TNFα-induced VCAM-1 expression and that this action is modulated at least in part through NO. 17-ॆ estradiol (17-ॆ E2) attenuates the expression of vascular cell adhesion molecule 1 (VCAM-1)in vivo at physiological levels (pg/ml), whereas supraphysiological concentrations of 17-ॆ E2 (ng/ml) are required in vitro. We assessed whether a metabolite of estrogen, which could only be generated in vivo, might be a more potent inhibitor of VCAM-1 expression and thereby explain this discrepancy. We report here that 17-epiestriol, an estrogen metabolite and a selective estrogen receptor (ER) ॆ agonist, is ∼400× more potent than 17-ॆ E2 in suppressing tumor necrosis factor (TNF) α-induced VCAM-1 mRNA as well as protein expression in human umbilical vein endothelial cells. Genistein, an ERॆ agonist, at low concentrations (1 and 10 nm) also suppressed TNFα-induced VCAM-1 mRNA expression. These actions of 17-epiestriol and genistein were significantly attenuated in the presence of the estrogen receptor antagonist ICI-182780. Other estrogenic compounds such as ethinyl estradiol and estrone did not have any effect on TNFα-induced VCAM-1 expression at the concentrations tested. We further show that, 1) 17-epiestriol induces the expression of endothelial nitric-oxide synthase mRNA and protein, 2) 17-epiestriol prevents TNFα-induced migration of NFκB into the nucleus, 3) NG-nitro-l-arginine methyl ester, an inhibitor of NO synthesis, abolishes 17-epiestriol-mediated inhibition of TNFα-induced VCAM-1 expression and migration of NFκB from the cytoplasm to the nucleus. Our results indicate that 17-epiestriol is more potent than 17-ॆ E2 in suppressing TNFα-induced VCAM-1 expression and that this action is modulated at least in part through NO. vascular cell adhesion molecule estrogen receptors ethinyl estradiol NG-nitro-l-arginine methyl ester tumor necrosis factor fetal bovine serum human umbilical vein endothelial cells endothelial nitric-oxide synthase glyceraldehyde-3-phosphate dehydrogenase dithiothreitol phenylmethylsulfonyl fluoride enzyme-linked immunosorbent assay The mechanism by which estrogens attenuate the development of atherosclerosis is not known, although various actions of estrogens have been suggested to mediate this effect (1Nathan L. Chaudhuri G. Annu. Rev. Pharmacol. Toxicol. 1997; 37: 477-515Google Scholar). We have previously demonstrated that 17-ॆ estradiol (17-ॆ E2) in vivo inhibits the adhesion of monocytes to endothelial cells of the rabbit aorta (2Nathan L. Pervin S. Singh R. Rosenfeld M. Chaudhuri G. Circ. Res. 1999; 85: 377-385Google Scholar). We also demonstrated that following a cholesterol-enriched diet to ovariectomized rabbits, expression of VCAM-11 protein was induced in the aorta, and this was attenuated by administration of 17-ॆ E2 (2Nathan L. Pervin S. Singh R. Rosenfeld M. Chaudhuri G. Circ. Res. 1999; 85: 377-385Google Scholar). Treatment of cultured rabbit aortic endothelial cells with 17-ॆ E2 also attenuated the lysophosphatidylcholine-induced expression of VCAM-1 protein. However, the concentrations of 17-ॆ E2 required to suppress VCAM-1 expression in vitro were in the supraphysiological range when compared with the physiological levels required for in vivo studies. We therefore postulated that one mechanism by which only relatively low concentrations of 17-ॆ E2 were required for in vivo studies when compared with in vitro studies was that 17-ॆ E2 might be converted to a more potent metabolite in vivo (2Nathan L. Pervin S. Singh R. Rosenfeld M. Chaudhuri G. Circ. Res. 1999; 85: 377-385Google Scholar). Some effects of estrogens are mediated through non-genomic mechanisms, whereas others require transcriptional activation of genes (3Haynes M.P. Russell K.S. Bender J.R. J. Nucl. Cardiol. 2000; 7: 500-508Google Scholar). These latter actions usually require that estrogens combine with specific receptors. Two types of estrogen receptors (ER) have been described. They are the 舠classical舡 ER, ERα and the 舠novel舡 more recently described ERॆ (4Kuiper G.G. Carlsson B. Grandien K. Enmark E. Haggblad J. Nilsson S. Gustafsson J.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 90: 5925-5930Google Scholar). Both ERα and ERॆ have been detected in endothelial cells and in vascular smooth muscle cells (5Register T.C. Adams M.R. J. Steroid Biochem. Mol. Biol. 1998; 64: 187-191Google Scholar, 6Lindner V. Kim S.K. Karas R.H. Kuiper G.G. Gustafsson J.A. Mendelsohn M.E. Circ. Res. 1998; 83: 224-229Google Scholar). The affinity of various estrogenic compounds for the two ER subtypes markedly differ. 17-ॆ E2 binds to ERα and ERॆ with similar affinity. 17-α ethinyl estradiol (EE), a synthetic estrogen widely used in oral contraceptive formulations, has an ERα-selective agonist potency, whereas 17-epiestriol, an estrogen metabolite, and genistein, a phytoestrogen, at nmconcentrations have ERॆ-selective agonist potency (7Barkhem T. Carlsson B. Nilsson Y. Enmark E. Gustafsson J. Nilsson S. Mol. Pharmacol. 1998; 54: 105-112Google Scholar). The present work was therefore undertaken to test whether certain estrogenic compounds, including an estrogen metabolite, with different agonistic potencies for the two types of estrogen receptors were more potent than 17-ॆ E2 in suppressing VCAM-1 mRNA expression and the potential mechanism(s) involved. Information obtained from these studies could lead to the development of compounds that attenuate atherogenesis with fewer side effects than those observed with 17-ॆ E2. The culture medium M199, HEPES buffer, and collagenase-type 1 from porcine skin were obtained from Invitrogen. Gelatin, endothelial-derived growth factor, genistein, TNFα, NG-nitro-l-arginine methyl ester (l-NAME), diethyl pyrocarbonate, salmon testes DNA, Denhardt's solution, and SDS were obtained from Sigma; the steroids 17-ॆ E2, EE, and 17-epiestriol were also obtained from Sigma with ∼997 purity as assessed by thin layer chromatography. Fetal bovine serum (FBS) charcoal/dextran-treated was obtained from HyClone Laboratories. FBS was obtained from Atlas, Fort Collins, CO, and ICI-182780 was obtained from Tocris Cookson Ltd., Ballwin, MO. The human NFκB p65 Nushift kit (2006760) was obtained from Geneka Biotechnology (Montreal, Canada). For isolation of human umbilical vein endothelial cells (HUVEC), umbilical cords from female fetus were selected as we observed increased expression of ERα and ERॆ mRNA when compared with those obtained from male fetus. HUVEC were isolated from freshly collected umbilical cords (female fetuses) as previously described (8Caulin-Glaser T. Watson C.A. Pardi R. Bender J.R. J. Clin. Invest. 1996; 98: 36-42Google Scholar) and cultured on 0.17 gelatin-coated 75-mm flasks in M199 medium supplemented with 207 FBS, 5 ॖg/ml endothelial-derived growth factor, 100 ॖg/ml heparin, 100 units of penicillin G sodium, 100 ॖg/ml streptomycin sulfate, 0.25 ॖg/ml amphotericin-B and 10 mm HEPES buffer, pH ≈ 7.5. Prior to the experiments, cells were shifted to phenol red-free M199 medium supplemented with 27 charcoal/dextran-treated FBS and the antibiotics mentioned above. Only second or third passage cells were utilized in all the current studies. RNA extraction and purification were done using RNeasy mini kit as described by the manufacturer's protocol (Qiagen, Chatsworth, CA). Total RNA (3 ॖg per sample) was subjected to reverse transcription reaction with 50 units of Moloney murine leukemia virus reverse transcriptase at 42 °C for 25 min as previously described (9Hodges Y.K. Tung L. Yan X.D. Graham J.D. Horwitz K.B. Horwitz L.D. Circulation. 2000; 101: 1792-1798Google Scholar). The resulting cDNA samples were PCR-amplified using Gene Amp RNA PCR kit (PerkinElmer Life Sciences) according to the manufacturer's protocol. Oligonucleotide primers were designed for simultaneous PCR amplification (9Hodges Y.K. Tung L. Yan X.D. Graham J.D. Horwitz K.B. Horwitz L.D. Circulation. 2000; 101: 1792-1798Google Scholar) of specific DNA fragments contained in both ERα and ERॆ using the following set of primers: forward primer sequence, 5′-AAG AGC TGC CAG GCC TGC CG-3′; reverse primer sequence, 5′-GCC CAG CTG ATC ATG TGA ACC A-3′. 10 ॖg of total RNA was loaded on 17 agarose-formaldehyde gel, electrophoresed, and transferred to Hybond membrane (Amersham Biosciences) overnight by capillary action. The RNA was UV-cross-linked using GS Gene Linker (BioRad). VCAM-1 (Research Genetics) and eNOS (a gift from Dr. Thomas Michel, Brigham and Women's Hospital, Boston, MA) cDNAs were labeled with [α-32P]dCTP (ICN Biochemicals) using the random priming method. Membranes were prehybridized at 42 °C overnight followed by hybridization with respective labeled probes for another 24 h at 42 °C. The membranes were washed twice at room temperature with 2× sodium chloride and sodium citrate (SSC) buffer and 0.57 SDS followed by washing at 65 °C for 30 min twice with 0.5× SSC and 0.17 SDS, and respective bands were quantitated using a Phosphoimager (Molecular Dynamics). The VCAM-1 band was normalized with the GAPDH band as an internal control. Following the treatments, cells were lysed in a buffer containing 50 mm HEPES (pH 7.5), 1 mm DTT, 150 mm NaCl, 1 mm EDTA, 0.17 Tween 20, 107 glycerol, 10 mm ॆ-glycerophosphate, 1 mm NaF, 0.1 mm orthovanadate, 10 ॖg/ml leupeptin, 10 ॖg/ml aprotinin, and 0.1 mm phenylmethylsulfonyl fluoride (10Singh R. Pervin S. Karimi A. Cederbaum S. Chaudhuri G. Cancer Res. 2000; 60: 3305-3312Google Scholar). 10 ॖg of total protein were subjected to SDS-PAGE and transferred to polyvinylidene difluoride membrane using a semi-dry transfer apparatus. The membranes were incubated at room temperature with eNOS monoclonal antibody (1:1000) or GAPDH (1:5000) antibody for 60 min. The membranes were then incubated in diluted (1:1000) horseradish peroxidase-linked secondary antibody (1:1000) for 60 min at room temperature. The proteins were detected using ECL Western blotting kit following exposure of the membrane to autoradiography film (Hyperfilm-ECL,Amersham Biosciences). The relative intensities were quantified by densitometric analysis (Personal Densitometer, SI, Molecular Dynamics). Membrane-bound VCAM-1 protein was measured by ELISA as described previously (11Mukherjee T.K. Dinh H. Chaudhuri G. Nathan L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4055-4060Google Scholar). After appropriate treatments, 108 cells were washed twice with ice-cold phosphate-buffered in 1 of ice-cold buffer mm pH 5 mm 10 mm NaCl, 0.1 mm mm DTT, mm 1 ॖg/ml of aprotinin, and in a and for 30 at The was in of extraction buffer mm pH mm mm glycerol, mm DTT, mm at °C for 30 and for 30 min at at °C. proteins in the were pH 0.1 mm EDTA, 207 glycerol, mm DTT, mm overnight at °C. The was by and proteins in the were quantitated by (BioRad). 10 ॖg of from NFκB assay was as described in the manufacturer's protocol NFκB p65 Nushift kit are as obtained from in were assessed by analysis of and test were as and third passage which were utilized for all both ERα and ERॆ mRNA not VCAM-1 mRNA expression was detected in the cells or those with the different estrogenic compounds The of the different estrogens are in that HUVEC when to 10 of for h to VCAM-1 mRNA expression. and were utilized for all the The effects of of HUVEC with different concentrations of 17-ॆ E2, and 17-epiestriol and 10 and genistein (1 and 10 nm) on VCAM-1 mRNA expression are in 17-ॆ E2 at concentrations from 1 to the TNFα-induced VCAM-1 whereas concentrations did not have any effect when compared with that observed with did not have any effect on VCAM-1 mRNA expression and 17-epiestriol a that was different from that of 17-ॆ concentrations was of VCAM-1 mRNA expression when compared with that observed with The was observed with 100 and The for 17-ॆ E2 and 17-epiestriol were at concentrations (1 and 10 the effect of 17-epiestriol was and the VCAM-1 expression to However, the of VCAM-1 expression at of 17-epiestriol was significantly than that observed with at similar concentrations to that of 17-epiestriol effect not (1 and 10 nm) attenuated VCAM-1 mRNA expression The for genistein was The effects of all these estrogen receptor were significantly attenuated in the presence of the ER antagonist ICI-182780 (1 and the results for 17-epiestriol are in of VCAM-1 mRNA expression using the Phosphoimager of the various in is as densitometric and show the effects of 17-ॆ E2 and on TNFα-induced VCAM-1 mRNA expression. from only 2) of a analysis the effects of different concentrations of 17-epiestriol on TNFα-induced VCAM-1 mRNA expression and by Phosphoimager is as of densitometric units obtained from GAPDH was used as internal control. from only 2) of a analysis the effects of different concentrations of genistein on TNFα-induced VCAM-1 mRNA expression and by Phosphoimager is as of densitometric units obtained from GAPDH was used as internal control. from 2) of analysis the effect of 17-epiestriol VCAM-1 and expression in the presence and of an ER antagonist ICI-182780 (1 The ER antagonist was to the culture medium containing HUVEC 60 min the of We the effect of 17-epiestriol on TNFα-induced VCAM-1 protein expression. HUVEC were with different concentrations of 17-epiestriol for the of TNFα, the cells were and for VCAM-1 protein by ELISA as described previously (11Mukherjee T.K. Dinh H. Chaudhuri G. Nathan L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4055-4060Google Scholar). in VCAM-1 protein was observed at a of 17-epiestriol with the to VCAM-1 TNFα-induced VCAM-1 protein expression also a to whether NO an in TNFα-induced VCAM-1 we the effect of 17-epiestriol on eNOS expression. Treatment of HUVEC with 17-epiestriol a on eNOS protein expression. The was observed at for both eNOS protein and mRNA expression not We assessed the effect of different concentrations of 17-epiestriol on TNFα-induced VCAM-1 mRNA expression in the and presence of (3 an inhibitor of NO of has been previously demonstrated by to significantly attenuate and NO by endothelial cells T. Chaudhuri G. Proc. Natl. Acad. Sci. U. S. A. Scholar). or with did not VCAM-1 expression. 17-epiestriol and and 1 nm) attenuated VCAM-1 mRNA and this effect was not observed in the presence of an inhibitor of NO of a analysis the effects of different concentrations of 17-epiestriol and and 1 nm) VCAM-1 mRNA expression in the and presence of an inhibitor of synthase The various in of the are also GAPDH was used as the internal whether 17-epiestriol TNFα-induced VCAM-1 expression by NFκB migration to the nucleus, we using to the on the VCAM-1 We also assessed whether NO an in this action by 17-epiestriol at concentrations of 100 not and significantly the of the shifted band by obtained from HUVEC with However, 10 of 17-epiestriol was in the of the shifted band when compared with of the was by with an of and with a labeled when band was observed The was further by analysis 3) with an to of NFκB following with 17-epiestriol in the presence was similar to that observed in the of 17-epiestriol and in the presence of and that the effect of 17-epiestriol on the of NFκB to the following of HUVEC with was on NO The of this was to whether selected estrogenic compounds, including a phytoestrogen, a synthetic estrogen, and an estrogen metabolite with different agonistic potencies for ERα and ERॆ were more potent than 17-ॆ E2 in TNFα-induced VCAM-1 expression of HUVEC in vitro. 17-ॆ E2 is the estrogen in binds to human ERα and ERॆ with similar affinity (7Barkhem T. Carlsson B. Nilsson Y. Enmark E. Gustafsson J. Nilsson S. Mol. Pharmacol. 1998; 54: 105-112Google Scholar) and was therefore used as the with which the to the estrogens were EE, a synthetic estrogen, was selected is widely used as the estrogenic in oral and has ERα-selective agonistic potency (7Barkhem T. Carlsson B. Nilsson Y. Enmark E. Gustafsson J. Nilsson S. Mol. Pharmacol. 1998; 54: 105-112Google Scholar). was selected is an estrogen metabolite M. S. A. 1996; which has an ERॆ-selective agonist potency (7Barkhem T. Carlsson B. Nilsson Y. Enmark E. Gustafsson J. Nilsson S. Mol. Pharmacol. 1998; 54: 105-112Google Scholar). was selected as markedly and is similar to that the in is in the 17-ॆ whereas that of is in the a agonist for both ERα and ERॆ (7Barkhem T. Carlsson B. Nilsson Y. Enmark E. Gustafsson J. Nilsson S. Mol. Pharmacol. 1998; 54: 105-112Google Scholar). The genistein binds to ERॆ as well as 17-ॆ E2, whereas affinity to ERα is than that of 17-ॆ E2 G.G. Carlsson B. B. Gustafsson J.A. 1998; Scholar). Our results indicate that 17-ॆ E2 did not VCAM-1 mRNA expression at concentrations and whereas at concentrations nm) VCAM-1 mRNA in a and action of 17-ॆ vitro has been by the results are studies using 17-ॆ E2 suppressed the of VCAM-1 mRNA expression induced by (2Nathan L. Pervin S. Singh R. Rosenfeld M. Chaudhuri G. Circ. Res. 1999; 85: 377-385Google Scholar, T. S. G. G. R. Circ. Res. 2000; Scholar) or by (8Caulin-Glaser T. Watson C.A. Pardi R. Bender J.R. J. Clin. Invest. 1996; 98: 36-42Google Scholar, K. K. T. M. K. Life Sci. 54: Scholar). J. Clin. Invest. Scholar) observed that 17-ॆ E2 did not VCAM-1 expression a in the presence of and effect when HUVEC were with has also been that 17-ॆ E2 effect on the TNFα-induced expression of VCAM-1 in cultured endothelial cells 1996; Scholar). These be to in the to which the endothelial cells used in the different studies the estrogen in the of 17-ॆ E2 or to in the used to VCAM-1 expression. this in both the present and in those previously (8Caulin-Glaser T. Watson C.A. Pardi R. Bender J.R. J. Clin. Invest. 1996; 98: 36-42Google Scholar, T. S. G. G. R. Circ. Res. 2000; Scholar, K. K. T. M. K. Life Sci. 54: the concentrations of 17-ॆ for of VCAM-1 expression in vitro were in the supraphysiological range (1 The physiological of 17-ॆ E2 is in the whereas that of 17-epiestriol has not been in EE, which is a more potent and selective agonist for ERα when compared with 17-ॆ E2, did not attenuate TNFα-induced VCAM-1 mRNA expression at any of the concentrations and potential for the of any effect of be that ERॆ the in VCAM-1 mRNA whereas ERα has a be the as 17-epiestriol, which has an ERॆ-selective agonistic potency, VCAM-1 mRNA expression and the was ∼400× than that observed with 17-ॆ E2. we explain concentrations of 17-epiestriol were similar effect of 17-epiestriol on VCAM-1 protein expression and a of NFκB to the was also which is a agonist for both ERα and ERॆ (7Barkhem T. Carlsson B. Nilsson Y. Enmark E. Gustafsson J. Nilsson S. Mol. Pharmacol. 1998; 54: 105-112Google did not VCAM-1 mRNA expression. that the of the in the is an for 17-epiestriol for selective ERॆ agonistic The of the various estrogens to attenuate VCAM-1 mRNA expression was not observed in the presence of the that the action of the estrogens were and more specific of the ER in the VCAM-1 mRNA expression by estrogens have to the of specific ERα and ERॆ receptor at concentrations has affinity to which is similar to that of 17-ॆ E2, whereas affinity to ERα is G.G. Carlsson B. B. Gustafsson J.A. 1998; Scholar). Our that genistein at nmconcentrations (1 and 10 nm) VCAM-1 mRNA expression the that ERॆ is in the of VCAM-1 expression in endothelial cells and that ERα has in this these genistein has G.G. Carlsson B. B. Gustafsson J.A. 1998; Scholar) the effects are to action on the in the of the VCAM-1 has been J. Biol. and to be The VCAM-1 for the transcription factor the of transcription and an T. S. G. G. R. Circ. Res. 2000; Scholar). NFκB is a transcription factor genes in endothelial activation R. R. S. and Treatment in Scholar). has recently been demonstrated that a of 17-ॆ suppressed VCAM-1 expression by the and DNA of and this was also with a of and transcription to the VCAM-1 T. S. G. G. R. Circ. Res. 2000; Scholar). These also demonstrated that in human endothelial NFκB inhibition by E2 was with this we to the that estrogens have increased the of which in have to the of DNA of transcription thereby to inhibition of VCAM-1 expression. is a of the actions of estrogens and NO on various transcription that VCAM-1 expression. NO expression of VCAM-1 and VCAM-1 transcription in part by protein NFκB R. J. Clin. Invest. Scholar, M. H. K. J. Biol. 1998; Scholar). NO also the NFκB K. M. Y. 1998; and with NO compounds also to DNA A. K. E. T. M. Scholar). We previously demonstrated that estrogens NO T. Chaudhuri G. Proc. Natl. Acad. Sci. U. S. A. and others have demonstrated that through both S. Proc. Natl. Acad. Sci. U. S. A. Scholar) and non-genomic T. G. Bender J.R. Circ. Res. 1997; Scholar, Karas R.H. Mendelsohn M.E. J. Clin. Invest. 1999; Scholar) We therefore to whether 17-epiestriol at concentrations that attenuated VCAM-1 mRNA expression also increased eNOS protein and whether this was at least in part to increased eNOS Our results that 17-epiestriol also a effect on eNOS protein expression. at a that suppressed TNFα-induced VCAM-1 mRNA expression also increased eNOS protein and that NO an in this action of was by that the of VCAM-1 mRNA expression by 17-epiestriol was not observed following inhibition of NO Our results also that NO an in inhibition of the and DNA of NFκB in HUVEC as this effect of 17-epiestriol was significantly attenuated in the presence of an inhibitor of NO concentrations (1 and 10 nm) of 17-epiestriol attenuated the eNOS protein expression when compared with and this explain the of 17-epiestriol on VCAM-1 mRNA and protein expression. Our did not the mechanism(s) by which NO by 17-epiestriol the of NFκB to the nucleus. is also that the in actions observed estrogenic compounds with different for ERα and ERॆ receptors could be mediated by with the The of ERα and ERॆ with the could lead to in and in K. Kuiper Nilsson S. Gustafsson J. 1997; Scholar). also be to whether the of and to the VCAM-1 by estrogens is also mediated by an in NO thereby an of NO in the of transcription by results indicate that estrogens VCAM-1 mRNA and protein expression by an action mediated by ERॆ and not by ERα and that NO an in this Our results further that 17-epiestriol, an estrogen metabolite, is more potent than 17-ॆ E2. be one potential as to concentrations of 17-ॆ E2 are required to suppress VCAM-1 vivo various of estrogens are when compared with those required in vitro. These results have of with to estrogen is the of ERα and ERॆ in the action of has been demonstrated that 17-ॆ E2 to vascular in an in ovariectomized Karas R.H. M. Kim S. K.S. Mendelsohn M.E. 1997; Scholar) and R.H. M. M. Gustafsson J.A. K.S. Mendelsohn M.E. Proc. Natl. Acad. Sci. U. S. A. 1999; Scholar) to the as in this has been suggested that ERα and ERॆ in vivo to mediate the action of estrogens following vascular has been demonstrated that genistein, which has selective affinity to actions that were to that observed with 17-ॆ E2 in an and were of effects on the which have ERα receptors S. H. E. M. L. E. Gustafsson J.A. Proc. Natl. Acad. Sci. U. S. A. 1999; Scholar). the in to the atherosclerosis the endothelial cells are and therefore be to the actions of estrogens the endothelial cells. therefore be to whether 17-epiestriol is as or more potent than 17-ॆ E2 in atherogenesis and is of effects on the when compared with 17-ॆ E2. studies are in to this in vivo in an Our results the action of 17-epiestriol in VCAM-1 and protein expression indicate that estrogens have effects in be one potential for the in to the effects of estrogen on for the Women's 2002; Scholar). We and for

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