Donate Help Contact The AHA Sign In Home
American Heart Association
Circulation
Search: search_blue_button Advanced Search
« Previous Article | Table of Contents | Next Article »
Circulation. 1996;94:727-733

This Article
Right arrow Abstract Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Venkov, C. D.
Right arrow Articles by Vaughan, D. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Venkov, C. D.
Right arrow Articles by Vaughan, D. E.

(Circulation. 1996;94:727-733.)
© 1996 American Heart Association, Inc.

Articles

Identification of Authentic Estrogen Receptor in Cultured Endothelial Cells

A Potential Mechanism for Steroid Hormone Regulation of Endothelial Function

Christo D. Venkov, PhD; Alan B. Rankin, BS; Douglas E. Vaughan, MD

the Cardiovascular Divisions (C.D.V., A.B.R., D.E.V.), Vanderbilt University Medical Center, and Nashville Veterans Affairs Medical Center (D.E.V.), Nashville, Tenn.

Correspondence to Douglas E. Vaughan, MD, Vanderbilt University Medical Center, Division of Cardiology, Rm 315 MRB II, Nashville, TN 37232-6300. E-mail Doug.Vaughan@mcmail.vanderbilt.edu.


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Background Estrogen plays a major role in the delayed expression of coronary heart disease (CHD) in women, and recent data indicate that postmenopausal estrogen therapy reduces the incidence of CHD by >40%. The mechanism or mechanisms through which estrogen exerts this benefit are unknown, although effects on blood pressure, carbohydrate and lipid metabolism, and coagulation have been suggested. We hypothesized that at least part of the effect of estrogen in reducing the incidence of CHD is due to an effect on endothelial cell function.

Methods and Results In the present study, we examined human aortic and umbilical vein endothelial cells and bovine aortic endothelial cells for the presence of estrogen receptors (ERs) through immunoblot and mRNA analyses. Electrophoretic mobility shift assays were also performed to determine the DNA-binding properties of the putative ERs. Nuclear extracts from all three endothelial cell types were found to contain a 67-kD protein that reacted with anti-ER monoclonal antibodies specific to different domains of the ERs. Each of these types of cells expresses proteins that bind specifically to consensus estrogen-responsive elements. Finally, Northern blots verified that endothelial cells express abundant amount of mRNA for the ER.

Conclusions These data indicate that endothelial cells constitutively possess the potential for transcriptional regulation of target genes by estrogen. The evolutionary conservation of this receptor in bovine and human endothelial cells suggests a common mechanism for estrogen regulation of endothelial function.


Key Words: endothelium • hormones • receptors • coronary disease


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
The protective effect of endogenous sex hormones is commonly believed to explain the gender gap in the risk of coronary heart disease. Estrogen deprivation results in alterations of plasma lipids, a drop in HDL level, and a rise in LDL level1 ; estrogen replacement therapy reverses these effects by accelerating the metabolism of LDL,2 possibly through ER-mediated transcriptional activation of LDL receptor gene expression.3

The epidemiological data suggest, however, that all of the atheroprotective effects of estrogen cannot be accounted for by alterations in serum lipid profiles.4 5 Recent data point to a positive correlation between ER expression and the absence of atherosclerosis in human coronary arteries.6 Estrogen modulates endothelium-dependent responses of arteries ex vivo,7 8 and its acute administration improves endothelium-dependent vasodilatation in healthy postmenopausal women.9 10 This direct vasodilatory effect of estrogen is rapid in onset and is unlikely to be caused by alterations in gene expression.11 12 Conversely, estrogen may modulate the endothelial expression of rate-limiting enzymes in the biosynthesis of the two important vasodilators prostacyclin and endothelium-derived relaxation factor (NO). A preliminary study indicates that ER causes a 50% increase in constitutive eNOS mRNA levels in HUVEC and induces a sixfold increase in their basal NO production.13 Estrogen also potentiates the effect of endothelin-1 on prostacyclin production in HUVEC.14

The transcriptional regulation of target genes by estrogen requires binding of the ligand to its intracellular receptor, which initiates a sequence of events leading to modulation of gene expression. The estrogen receptor is a hormone-dependent transcription factor that induces or represses transcription of selected genes containing consensus regulatory sequences, referred to as EREs. The classic ER is a 67-kD protein with separate and highly conserved DNA and hormone-binding domains and is coded for by a cDNA that is 6.2 kb long. Shorter and commonly nonfunctional variants of the ER have been described in breast tumor cells15 and, recently, in normal tissues.16 17 It is generally assumed that in vivo, estrogen induces structural changes in its receptor and thus enhances binding to consensus DNA sequences (EREs) and promotes subsequent transcriptional activation. The precise binding of ER to DNA is realized through two Zn2+ binding motifs folded to form a single structural unit.18 EREs are highly conserved palindromic sequences with the characteristic motif GGTCAC(N)3GTGACC.

With regard to vascular tissues, functional ER has been identified in vascular smooth muscle cells,19 and ER presence in endothelial cells had been suggested by estrogen-binding studies.20 21 However, the simple measurement of ER presence through ligand binding does not provide conclusive evidence of its expression or functional potential. In fact, the existence of sex steroid hormone receptors in endothelial cells has been disputed.22 The present study was designed to determine whether ERs can be identified in endothelial cells through the use of specific monoclonal antibodies and molecular probes. Our results confirm the presence of a classic ER in endothelial cells that can bind specifically to a consensus DNA ERE.


*    Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Cell Cultures
HAEC (Cell Systems Co) were grown and maintained in the specific medium supplied by the manufacturer and supplemented with 10% FBS (Hyclone Laboratories). HUVEC and BAEC were prepared as primary cultures and cultivated in Medium 199 (GIBCO-BRL), supplemented with 15% FBS. For treatment with estrogen (10 nmol/L 17-ß estradiol, Sigma Chemical Co), cells were cultivated for >=2 days in a phenol red–free medium supplemented with charcoal/dextran–treated serum. The estrogen-responsive rat pituitary cells GH323 (kindly provided by Dr Mark Seyfred, Vanderbilt University) were maintained in DMEM with 10% bovine calf serum. The ER-negative human adenocarcinoma breast cells BT-20 were kindly provided by Dr Fritz Parl (Vanderbilt University).

Protein Extracts
For separation of nuclear and cytosolic fractions, cells were washed once with cold PBS; washed once with buffer containing 20 mmol/L PIPES, pH 6.8, 5 mmol/L MgCl, 1 mmol/L CaCl, 80 mmol/L KCl, 5% sucrose, and the ex tempore added proteinase inhibitors 0.2 mmol/L phenylmethylsulfonyl fluoride, 50 µg/mL aprotinin, 5 µg/mL leupeptin, and 5 µg/mL pepstatin A; resuspended into the same buffer with 0.5% Nonidet P-40; and gently stirred on ice for 5 minutes. Nuclear pellets were collected through centrifugation at low speed, washed once with the same buffer and once without the nonionic detergent, and extracted in a buffer containing 50 mmol/L Tris-HCl, 400 mmol/L NaCl, 10% glycerol, and 1 mmol/L dithiothreitol. The extracts were clarified through a 30-minute centrifugation at 10 000g and stored at -80°C. The cytosolic protein supernatant was concentrated with the use of a Centricon-30 concentrator (Amicon) and stored at -80°C.

Immunoblotting
Proteins were resolved through the use of standard SDS-PAGE24 on 10% gels and transferred to a polyvinylidene difluoride Immobilon membrane (Millipore) with the use of a Mini Trans-Blot system (Bio-Rad). Membranes were blocked in a 10% NFDM-TBST solution (consisting of 10% nonfat dry milk in 0.1 mol/L Tris-HCl, 0.15 mmol/L NaCl, and 0.05% Tween-20, pH 7.5) and probed with the anti-ER monoclonal antibodies H-222, D-75 (kindly provided by Dr G. Greene, University of Chicago [Illinois]) or with 1D5 (DAKO Co). The anti–urokinase plasminogen activator receptor monoclonal antibody used as a control was from American Diagnostica Inc. For detection of reactive proteins, an enhanced chemiluminescence detection kit with anti-mouse horseradish peroxidase–linked IgG was used (ECL kit, Amersham).

Electrophoretic Mobility Shift Assay
The reactions were performed as described previously.25 Nuclear extracts (typically 5 to 10 µg of total protein) from endothelial or positive and negative control cells were preincubated for 20 minutes on ice with 2 µg of poly(dI/dC) (Pharmacia Biotech) and 1 µg of single-stranded nonspecific oligonucleotide in a reaction containing 100 mmol/L NaCl, 2 mmol/L dithiothreitol, 10% glycerol, and 25 mmol/L HEPES buffer, pH 7.4, before the addition of the radiolabeled probe. The unlabeled competitor oligonucleotides (25 ng) or either of the anti-ER antibodies 1D5 or H-222 (1 µg) was added during preincubation. Two slightly different consensus oligonucleotides with characteristic ERE palindromic motifs were used: a 19-bp oligomer, 5'-AATTCGGTCACGCTGACCA-3', or a 27-bp oligomer from the vitellogenin A2 ERE, 5'-GATCCTAGAGGTCACAGTGACCTACGA-3' (kindly provided by Dr G. Greene). The oligonucleotides were end-labeled with [{gamma}-32P]dATP through the use of T4 polynucleotide kinase, adjusted to {approx}30 000 cpm/0.25 ng/µL, and 1 µL/19-µL sample was added. After additional incubation for 30 minutes at room temperature, the samples were directly loaded on a prerun 4.6% polyacrylamide gel in 0.5x TBE (0.5x: 0.045 mol/L Tris/borate, 0.002 mL EDTA) and run at 10 mA for 3 hours. The gel was fixed in 10% acetic acid plus 20% methanol, vacuum dried, and exposed to x-ray film.

mRNA Analysis
Total RNA was extracted directly in the culture plates with the use of RNAzol (Biotecx Labs) from endothelial cells washed with cold PBS. The RNA was kept under ethanol at -20°C until use. Radiolabeled antisense RNA probes were synthesized (Maxiscript, Ambion Inc) from a lambda OR3 cDNA clone as a template (POR3, American Type Culture Collection) coding for a region in the DNA-binding domain of the ER,26 which was subcloned into a pBluescript II SK± (Stratagene). For Northern hybridization analysis, total RNA samples were subjected to denaturing agarose formaldehyde electrophoresis,27 transferred to membranes (Z-blot, Bio-Rad), and hybridized to the ER riboprobe for 20 hours at 63°C. The stringent wash conditions for membranes included 63°C in 0.2x SSC (1x: 0.15 mL sodium chloride, 0.015 mol/L sodium citrate) plus 0.1% SDS for 30 minutes.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 *Results
down arrowDiscussion
 down arrowReferences
 
Identification of ERs in Endothelial Cell Nuclear Extracts by Immunoblotting
Protein extracts from separated nuclear and cytosolic fractions were subjected to standard SDS electrophoresis, transferred to a membrane, and probed with various antibodies against the ER. The results shown in Fig 1ADown demonstrate the presence of an immunoreactive protein fraction with an apparent molecular mass of 67 kD recognized by the anti-ER antibody H-222 in nuclear extracts from BAEC (lane B) and HUVEC (lane C) that corresponds to a band in the nuclear extracts from the estrogen-responsive GH3 cells (lane A). This finding was confirmed with the use of other anti-ER monoclonal antibodies specific to different epitopes. The results shown on Fig 1BDown verify the presence of a D-75–immunoreactive band in nuclear extracts from HAEC that corresponds in mobility to the positive control and to the band recognized by H-222 on Fig 1ADown. The same pattern of immunostaining in all types of endothelial cells studied was also obtained with another anti-ER monoclonal antibody, 1D5, that is specific to the amino-terminal region of the receptor (Fig 1CDown). The immunostaining patterns obtained with the three antibodies were highly reproducible and did not depend on the type of antibody used. Parallel experiments with a monoclonal antibody from the same class but specific to the anti–urokinase plasminogen activator receptor (see "Methods") failed to produce a distinctive band in this region and confirmed the specificity of the 67-kD protein toward the anti-ER antibodies used. The cytosolic fractions from all cell types equally lacked any distinctive immunostained band (not shown). The presence of the ER-immunoreactive band did not change in endothelial cells cultivated up to passage 8.





View larger version (355K):
[in this window]
[in a new window]
  		Figure 1. Immunoblotting of nuclear extracts from different endothelial and positive control cells probed with anti-ER monoclonal antibodies. A, Approximately 10 µg of nuclear protein extracts from BAEC (B) or HUVEC (C) and 5 µg of GH3 (A) were run on SDS-polyacrylamide gels, transferred to a membrane, and immunostained with H-222 as described in "Methods." B, Increasing amounts of protein (6, 12, and 24 µg) from HAEC and GH3 cells were immunostained with D-75. C, 10 µg protein from GH3 (lane A), HAEC (lane B), HUVEC (lane C), and BAEC (lane D) was similarly probed with 1D5 monoclonal antibodies.

Proteins From Endothelial Cells Form Specific Complexes With the ERE
Nuclear extracts from early passage endothelial cells were probed for their ability to interact in vitro with ER consensus oligonucleotide sequences. Two complexes with considerable differences in mobility were clearly observed (Fig 2ADown). The complexes formed with endothelial proteins correspond in mobility and specificity to complexes formed with extracts from GH3 estrogen-responsive cells. Nuclear extracts from the ER-negative breast tumor cell line BT-20 were able to form only one complex that was unaffected by the addition of unlabeled ERE. Titration analysis with nuclear and cytosolic extracts and unlabeled specific and nonspecific oligonucleotides (Fig 2BDown) indicated that the two complexes are affected by the consensus ERE but not by the nonspecific oligonucleotide and therefore represent specific DNA/protein complexes. Also, these complexes are formed only with nuclear proteins, which again emphasizes their authenticity. The same pattern of specific complex formation was observed with protein extracts from transfected COS cells expressing high levels of ER (kindly provided by Dr G. Greene). The mobility and intensity of these complexes did not depend on the presence of estrogen in either the reactions or the culture medium independent of the cell type: GH3 extracts displayed the same pattern as endothelial ones. The complexes shown in Fig 2A through 2CDown were formed with the use of the vitellogenin A2 27-mer ERE, but similar results were obtained with the 19-mer oligomer.





View larger version (215K):
[in this window]
[in a new window]
  		Figure 2. Electrophoretic mobility shift assay of nuclear extracts from endothelial cells and positive (GH3) or negative control (BT-20) cells. Complexes formed with the radiolabeled 27-mer vitellogenin A2 ERE were challenged with a 100x molar excess of either unlabeled ER or nonspecific oligonucleotide (A and C) or as indicated (B) and with 1 µg of the anti-ER antibody 1D5 (C). The positions of complexes are marked. The experiment in B was performed with nuclear extracts from HUVEC, but similar results were obtained with extracts from the other cell types shown in A and C.

To confirm that authentic ER was a component of these complexes, electrophoretic mobility shift assays were performed in the presence of the anti-ER monoclonal antibody 1D5 (Fig 2CUp). In HAEC and HUVEC, the antibody induced a nearly complete supershift of the slow moving complex, whereas in GH3 and BAEC, the shift was not total, and a certain amount of this complex remained. Furthermore, in BAEC, the shifted complex had a different mobility. The faster moving complex was affected to a lesser extent in the supershift experiments. Similar results were obtained with another ER-specific antibody, H-222. Otherwise, we did not find any cell type or species specificity of the complexes: human arterial, human umbilical vein, and bovine endothelial nuclear extracts were equally able to form specific complexes and showed identical affinity toward each of the EREs used.

Endothelial Cells Express a Full-Length mRNA for the ER
In these experiments, total RNA was extracted from early passage cell cultures and analyzed through Northern blot analysis with an {approx}0.4-kb antisense RNA spanning the region between nucleotides 1170 and 1600 of the ER cDNA (see "Methods"). The results shown in Fig 3Down demonstrate the presence of abundant mRNA for the ER with the expected 6.2-kb length in all endothelial cell types studied. The presence of ER mRNA in endothelial cells was also verified through RNase protection assays with the same radiolabeled probe. RNA extracts from endothelial cells were able to protect the 0.4-kb antisense ER RNA from degradation by RNases A and T1 (data not shown). We did not find any site- or species-specific differences with regard to the level or size of ER mRNA from endothelial cells. Furthermore, this message did not appear to be affected by the treatment of cells with estrogen, which is known to modulate the expression of ER mRNA in breast tumor cells.28




View larger version (143K):
[in this window]
[in a new window]
  		Figure 3. Presence of ER mRNA in endothelial cells. Increasing amounts of RNA from BAEC (A) or HUVEC (B) were separated on agarose denaturing gels, transferred to a membrane, and hybridized to an antisense transcript of the ER cDNA. The relative amounts of RNA in each lane were visualized with the use of ethidium bromide staining and photographed (bottom). The positions of ER mRNA and rRNA are marked.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
The concept that ovarian hormones may influence the endothelial functions in various physiological and pathophysiological conditions is not new.20 Data continue to accumulate pointing to a positive correlation between estrogen plasma levels and fibrinolysis,29 estrogen modulation of the biosynthesis of endothelium-derived vasodilators,13 14 and enhancement by estrogen of endothelial cell activities that are important in neovascularization.30 Still, the mechanism or mechanisms of these effects have not been adequately explained. Since the effect of estrogen is realized in most cases through its intracellular receptor, attempts have been made to detect ER in vascular tissues, and it has been positively identified in vascular smooth muscle cells.19 Although previous studies have suggested the presence of ER in endothelial cells,20 21 a definitive characterization of endothelial ER has not been published, and its expression in endothelial cells has been disputed.22

With a panel of anti-ER antibodies specific for different regions of the receptor, we were able to establish the presence of a 67-kD protein in nuclear extracts of HAEC, HUVEC, and BAEC. The nuclear localization of the receptor found in endothelial cells is in agreement with the existing data for other cell types, which point to nuclear localization of ER even without ligand binding.31 32 The three monoclonal antibodies used in this study recognize distinct epitopes in the ER molecule: near the hormone-binding region of the ER (H-222),33 the "hinge" region between the hormone- and DNA-binding domains (D-75),33 and the amino-terminal domain (A/B region) of the ER (1D5).34 The fact that all three highly specific antibodies recognize the same protein fraction in all endothelial extracts confirms its authenticity and suggests that this protein may have the capacity to function as a bona fide ER.

The high affinity of ER binding to ERE has been exploited in electrophoretic mobility shift assays for studies of the mechanisms of estrogen-activated transcriptional regulation.35 36 37 Using this technique, we were able to obtain evidence that the 67-kD nuclear protein recognized by anti-ER antibodies in endothelial cell extracts can bind radiolabeled ERE with high affinity, as expected for classic ERs. The two complexes formed with ERE and nuclear extracts display different affinities: a 100-molar excess of unlabeled specific oligonucleotide completely abolished the slower moving complex, whereas the faster moving one was considerably, but not completely, reduced. Since a 100-molar excess of nonspecific oligonucleotide did not affect its intensity, it is unlikely that the latter represents nonspecific interactions. In the supershift experiments, the faster migrating complex was only marginally affected by the anti-ER antibody. One possible explanation for this divergent pattern is that the two complexes differ in the composition of additional nuclear proteins, which may account for different conformations and, therefore, different affinities toward ERE. It has been suggested that the multiple ER/ERE complexes reported in other studies were caused by different conformations38 arising probably from diversity in ER-associated nuclear proteins.37 This could also explain the cell type– and species-specific differences in the pattern of complexes formed in the presence of anti-ER antibody observed in our experiments. ER is reported to form complexes with other proteins before binding with estrogen39 or when in complex with ERE,40 41 and it is hypothesized that the number and mobility of such complexes are tissue specific. Indeed, specific control of gene transcription requiring the participation of multiple tissue-specific DNA-binding proteins has been reported,42 and a steroid hormone receptor coactivator protein has been described.43

Two similar (but not identical) EREs were used in these studies to confirm specificity of the complexes regardless of the source of the nuclear extracts. The use of different ERE consensus oligomers in the electrophoretic mobility shift assay studies did not influence the pattern of complex formation, although the 27-mer vitellogenin A2 oligonucleotide yielded consistently better autoradiographic patterns. The finding that estrogen treatment was not a requirement for the formation of specific complexes between ERE and endothelial proteins or even with proteins from the positive control cells was not surprising. Estrogen regulates the expression of target genes by binding to its receptor, which in turn is thought to dimerize, bind to EREs, and activate consensus DNA sequences. However, a distinction should be made between in vitro and in vivo situations, and there is growing evidence that estrogen is not required for specific DNA binding in vitro.35 36 37 44 45 On the other hand, ER-induced alterations in chromatin structure and trans-activation in vivo are reported to be completely dependent on the presence of estradiol.46 It has been suggested recently that the in vivo ER is bound to target DNA sequences regardless of the hormonal status of the cell but that steroid binding is required for interaction of the receptor with nuclear proteins and activation of the transcriptional machinery.47

The comparatively high amount of ER mRNA in endothelial cells observed by Northern hybridization analysis was confirmed through RNase protection assays and reflected at the protein level by immunoblot studies. Taken together, these data suggest that endothelial cells express ER at a high constitutive rate. Based on the results of immunoblotting and electrophoretic mobility shift assay experiments, this receptor is localized only in the nucleus. It also appears to be authentic and identical to the classic one described in human estrogen-responsive cells.26 However, it does not seem to be regulated in the same way as in breast cancer cells since ER mRNA level was not changed by treatment of endothelial cells with estrogen, which is known to downregulate ER expression.33

The finding of the ER in both human and bovine endothelial cells suggests conservation of estrogen responsiveness in endothelium and attests to the importance of this kind of regulation. The next step in these studies will involve a systematic characterization of the genes regulated by estrogen in endothelial cells. Several candidate genes have been identified. Estrogen is known to influence the expression of plasminogen activators tissue-type plasminogen activator and urokinase plasminogen activator and their major inhibitor, plasminogen activator inhibitor type 1, in endometrial cells.48 Furthermore, estrogen stimulates the secretion of plasminogen activators by mammary tumor cells49 50 and tissue-type plasminogen activator–producing melanoma cells.51 Estrogen administration in ovariectomized rats modulates aortic gene expression of angiotensinogen,52 which may be expressed in endothelial cells.53 Putative EREs have been described in the upstream regulatory regions of several genes of the mammalian fibrinolytic system,22 and it is possible that estrogen can directly or indirectly influence vascular fibrinolytic balance. Recently, estrogenic modulation of the biosynthesis of endothelium-derived relaxation factor (NO) in endothelial cells was reported,13 and both pregnancy and estradiol treatment increase the levels of mRNA for the calcium-dependent eNOS in the guinea pig.54 Estrogen has also been shown to modulate the expression of mRNA for intercellular adhesion proteins, E-selectin, intercellular adhesion molecule type 1, and vascular cell adhesion molecule type 155 and thus prevent monocyte adhesion during atherogenesis. Estrogen therapy has been reported to prevent retention and oxidation of lipids, to accelerate the metabolism of LDL, and to decrease cholesterol levels in blood. A summary of endothelial genes identified as candidates for estrogen regulation in vascular endothelial cells is presented in the TableDown. The presence of an immunoreactive and ERE-specific estrogen receptor in vascular endothelial cells, together with the finding of DNA sequences similar to EREs in the regulatory regions of several endothelial genes, argues for a role of estrogen in transcriptional regulation of endothelium.


View this table:
[in this window]
[in a new window]
  		Table 1. Endothelial Genes Potentially Regulated by Estrogen

On the other hand, estrogen may participate in transcriptional regulation of genes lacking consensus EREs via interactions with other DNA response elements. Thus, a half-palindromic ERE may compose part of a phorbol ester–responsive element, which in turn differs in only one nucleotide from a canonical transcription factor AP-1 recognition site.40 Indeed, ER-mediated activation of different genes involving Fos/Jun complexes has been described,40 56 and conversely, the regulation of c-fos gene expression through ER has been suggested.57 In addition, estrogen activates the binding of a nuclear factor-{kappa}B enhancer–specific protein to its consensus DNA site in uterine tissue.58 The recently reported serum-induced activation of ER in vascular smooth muscle cells59 and estrogen-stimulated tyrosine kinase activation in cultured aortic endothelial cells60 support the concept of cross-talk between mitogen- and steroid hormone–signaling pathways in vascular tissue. These and other data61 62 indicate a convergence of hormonal induction and activation of transduction pathways at the transcriptional level and suggest alternative mechanisms of estrogen action on endothelium.


*    Selected Abbreviations and Acronyms
 
BAEC = bovine arterial endothelial cells
eNOS = nitric oxide synthase (endothelial)
ER = estrogen receptor
ERE = estrogen-responsive element
HAEC = human arterial endothelial cells
HUVEC = human umbilical vein endothelial cells
NO = nitric oxide


*    Acknowledgments
 
This work was supported by National Institutes of Health grant HL-50878 and by a Merit Award from the Department of Veterans Affairs Research Service. Dr Vaughan is the recipient of a Clinical Investigator Award from the Department of Veterans Affairs Research Service. We are grateful to Dr Fritz Parl (Vanderbilt University Medical Center) for his contribution to the study and critical review of the manuscript. We greatly appreciate the help of Dr Geoffrey Greene (University of Chicago [Illinois]) in providing some of the antibodies, ER-containing protein extracts, and sequence for vitellogenin A2 ERE used in the study. The generous gift of ICI 164,384 by Dr A.E. Wakeling (Zeneca Pharmaceuticals, UK) is gratefully acknowledged. We also acknowledge the helpful discussions with Drs Allen Naftilan and Mary A. Brown.

Received December 18, 1995; revision received January 31, 1996; accepted February 1, 1996.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 up arrowDiscussion
 *References
 

  1. Barrett-Conor E, Wingard DL, Criqui MH. Postmenopausal estrogen use and heart disease risk factors in the 1980s. JAMA. 1989;261:2095-2100.[Abstract]
  2. Walsh BW, Schiff I, Rosner B, Greenberg L, Ravnikar V, Sacks FM. Effects of postmenopausal estrogen replacement on the concentrations and metabolism of plasma lipoproteins. N Engl J Med. 1991;325:1196-1204.[Abstract]
  3. Veldhuis JD, Gwyne JT. Estrogen regulates low density lipoprotein metabolism by cultured swine granulose cells. Endocrinology. 1985;117:1321-1327.[Abstract]
  4. Grady D, Rubin SM, Petitti DB, Fox CS, Black D, Ettinger B, Ernster VL, Cummings SR. Hormone therapy to prevent disease and prolong life in postmenopausal women. Ann Intern Med. 1992;117:1016-1037.
  5. Stevenson JC, Crook D, Godsland IF, Collins P, Whitehead MI. Hormone replacement therapy and the cardiovascular system: nonlipid effects. Drugs. 1994;47(suppl 2):35-41.
  6. Losordo DW, Kearney M, Kim EA, Jekanowski J, Isner JM. Variable expression of the estrogen receptor in normal and atherosclerotic coronary arteries of premenopausal women. Circulation. 1994;89:1501-1510.[Abstract/Free Full Text]
  7. Gisclard V, Miller VM, Vanhoutte PM. Effect of 17-beta estradiol on endothelium-dependent responses in the rabbit. J Pharmacol Exp Ther. 1988;244:19-22.[Abstract/Free Full Text]
  8. Kauser K, Sonnenberg D, Henrichmann F, Rubanyi GM. 17-Beta estradiol inhibits IL-1-beta induced suppression of contractile reactivity in isolated rat thoracic aorta. Circulation. 1994;90(suppl I):I-577. Abstract.
  9. Gehard MD, Roddy MA, Knab ST, Shelly J, Creager SJ, Creager MA. Endothelium-dependent vasodilation in postmenopausal women. Circulation. 1994;90(suppl I):I-86. Abstract.
  10. Reis SE, Gloth ST, Blumenthal RS, Resar JR, Zacur HA, Gerstenblith G, Brinker JA. Ethinyl estradiol acutely attenuates abnormal coronary vasomotor responses to acetylcholine in postmenopausal women. Circulation. 1994;89:52-60.[Abstract/Free Full Text]
  11. Touchette N. Man bites dogma: a new role for steroid hormones. J NIH. 1990;2:71.
  12. Mendelsohn ME, Karas RH. Estrogen and the blood vessel wall. Curr Opin Cardiol. 1994;9:619-626.[Medline] [Order article via Infotrieve]
  13. Gaulin-Glaser TL, Sessa W, Sarrel P, Bender J. The effect of 17-beta estradiol on human endothelial cell nitric oxide production. Circulation. 1994;90(suppl I):I-30. Abstract.
  14. Muck AO, Seeger H, Korte K, Lippert TH. The effect of 17-beta estradiol and endothelin-1 on prostacyclin and thromboxane production in human endothelial cell cultures. Clin Exp Obstet Gynecol. 1993;20:203-206.[Medline] [Order article via Infotrieve]
  15. Fuqua SAW, Chamness GC, McGuire WL. Estrogen receptor mutations in breast cancer. J Cell Biochem. 1993;51:135-139.[Medline] [Order article via Infotrieve]
  16. Karas RH, Baur WE, Mendelsohn ME. Human vascular smooth muscle cells express a novel estrogen receptor isoform. Circulation. 1994;90(suppl I):I-462. Abstract.
  17. Skipper JK, Young LJ, Bergen JM, Tetzlaff MT, Osborn CT, Crews D. Identification of an isoform of the estrogen receptor nessenger RNA lacking exon four and present in the brain. Proc Natl Acad Sci U S A. 1993;90:7172-7175.[Abstract/Free Full Text]
  18. Schwabe JWR, Chapman L, Finch JT, Rhodes D. The crystal structure of the estrogen receptor DNA-binding domain bound to DNA: how receptors discriminate between their response elements. Cell. 1993;75:567-568.[Medline] [Order article via Infotrieve]
  19. Karas RH, Patterson BL, Mendelsohn ME. Human vascular smooth muscle cells contain functional estrogen receptor. Circulation. 1994;89:1943-1950.[Abstract/Free Full Text]
  20. Colburn P, Buonassisi V. Estrogen-binding sites in endothelial cell cultures. Science. 1978;201:817-819.[Abstract/Free Full Text]
  21. Alexander JJ, Hoenig M, Graham D, Imbembo A. Effect of estradiol on low density lipoprotein uptake by bovine aortic endothelial cells. J Surg Res. 1989;46:537-542.[Medline] [Order article via Infotrieve]
  22. Kooistra T, Bosma PJ, Jespersen J, Kluft C. Studies on the mechanism of action of oral contraceptives with regard to fibrinolytic variables. Am J Obstet Gynecol. 1990;163:404-413.[Medline] [Order article via Infotrieve]
  23. Rhode PR, Gorski J. Growth and cell cycle regulation of mRNA levels in GH3 cells. Mol Cell Endocrinol. 1991;82:1-9.[Medline] [Order article via Infotrieve]
  24. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227:680-685.[Medline] [Order article via Infotrieve]
  25. Ausubel FM, Brent R, Kingston R, Moore D, Seidman JG, Smith J, Struhl K, eds. Current Protocols in Molecular Biology. Vol 1. New York, NY: John Wiley & Sons; 1994:4.1.2-4.10.10.
  26. Walter P, Green S, Greene G, Krust A, Bornert JM, Jeltisch JM, Staub A, Jensen E, Scrace G, Waterfield M, Chambon P. Cloning of the human estrogen receptor cDNA. Proc Natl Acad Sci U S A. 1985;82:7889-7893.[Abstract/Free Full Text]
  27. Ausubel FM, Brent R, Kingston R, Moore D, Seidman JG, Smith J, Struhl K, eds. Current Protocols in Molecular Biology. Vol 1. New York, NY: John Wiley & Sons; 1994:4.9.2.
  28. Borras M, Hardy L, Lempereur F, ElKhissiin AH, Legros N, Gol-Winkler R, Leclercq G. Estradiol-induced down-regulation of estrogen receptor: effect of various modulators of protein synthesis and expression. J Steroid Biochem Mol Biol. 1994;48:325-336.[Medline] [Order article via Infotrieve]
  29. Gebara OCE, Mittleman MA, Sutherland P, Lipinska I, Matheney T, Xu P, Welty FK, Wilson PWF, Levy D, Muller JE, Tofler GH. Association between increased estrogen status and increased fibrinolytic potential in the Framingham Offspring study. Circulation. 1995;91:1952-1958.[Abstract/Free Full Text]
  30. Morales DE, McGowan KA, Grant DS, Maheshwari S, Bhartiya D, Cid MC, Kleinman HK, Schnaper HW. Estrogen promotes angiogenic activity in human umbilical vein endothelial cells in vitro and in a murine model. Circulation. 1995;91:755-763.[Abstract/Free Full Text]
  31. King WJ, Greene GL. Monoclonal antibodies localize estrogen receptor in the nuclei of target cells. Nature. 1984;307:745-749.[Medline] [Order article via Infotrieve]
  32. Welshons WV, Lieberman ME, Gorski J. Nuclear localization of unoccupied oestrogen receptors. Nature. 1984;307:747-749.[Medline] [Order article via Infotrieve]
  33. Greene GL, Sobel NB, King WJ, Jensen EV. Immunochemical studies of estrogen receptors. J Steroid Biochem. 1984;20:51-56.[Medline] [Order article via Infotrieve]
  34. Saati TA, Clamens S, Cohen-Knafo E, Faye JC, Prats H, Coindre JM, Wafflart J, Caveriviere P, Bayard F, Delsol G. Production of monoclonal antibodies to human estrogen-receptor protein (ER) using recombinant ER (RER). Int J Cancer. 1993;55:651-654.[Medline] [Order article via Infotrieve]
  35. Reese JC, Katzenellenbogen BS. Differential DNA-binding abilities of estrogen receptor occupied with two classes of antiestrogens: studies using human estrogen receptor overexpressed in mammalian cells. Nucleic Acids Res. 1991;19:6595-6602.[Abstract/Free Full Text]
  36. Nardulli AM, Greene GL, Shapiro DJ. Human estrogen receptor bound to an estrogen response element bends DNA. Mol Endocrinol. 1993;7:331-340.[Abstract]
  37. Curtis SW, Korach KS. Uterine estrogen receptor-DNA complexes: effects of different ERE sequences, ligands and receptor forms. Mol Endocrinol. 1990;4:276-286.[Abstract]
  38. Brown M, Sharp PA. Human estrogen receptor forms multiple protein-DNA complexes. J Biol Chem. 1990;265:11238-11243.[Abstract/Free Full Text]
  39. Chambraud B, Berry M, Redeuiln G, Chambon P, Baulieu EE. Several regions of human estrogen receptor are involved in the formation of receptor-heat shock protein 90 complexes. J Biol Chem. 1990;265:20686-20691.[Abstract/Free Full Text]
  40. Gaub MP, Bellard M, Scheuer I, Chambon P, Sassone-Corsi P. Activation of the ovalbumin gene by the estrogen receptor involves the Fos-Jun complex. Cell. 1990;63:1267-1276.[Medline] [Order article via Infotrieve]
  41. Mukherjee R, Chambon P. A single-stranded DNA-binding protein promotes the binding of the purified estrogen receptor to its responsive element. Nucleic Acids Res. 1990;18:5713-5716.[Abstract/Free Full Text]
  42. Cogan JG, Sun S, Stoflet ES, Schmidt LJ, Getz MJ, Strauch AR. Plasticity of vascular smooth muscle a-actin gene transcription. J Biol Chem. 1995;270:11310-11321.[Abstract/Free Full Text]
  43. Onate SA, Tsai SY, Tsai MJ, O'Malley BW. Sequence and characterization of a coactivator for the steroid hormone receptor family. Science. 1995;270:1354-1357.[Abstract/Free Full Text]
  44. Curtis SW, Korach KS. Uterine estrogen receptor interaction with estrogen-responsive DNA sequences in vitro: effects of ligand binding on receptor-DNA complexes. Mol Endocrinol. 1990;280:276-286.
  45. Murdoch FE, Meier DA, Furlow D, Grunwald KAA, Gorski J. Estrogen receptor binding to a DNA response element in vitro is not dependent upon estradiol. Biochemistry. 1990;29:8377-8385.[Medline] [Order article via Infotrieve]
  46. Gilbert DM, Losson R, Chambon P. Ligand dependence of estrogen receptor induced changes in chromatin structure. Nucleic Acids Res. 1992;20:4525-4531.[Abstract/Free Full Text]
  47. Murdoch FE, Gorski J. The role of ligand in estrogen receptor regulation of gene expression. Mol Cell Endocrinol. 1991;78:C103-C108.[Medline] [Order article via Infotrieve]
  48. Chen GA, Feng Q, Zhang LZ, Liu YX. Plasminogen activators and PAI-1 in human endometrium. Acta Physiol Sinica. 1992;44:502-509.
  49. Yamashita J, Inada K, Yamashita S, Matsuo S, Nakashima Y, Ogawa M. Specific stimulation by estradiol on tissue-type plasminogen activator production in 7,12-dimethylbenz(a)anthracene-induced rat mammary turmor cells. Horm Metab Res. 1992;24:565-569.[Medline] [Order article via Infotrieve]
  50. Mira-y-Lopez R, Ossowski L. Hormonal modulation of plasminogen activator: an approach to prediction of human breast tumor responsiveness. Cancer Res. 1987;47:3558-3564.[Abstract/Free Full Text]
  51. Kjaeldgaard A, Larsson B, Astedt B. Estrogen regulation of tissue plasminogen activator in a human melanoma cell line. Thromb Res. 1986;42:397-406.[Medline] [Order article via Infotrieve]
  52. Gordon MS, Chin WW, Shupnik MA. Regulation of angiotensinogen gene expression by estrogen. J Hypertens. 1992;10:361-366.[Medline] [Order article via Infotrieve]
  53. Kifor I, Dzau V. Endothelial renin-angiotensin pathway: evidence for intracellular synthesis and secretion of angiotensin. Circulation. 1987;60:422-428.
  54. Weiner CP, Lizasoain I, Baylis SA, Knowles RG, Charles IG, Moncada S. Induction of calcium-dependent nitric oxide synthases by sex hormones. Proc Natl Acad Sci U S A. 1994;91:5212-5216.[Abstract/Free Full Text]
  55. Cid MC, Kleinman HK, Grant DS, Schnaper HW, Fauci AS, Hoffman GS. Estradiol enhances leukocyte binding to tumor necrosis factor (TNF)-stimulated endothelial cells via an increase in TNF-induced adhesion molecules E-selectin, intercellular adhesion molecule type 1 and vascular cell adhesion molecule type 1. J Clin Invest. 1994;93:17-25.
  56. Umayahara Y, Kawamori R, Watada H, Imano E, Iwama N, Morishima T, Yamasaki Y, Kajimoto Y, Kamada T. Estrogen regulation of the insulin-like growth factor I gene transcription involves an AP-1 enhancer. J Biol Chem. 1994;269:16433-16442.[Abstract/Free Full Text]
  57. Hyder SM, Stancel GM. In vitro interaction of uterine estrogen receptor with the estrogen response element present in the 3'-flanking region of the murine c-fos protooncogene. J Steroid Biochem Mol Biol. 1994;48:69-79.[Medline] [Order article via Infotrieve]
  58. Shyamala G, Guiot MC. Activation of kappa B-specific proteins by estradiol. Proc Natl Acad Sci U S A. 1992;89:10628-10632.[Abstract/Free Full Text]
  59. Karas RH, Baur WE, Mendelsohn ME. Crosstalk between growth regulatory and estrogen signaling pathways in human vascular smooth muscle cells. Circulation. 1994;90(suppl I):I-291.
  60. Gips SJ, Goldschmict-Clermont PJ. Short term exposure to 17beta-estradiol stimulates tyrosine phosphorylation of pp125 focal adhesion kinase in cultured endothelial cells. Circulation. 1994;90(suppl I):I-87. Abstract.
  61. Ignar-Trowbridge DM, Nelson KG, Bidwell MC, Curtis SW, Washburn TF, McLachlan JA, Korach KS. Coupling of dual signaling pathways: epidermal growth factor action involves the estrogen receptor. Proc Natl Acad Sci U S A. 1992;89:4658-4662.[Abstract/Free Full Text]
  62. Cho H, Katzenellenbogen B. Synergistic activation of estrogen receptor-mediated transcription by estradiol and protein kinase activators. Mol Endocrinol. 1993;7:441-452.[Abstract]
  63. Uehara K, Matsubara S, Kadomatsu K, Tsutsui J, Muramatsu T. Genomic structure of human midkine (MK), a retinoic acid-responsive growth/differentiation factor. J Biol Chem. 1992;111:563-567.
  64. Miyahara K, Kawamoto T, Sase K, Yui Y, Toda K, Yang LX, Hattori R, Aoyama T, Yamamoto Y, Doi Y, Ogoshi S, Hashimoto K, Kawai C, Sasayama S, Shizuta Y. Cloning and structural characterization of the human endothelial nitric-oxide-synthase gene. Eur J Biochem. 1994;223:719-726.[Medline] [Order article via Infotrieve]



This article has been cited by other articles:


Home page
 Arterioscler. Thromb. Vasc. Bio.Home page
M. E. Kleinman, M. R. Greives, S. S. Churgin, K. M. Blechman, E. I. Chang, D. J. Ceradini, O. M. Tepper, and G. C. Gurtner
Hypoxia-Induced Mediators of Stem/Progenitor Cell Trafficking Are Increased in Children With Hemangioma
Arterioscler. Thromb. Vasc. Biol., December 1, 2007; 27(12): 2664 - 2670.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
D. Sun, C. Yan, A. Jacobson, H. Jiang, M. A. Carroll, and A. Huang
Contribution of epoxyeicosatrienoic acids to flow-induced dilation in arteries of male ER{alpha} knockout mice: role of aromatase
Am J Physiol Regulatory Integrative Comp Physiol, September 1, 2007; 293(3): R1239 - R1246.
[Abstract] [Full Text] [PDF]

Home page
 Arterioscler. Thromb. Vasc. Bio.Home page
Y. Yuan and J. Xu
Loss-of-Function Deletion of the Steroid Receptor Coactivator-1 Gene in Mice Reduces Estrogen Effect on the Vascular Injury Response
Arterioscler. Thromb. Vasc. Biol., July 1, 2007; 27(7): 1521 - 1527.
[Abstract] [Full Text] [PDF]

Home page
 Eur J EndocrinolHome page
M Alevizaki, K Saltiki, A Cimponeriu, I Kanakakis, N Xita, C C Alevizaki, I Georgiou, and H-L Sarika
Severity of cardiovascular disease in postmenopausal women: associations with common estrogen receptor {alpha} polymorphic variants
Eur. J. Endocrinol., April 1, 2007; 156(4): 489 - 496.
[Abstract] [Full Text] [PDF]

Home page
 CirculationHome page
H. Hamada, M. K. Kim, A. Iwakura, M. Ii, T. Thorne, G. Qin, J. Asai, Y. Tsutsumi, H. Sekiguchi, M. Silver, et al.
Estrogen Receptors {alpha} and {beta} Mediate Contribution of Bone Marrow-Derived Endothelial Progenitor Cells to Functional Recovery After Myocardial Infarction
Circulation, November 21, 2006; 114(21): 2261 - 2270.
[Abstract] [Full Text] [PDF]

Home page
 Endocr. Rev.Home page
J. L. Turgeon, M. C. Carr, P. M. Maki, M. E. Mendelsohn, and P. M. Wise
Complex Actions of Sex Steroids in Adipose Tissue, the Cardiovascular System, and Brain: Insights from Basic Science and Clinical Studies
Endocr. Rev., October 1, 2006; 27(6): 575 - 605.
[Abstract] [Full Text] [PDF]

Home page
 Arterioscler. Thromb. Vasc. Bio.Home page
C. Bolego, E. Vegeto, C. Pinna, A. Maggi, and A. Cignarella
Selective Agonists of Estrogen Receptor Isoforms: New Perspectives for Cardiovascular Disease
Arterioscler. Thromb. Vasc. Biol., October 1, 2006; 26(10): 2192 - 2199.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S. Joy, R. C. M. Siow, D. J. Rowlands, M. Becker, A. W. Wyatt, P. I. Aaronson, C. W. Coen, I. Kallo, R. Jacob, and G. E. Mann
The Isoflavone Equol Mediates Rapid Vascular Relaxation: Ca2+-INDEPENDENT ACTIVATION OF ENDOTHELIAL NITRIC-OXIDE SYNTHASE/Hsp90 INVOLVING ERK1/2 AND Akt PHOSPHORYLATION IN HUMAN ENDOTHELIAL CELL
J. Biol. Chem., September 15, 2006; 281(37): 27335 - 27345.
[Abstract] [Full Text] [PDF]

Home page
 J EndocrinolHome page
A. D.-Y. Lin, A. Mannikarottu, B. A Kogan, C. Whitbeck, P. Chichester, R. E Leggett, and R. M Levin
Estrogen induces angiogenesis of the female rabbit bladder.
J. Endocrinol., August 1, 2006; 190(2): 241 - 246.
[Abstract] [Full Text] [PDF]

Home page
 CirculationHome page
A. Iwakura, S. Shastry, C. Luedemann, H. Hamada, A. Kawamoto, R. Kishore, Y. Zhu, G. Qin, M. Silver, T. Thorne, et al.
Estradiol Enhances Recovery After Myocardial Infarction by Augmenting Incorporation of Bone Marrow-Derived Endothelial Progenitor Cells Into Sites of Ischemia-Induced Neovascularization via Endothelial Nitric Oxide Synthase-Mediated Activation of Matrix Metalloproteinase-9
Circulation, March 28, 2006; 113(12): 1605 - 1614.
[Abstract] [Full Text] [PDF]

Home page
 J. Physiol.Home page
M. J. Byers, A. Zangl, T. M. Phernetton, G. Lopez, D.-b. Chen, and R. R. Magness
Endothelial vasodilator production by ovine uterine and systemic arteries: ovarian steroid and pregnancy control of ER{alpha} and ER{beta} levels
J. Physiol., May 15, 2005; 565(1): 85 - 99.
[Abstract] [Full Text] [PDF]

Home page
 J. Lipid Res.Home page
L. H. Smith, M. S. Petrie, J. D. Morrow, J. A. Oates, and D. E. Vaughan
The sterol response element binding protein regulates cyclooxygenase-2 gene expression in endothelial cells
J. Lipid Res., May 1, 2005; 46(5): 862 - 871.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
C. M. Klinge, K. A. Blankenship, K. E. Risinger, S. Bhatnagar, E. L. Noisin, W. K. Sumanasekera, L. Zhao, D. M. Brey, and R. S. Keynton
Resveratrol and Estradiol Rapidly Activate MAPK Signaling through Estrogen Receptors {alpha} and {beta} in Endothelial Cells
J. Biol. Chem., March 4, 2005; 280(9): 7460 - 7468.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
W. X. Liao, R. R. Magness, and D.-b. Chen
Expression of Estrogen Receptors-{alpha} and -{beta} in the Pregnant Ovine Uterine Artery Endothelial Cells In Vivo and In Vitro
Biol Reprod, March 1, 2005; 72(3): 530 - 537.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
Q. Lu, D. C. Pallas, H. K. Surks, W. E. Baur, M. E. Mendelsohn, and R. H. Karas
Striatin assembles a membrane signaling complex necessary for rapid, nongenomic activation of endothelial NO synthase by estrogen receptor {alpha}
PNAS, December 7, 2004; 101(49): 17126 - 17131.
[Abstract] [Full Text] [PDF]

Home page
 Exp. Biol. Med.Home page
P. S. Cooke and A. Naaz
Role of Estrogens in Adipocyte Development and Function
Experimental Biology and Medicine, December 1, 2004; 229(11): 1127 - 1135.
[Abstract] [Full Text] [PDF]

Home page