Journal of Applied Physiology Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

J Appl Physiol 92: 1064-1073, 2002. First published November 2, 2001; doi:10.1152/japplphysiol.00374.2001
8750-7587/02 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow All Versions of this Article:
92/3/1064    most recent
00374.2001v1
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
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 ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (12)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Villablanca, A. C.
Right arrow Articles by Rutledge, J. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Villablanca, A. C.
Right arrow Articles by Rutledge, J. C.
Vol. 92, Issue 3, 1064-1073, March 2002

Time- and dose-dependent differential upregulation of three genes by 17beta -estradiol in endothelial cells

Amparo C. Villablanca1, Kristine A. Lewis1, and John C. Rutledge2

1 Division of Cardiovascular Medicine and 2 Division of Endocrinology, Nutrition, and Vascular Medicine, University of California, Davis, California 95616-8636


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The purpose of this study was to identify genetic targets in the vasculature for estrogen by profiling genes expressed in female human aortic endothelial cells exposed to various doses of 17beta -estradiol at differing concentrations and for differing periods of time. Our approach employed a RT-PCR-based cloning strategy of DNA differential display analysis, with differential expression verified by semiquantitative PCR performed with gene-specific primers. A significant increase in mRNA expression in response to 17beta -estradiol was observed for the following three genes: aldose reductase (3.4-fold), caspase homologue-alpha protein (4.2-fold), and plasminogen activator inhibitor-1 intron e (2.3-fold). For all three upregulated genes, estradiol-induced upregulation occurred with a similar time course and temporally clustered to the first 24 h after hormone treatment. In addition, the effect of estradiol dose on gene expression was consistent and occurred at physiological concentrations. Our results describe previously uncharacterized estradiol-sensitive time- and dose-dependent regulation of genes with potential importance to vascular function in human endothelial cells.

estrogen; estradiol; endothelial; cell culture; gene expression; differential display; vascular; RT-PCR; mRNA; aldose reductase; caspase homologue; plasminogen activator inhibitor-1


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ESTROGEN IS AN IMPORTANT FEMALE sex steroid hormone with key actions on both physiological and pathophysiological conditions, including reproduction, neoplasia, and atherosclerosis. In addition, estrogens are increasingly appreciated as complex, powerful cardiovascular mediators with multifaceted mechanisms of action on vascular tissue (17, 48). The vasculature responds to estrogen via a number of mechanisms, including vasodilatation via increased production of nitric oxide, a potent vasodilator (15, 48), favorable effects on lipoprotein metabolism and cholesterol accumulation (16, 26), and regulation of apoptotic cell death (2, 44).

In endothelial cells, the most important physiologically relevant estrogen, 17beta -estradiol, regulates the expression of several genes having direct or potential relevance to vascular function, including endothelin-1 (6), nitric oxide synthase (20, 22, 29), vascular cell adhesion molecules, including VCAM-1 (9, 41, 43), angiotensinogen (19), cyclooxygenase-1 (jun), vertebrate trp genes (a family of channel proteins that form the structural basis for Ca2+ influx through the capacitative Ca2+ entry pathway) (10), 27-kDa heat-shock protein (36), and the substance P receptor (46). Furthermore, reports in which a variety of experimental systems were used indicate that enhanced estrogen production, such as that seen in ovarian hyperstimulation, is a potent signal in regulation of gene expression for vascular endothelial growth factor/vascular permeability factor (11, 33). Thus estradiol may be an essential trigger for significant genetic reprogramming of the vessel wall with selective gene induction and/or downregulation. Therefore, it is highly probable that the expression of many other genes that have not yet been reported to be regulated by female sex steroid hormones are regulated by estradiol in the vasculature.

The purpose of this study was to better understand the molecular actions of estradiol on vascular gene expression. Review of other systems that investigated hormonal regulation of gene expression demonstrated that exposure time and estradiol dose may be important factors in gene expression regulation. Therefore, we reasoned that, to better understand the physiological relevance of our findings and glean insight into potential mechanisms, an understanding of the time and dose dependency of differential gene expression by estradiol would be important. We also reasoned that, if cells were synchronized to growth quiescence so that the influences of differences in cell cycle were eliminated, new knowledge of the action of estradiol on endothelial cell gene expression would emerge. We therefore hypothesized that, in human aortic endothelial cells, estradiol induces vascular gene expression that is time and estradiol dose dependent, occurring at physiologically significant concentrations and clustering temporally.

Our experimental approach used the technique of mRNA gene display (27) to detect differentially expressed genes in response to 17beta -estradiol in normal human aortic endothelial cells. This strategy enabled us to identify genes that were differentially up- or downregulated by estradiol. Our results demonstrated that in human aortic endothelial cells estradiol treatment resulted in time- and dose-dependent upregulation of a number of genes important to apoptotic cell death, smooth muscle cell growth, and regulation of cellular glucose influx.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. For differential display, materials were obtained from the following sources: superscript II RNase human Moloney murine leukemia virus reverse transcriptase, 5× first strand buffer, 10 mM dNTP mix, and 0.1 M DTT from GIBCO BRL (Gaithersburg, MD); delta RNA fingerprinting kit containing oligo(dT) primer, PCR primers for RNA fingerprinting, and KlenTaq Taq polymerase from Clontech (Palo Alto, CA); acryl/bis-acrylamide (19:1) from Bio-Rad (Hercules, CA); temed, ammonium persulfate, and urea from Sigma Chemical (St. Louis, MO); [alpha -35S]dATP Easytide, 1,250 Ci/mmol, from New England Nuclear (Boston, MA); and Tris borate EDTA buffer components from Fisher Scientific (Fairlawn, NJ).

For reamplification, cloning and sequencing materials and reagents were obtained from the following sources: AmpliTaq Gold from Perkin-Elmer (Foster City, CA); T7 promoter and M13 reverse primers from Midland Certified Reagent (Midland, TX); original TA cloning kit containing 10× ligation buffer, T4 DNA ligase, SOC medium, beta -mercaptoethanol, INValpha F' E. coli competent cells, and pCR 2.1 vector from Invitrogen (Carlsbad, CA); Luria-Bertani medium and agar from the media room at University of California, Davis; ampicillin from Apothecon (Princeton, NJ); X-Gal from Boehringer Mannheim (Indianapolis, IN) in dimethylformamide; KH2PO4 and K2HPO4 from Fisher Scientific; and terrific broth containing tryptone, yeast extract, and glycerol from Difco (Detroit, MI).

Overview of experimental design and experimental approach. To identify genes whose expression was differentially regulated by 17beta -estradiol in human aortic endothelial cells and to determine the time- and dose-response pattern of differential gene expression, we incubated endothelial cells with estradiol, or vehicle control, at a range of exposure times and estradiol concentrations. Total RNA was then extracted from the cells, reversed transcribed using a total of four arbitrary 25- and 26-mer oligonucleotide primers in combination with three oligo(dT) primers, and then subjected to mRNA differential display analysis. Hundreds of gene transcripts were screened for differential expression among the two cell groups. After amplification, the products were analyzed on sequencing gels, and those bands that appeared to be differentially expressed were recovered, reamplified, cloned, sequenced, and identified using GenBank searches. Only cDNAs whose sequences corresponded to genes with potential physiological relevance to vascular function were selected for further verification. Finally, to verify differential expression of apparently differentially displayed cDNAs, their initial reverse transcription products were analyzed by semiquantitative RT-PCR using gene-specific primers.

Cell culture and estradiol treatment. Human endothelial cells from autopsy specimens of normal female adult aortas were obtained from Clonetics (San Diego, CA) at passage 4. The cells were derived from the same individual and not pooled from a variety of donors. Cells were grown in monolayer culture in endothelial growth media (EGM, a modified MCDB 131 formulation) supplemented with human recombinant epidermal growth factor (10 ng/ml), hydrocortisone (1 µg/ml), bovine brain extract (12 µg/ml), 2% fetal bovine serum (Hyclone), plus antibiotics (50 µg/ml gentamicin and 50 ng/ml amphotericin B) in 75-cm2 plastic tissue culture flasks (Fisher) in a humidified atmosphere of 5% CO2 in 95% air at 37°C. Cells were passaged weekly using trypsin (0.025%) and EDTA (0.01%), and the cell suspension was neutralized with HEPES balanced salt solution.

For experiments, cells were plated in fresh EGM in 35-mm plastic tissue culture dishes at 50,000 cells/dish and grown for 48 h to preconfluence, at which point the cell concentration per dish was ~1 × 106. Cells were then serum deprived for 19-24 h by incubating them with EGM devoid of FBS. Serum deprivation was used to synchronize all of the cells to growth quiescence so that any variability in cell-cycle-dependent gene expression would be minimized and equalized among the cultured cell population. Immediately before each experiment, the serum-free medium was removed from the wells, cells were washed with PBS, and 17beta -estradiol (prepared in ethanol; final concentration of ethanol in the wells of <0.01%) was added to the cells at various concentrations.

To determine whether gene expression was dependent on the concentration of estradiol and to examine the effect of a broad range of estradiol concentrations on gene expression, the cells were exposed to 1, 10, 100, and 1,000 nM estradiol in EGM containing low-estrogen serum. The low-estrogen medium contained 10% serum stripped of endogenous hormones by a 30-min incubation at 45°C with a vol/vol dextran-coated charcoal pellet solution (0.25% Norit A, 0.0025% dextran T-70 in 0.01 M Tris · HCl, pH 8.0, at 4°C), as previously described (7). This resulted in serum estradiol concentrations of 2-3 pg/ml as determined by radioimmunoassay. To determine whether there was a time dependence to changes in gene expression, the cells were incubated with estradiol for a broad range of incubation periods: 30 min and 1, 2, 4, and 24 h. Subsequently, the medium was removed from the dishes, and total RNA was immediately extracted from the cells. Identically prepared but hormone-untreated cells incubated with vehicle were used for controls.

RNA isolation. Total RNA was isolated from experimental and control cell dishes (one RNA sample per dish) using Tri-Reagent (Molecular Research, Cincinnati, OH), per the manufacturer's instructions. Each dish of endothelial cells yielded ~4-5 µg of RNA, the predicted yield amount for 106 cells per the manufacturer's estimates. The integrity of RNA with this method has been repeatedly ascertained in our laboratory and was confirmed by formaldehyde gel electrophoresis. DNase I treatment for contaminating genomic DNA was not necessary in accordance with the manufacturer's protocol and our prior experience with these techniques. Total RNA yields were determined by ultraviolet spectrophotometry (Uvikon), and samples with 260/280 nm optical density intensities of >1.5:1 were used. All labware and solutions used to isolate RNA were rendered RNase free before use. RNA samples were stored in Tris-EDTA buffer at -80°C until ready for use.

RT-PCR differential mRNA fingerprinting. The technique of Delta reverse transcription PCR fingerprinting (Clontech) is based on reverse transcription using oligo(dT) as a primer and amplification of 3'-terminal RNA sequences (5, 27). The method used in this study relies on a combination of thermophilic DNA polymerases with 3'-5' exonuclease (proofreading) activity to produce larger, easier to detect, higher fidelity, and not overcycled PCR products than possible with conventional PCR (32) to minimizing false-positive results and optimize sensitivity. For fingerprinting, only a single cDNA synthesis reaction is needed for each RNA sample. Samples of total RNA (2 µg total RNA per sample) were prepared in parallel from each of the estradiol-treated and untreated endothelial cell dishes, including appropriate positive RNA controls; these were then used as a template for reverse transcription to cDNA before differential display was performed. RNA was converted into first-strand cDNA using oligo(dT) primer and superscript II reverse transcriptase. Each 2 µg of total RNA sample yielded ~350 ng of full-length single-strand cDNA.

A series of combinations of arbitrary "P" primers and oligo(dT) or "T" primers provided in the DNA fingerprinting kit (Clontech) were used for differential display analysis. These long (25-nucleotide P primers and 29-nucleotide T primers) primer pair combinations will generally produce PCR products derived from the 3' end of the mRNA, vs. internal mRNA regions, and typically produce one or two differentially expressed bands per primer pair (up to 90 pair combinations possible). In the presence of radiolabeled nucleotide, sequences are amplified on the basis of chance homology to long arbitrary P primers. The primers have been designed to allow for higher stringency PCR, which in turn leads to greater reproducibility and yield of the resulting fingerprints. Each pair of primers used will produce a different fingerprint and may identify a different set of differentially expressed RNAs. We began with five pairs of P/T primers: P1/T4, P3/T5, P10/T4, P10/T5, and P8/T8. In our hands, two sets of these primer pairs, P1/T4 and P10/T5, yielded many differentially displayed bands. Therefore, the majority of fingerprinting experiments used these two sets of primer pairs. The primer sequences were as follows: 5'-ATT AAC CCT CAC TAA ATG CTGG GGA-3 for P1; 5'-CAT TAT GCT GAG TGA TAT CTT TTT TTT TCA-3' for T4; 5'-ATT AAC CCT CAC TAA AGC ACC GTC C-3' for P10; and 5'-CAT TAT GCT GAG TGA TAT CTT TTT TTT TCC-3' for T5.

Additional combinations of up to two T primers and three P primers (for a total of six fingerprints with each template) were used as needed.

Two dilutions of cDNA product from the reverse-transcribed RNA samples (1:10 dilution of RT cDNA product = 3.5 ng cDNA and 1:40 dilution of RT cDNA product = 0.875 ng cDNA), a negative H2O control, one total RNA control, and two positive control cDNAs previously established to produce a band with the P and T primer were amplified using P and T primers in the presence of MgCl2 (1.5 mM), KlenTaq Taq polymerase mix (Clontech), and 3.75 µCi [alpha -35S]dATP. The KlenTaq polymerase mix contains as the primary polymerase a combination of thermophilic DNA polymerases for long-distance PCR, which produces larger, easier to detect, higher fidelity, and not overcycled PCR products than possible with conventional PCR (21). In addition, the presence of a minor amount of a secondary DNA polymerase with 3' to 5' exonuclease (proofreading) activity was used to increase the PCR accuracy. Also, a TaqStart antibody, included in the KlenTaq Taq polymerase mix (Clontech), was used to provide an automatic form of "hot start" PCR and reduce synthesis from nonspecifically bound primers during PCR by binding and inactivating Taq DNA polymerase (native or truncated). PCR of experimental samples was performed according to the manufacturer's protocol with standard PCR techniques (26) in a Perkin Elmer 480 thermal cycler; the following program, designed to increase annealing stringency (critical for reproducibility and low background fingerprinting), was used: 1 cycle at 94°C for 5 min, 40°C for 5 min, and 68°C for 5 min; then 2 cycles at 94°C for 2 min, 40°C for 5 min, and 68°C for 5 min for annealing; then 25 cycles at 94°C for 1 min, 60°C for 1 min, and 68°C for 2 min; and a final extension step at 68°C for 7 min. cDNA samples were resolved on 5% polyacrylamide denaturing gels in 0.5× TBE buffer. The gels were dried under vacuum before exposure to Kodak XOMAT-AR film at room temperature for at least 2 days.

Recovery and reamplification of cDNAs. Gel autoradiographs were carefully examined by two independent observers for evidence of differentially expressed bands. Bands identified as being differentially expressed were those present in control (estradiol-untreated endothelial cells) but absent in estradiol-treated samples or absent in control but present in estradiol-treated samples. Figure 1 demonstrates an example of a differentially expressed band following autoradiography. Differentially expressed bands were excised from the corresponding gels, and the cDNA was eluted. Each band was then reamplified by PCR using the original fingerprinting P and T primers. PCR was performed in a Perkin Elmer 2400 thermal cycler with the following program: 20 cycles at 94°C for 30 s, 60°C for 30 s, and 68°C for 2 min. Amplification was gel verified by electrophoresis.


View larger version (76K):
[in this window]
[in a new window]
  		Fig. 1.   Representative example of autoradiograph results from differential display (DD) analysis. RNA extracted from endothelial cells of control and estradiol-treated cells (E) at the various concentrations shown (1, 10, 100, and 1,000 nM) was subjected to reverse transcription with 1 of 9 possible oligo(dT) primers (T1-T9). cDNAs were then PCR amplified with the use of the corresponding oligo(dT) primer used in the reverse transcription reaction and various arbitrary oligonucleotide primers (P1-P10), and the products were used as a template in differential display reactions to generate endothelial-specific gene expression fingerprints. In the presence of 35S- radiolabeled dATP, sequences were amplified on the basis of chance homology to arbitrary primers. After amplification and autoradiography, products were analyzed on 6% polyacrylamide sequencing gels. Selected bands distinctly upregulated or downregulated in estradiol-treated lanes on the DD gels were excised, and the cDNA was extracted, reamplified, cloned into vectors, and sequenced to identify differentially expressed gene sequences, as described in METHODS. An example is shown of a portion of a sequencing gel in which apparent differential expression is seen (i.e., band is absent in control but present in all 4 estradiol treatment lanes). For comparison, the gel also demonstrates examples of multiple other bands that do not demonstrate apparent differential expression (i.e., present in both control and estradiol treatment). Stronger bands present across samples represent relatively abundant gene expression, and lighter bands represent relatively less abundant gene expression.

Cloning and sequencing of cDNAs. For cloning, we ligated the differentially expressed PCR products into a pCR 2.1 vector after transformation of One-Shot INValpha F' E. coli-competent cells (Invitrogen) by using a TA cloning kit (27, 30) and standard molecular cloning techniques (39). Approximately six positive colonies were picked per plate and grown in terrific broth medium. DNA was recovered from an aliquot of transformed cells, and the PCR product insert was reamplified by PCR using T7 promoter and M13 reverse primers designed to match the flanking pCR 2.1 vector sequences to the PCR insert. Amplification of the correct insert was once again verified by gel electrophoresis. Bands that were identified as differentially expressed on gels and could be successfully amplified and cloned were subsequently sequenced. For cases in which cut bands had multiple clones characterized and sequenced (different sequences of the same size comigrating on the gel), we verified that these clones represented different gene products by resequencing them with M13 reverse primer.

Before we performed sequencing, the PCR-amplified cloned product was purified in a Centricon-100 concentrator. Samples were prepared for sequencing using an ABI prism dye terminator cycle sequencing ready reaction kit with AmpliTaq DNA polymerase fluorescent sequencing (Perkin Elmer) per the manufacturer's instructions. Samples were run on an ABI prism 377 DNA sequencer on a 4.25% acryl/bisacrylamide gel (5% LongRanger gel). Data were analyzed using ABI prism sequencing 2.1.1 software. Sequence data were matched against GenBank databases for human nucleotide sequences and against protein and expressed sequence tag (EST) databases using FASTA (FastA) and NetBLAST (National Center for Biotechnology Information in Bethesda, MD). FastA (35) searches for similarities between one sequence (the query) and any group of sequences of the same type (nucleic acid or protein) as the query sequence. BLAST (basic local alignment search tool) uses heuristic algorithms for searching protein and nucleic acid databases for similarities to query sequences (1). Sequences with high homology (>75%) to identified physiologically important human mRNAs were chosen for subsequent verification of differential expression in the estradiol-treated and untreated human aortic endothelial cells. All gene matches had FastA e scores (34) (the probability of observing a score greater than or equal to the observed score purely by chance when doing a search) close to e-40, indicating identity to a known gene.

Verification of differential expression by semiquantitative PCR. The most direct way to verify differential expression of bands identified by delta RNA fingerprinting is to use the reamplified bands as probes on Northern blots of poly(A)+ RNA from the original RNA tissue sources. However, this method could not be used due to the small amount of total RNA obtained from the endothelial cells. Also, because the cloned fragments from differential display include the 3' end untranslated regions, they were not ideal for use as probes in quantifying mRNA. To overcome these problems, mRNAs identical to our known and unknown genes were analyzed by quantitative RT-PCR. The cDNAs prepared from the total RNA of the control and estradiol-treated endothelial cells by reverse transcription that were used for the cDNA fingerprint reactions were also used for verification of apparently differentially expressed sequences.

Genes were chosen for primer design and relative quantitation by PCR based on sequences corresponding to known genes that were of interest due to their potential physiological relevance to cardiovascular function. The program Primer3 (37) (available online at http://www.genome.wi.mit.edu/cgi-bin/primer/primer3.cgi) was used to design PCR primers for amplifying cDNAs corresponding to physiologically relevant differentially displayed mRNAs that were identified by cloning and sequencing. Primers were designed for amplifying D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA, a "housekeeping" gene, to control for the amount of total cDNA template. To ensure that the analyzed mRNA was indeed from the specific genes we identified in GenBank searches, we designed primer pairs that contained sequences within the unique coding region for each of the identified genes of interest, extending beyond our cloned fragment, verifying that they had similar GC content to the primers for control GAPDH cDNA. In addition, we verified that our primer pairs yielded PCR products with the exact length as predicted from the known sequences. Custom-designed primers used for semiquantitative PCR analysis of three genes of interest and GAPDH (Midland Certified Reagent) had sequences as follows. For (clone ea2) caspase homologue (CASH), right primer = 5'-TCC AGA AGT ACA AGC AGT CTG TTC-3' and left primer = 5'-AAA CTC TGC TGT TCC AAT CAT ACA-3'. For (clone 4) plasminogen activator inhibitor-1 (PAI-1), right primer = 5'-GTC CTT GGA AGT GAT TTC TTT TGT-3' and left primer = 5'-CCC AGG TTG AAT TTC CCA GAT-3'. For (clone 41) aldose reductase, right primer = 5'-ACA AAA GCA CTT TTT ATT TGA GGC-3' and left primer = 5'-GGT GGA GAT GAT CTT AAA CAA ACC-3'. For GADPH, right primer = 5'-CTC AGT GTA GCC CAG GAT GC-3' and left primer = 5'-ACC ACC ATG GAG AAG GCT GG-3'.

Briefly, equal amounts of the cDNAs prepared by reverse transcription from the original estradiol-treated and control RNA samples used for fingerprinting were diluted 1:10, 1:20, 1:40, and 1:80 (corresponding to ~3.5, 1.75, 0.88, and 0.44 ng of DNA, respectively) and subsequently amplified by PCR. The optimal conditions for amplification (the proportion between the two pairs of primers, temperatures, and cycle number) were experimentally determined by preliminary experiments as follows. First, to ensure that both the specific gene cDNA and the GAPDH cDNA were amplified at similar efficiencies, we determined the ratio of the pair of primers for the specific gene cDNA to the pair of primers for the GAPDH cDNA. Because the amount of each specific gene mRNA or cDNA was different, we next used the same molarity of primers and adjusted the cycle number and depended on the template dilutions to be sure that we were in exponential amplification for both GAPDH and our gene of interest. We performed preliminary studies to determine the optimal number of PCR cycles appropriate for optimal semiquantitative PCR analysis. This was done by identifying the number of PCR cycles that permitted detection of signals from both the specific gene mRNAs and the control GAPDH mRNA, by demonstrating that the amount of both products increased proportionately with the cycle number and the ratio between the two genes remained the same. In this manner, amplification was stopped before the signal for one or both cDNAs reached saturation. Last, by using sequential dilutions of DNA template for the specific genes and for GAPDH, we verified that relative differences in gene expression remained constant over a wide template concentration range.

To reduce spurious priming during gene amplification, PCR was performed for 10 "touchdown" cycles (32) in a Perkin Elmer 2400 thermal cycler as follows. The first touchdown cycle was for 30 s at 94°C, 30 s at 61°C for annealing, and 2 min at 72°C. The next touchdown cycle was 30 s at 94°C, 30 s at 60.9°C for annealing, and 2 min at 72°C. The remaining eight "touchdown" cycles dropped 0.1°C in the annealing temperature after each cycle. These touchdown cycles were followed by 10-15 cycles of 30 s at 94°C, 30 s at 60°C, and 2 min at 72°C. Samples were held for a final 7-min extension cycle at 72°C and then cooled to 4°C. Correct amplification was verified by gel electrophoresis of the PCR products.

Quantification of bands from verification gels was performed with spot densitometry (Alpha Innotech System). Densitometry values were obtained from the PCR product for each primer-targeted cDNA from estradiol-treated and control samples at each of the four cDNA template dilutions described above. To confirm the use of equal amounts of cDNA and to allow PCR products to be quantitated comparatively, PCR amplifications were also performed with GAPDH. This also permitted the spot densitometry values of the estradiol-treated and control PCR product signals to be normalized to the GAPDH PCR product signal, at corresponding dilutions, to obtain normalized densitometry ratios. Normalized ratio data were then evaluated for differential expression (i.e., greater or less than a twofold difference in expression in the estradiol-treated vs. control samples).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Differential display of mRNAs from estradiol-treated vs. control human aortic endothelial cells. A total of 44 cDNA fragments apparently differentially regulated by estradiol treatment were identified in the autoradiographs and isolated from the gels. From these, we were eventually able to clone and sequence 30 gene fragments. Seven of the genes (clones 37, 38, 39, 40, 43, 44, and d) were downregulated by estradiol (present in control but absent with estradiol) (see Table 1). Because differential display fingerprinting is not a quantitative tool, but is best utilized as an absent or present screening, it is not possible to state that gene expression for the downregulated genes was zero after estradiol treatment. However, there was no detectable gene expression; this was demonstrated by an apparent absence of a band in the treatment condition compared with the control condition, as determined by two observers. The downregulated differentially displayed genes were not further characterized in this study because they all corresponded to EST fragments with unknown function and could not be assessed further for potential functional relevance to vascular pathophysiology.

                              
View this table:
[in this window]
[in a new window]
  		Table 1.   Summary of estradiol-downregulated clones identified by differential display in human aortic endothelial cells

A greater number of the apparently differentially regulated genes, 23 genes, were upregulated by estradiol because they appeared only in estradiol-treated cells and not in vehicle-treated control cells. By using GenBank searches, 10 of the 23 upregulated genes were found to have significant homology or identity (80-100% homology) to previously identified genes and were therefore considered for further analysis (clones 4, 5, 7a, 7b, 16, 21, 22, 41, c, and ae2). As anticipated from GenBank searches, none of the upregulated clones represented clearly novel genes. However, several had short and poor matches to known genes or matches to ESTs of unknown function, potentially representing novel genes. The remaining genes had lesser degrees of homology to known genes and were therefore not considered for further study.

Confirmation of the differentially expressed genes by semiquantitative RT-PCR. We selected 5 of the 10 upregulated differentially expressed genes with significant homology to known genes for further analysis and confirmation of differential expression by semiquantitative PCR (clones 4, 7b, 16, 41, and ea2) (Table 2). Once again, the main criterion utilized to determine gene selection for verification was the potential relevance of the gene's known function(s) to a physiologically important or interesting vascular process (e.g., vascular cell growth, vascular permeability, vascular inflammation, and so forth). On the basis of this criterion, we opted to not verify expression for the other five significantly homologous upregulated differentially expressed genes (clones 5, 7a, 21, 22, and c). To confirm that the expression of the five selected genes was indeed upregulated by estradiol treatment, differential display results were confirmed by semiquantitative RT-PCR (34) with the use of sequence-specific primers, after we verified that the primer pairs yielded PCR products with the exact length as predicted from the known sequences (Table 3).

                              
View this table:
[in this window]
[in a new window]
  		Table 2.   Summary of estradiol-upregulated clones identified by differential display in human aortic endothelial cells


                              
View this table:
[in this window]
[in a new window]
  		Table 3.   Specific oligonucleotide primers used in this study for semiquantitative PCR verification

The densitometry signals for the estradiol-treated and control endothelial cell samples, expressed as a ratio for four DNA template dilutions, were normalized to the GAPDH signal for the same dilution, yielding normalized densitometry ratios. The data for the normalized ratios for the specific genes verified are summarized in Fig. 2.


View larger version (27K):
[in this window]
[in a new window]
  		Fig. 2.   RT-PCR verification of mRNA differential gene expression for specific genes of interest. Reverse-transcribed cDNAs for each of the specific genes of interest (clone 4, clone 41, and clone ea2; see text) were coamplified with target gene and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers. For each mRNA sample of the specific gene of interest, we then calculated the densitometry signal for control and estradiol-treated endothelial cell samples and expressed them as the multiples of increase in the densitometry signal of estradiol-treated to control human aortic endothelial cells for a series of cDNA template dilutions (1:10 to 1:40). Calculated values were normalized to the GAPDH signal. Shown are the densitometry ratios of estradiol-treated to control, normalized for the GAPDH PCR product for the specific genes verified: averaged over 2 template dilutions, 1:20 and 1:40 template dilution (A), and 1:40 template dilution (B). CASH, caspase homologue; PAI-1, plasminogen activator inhibitor-1.

We verified that mRNAs for three genes of interest were significantly differentially expressed and upregulated over control (i.e., absent in control endothelial cells and present in estradiol-treated endothelial cells): PAI-1 intron e (clone 4), a gene involved in thrombogenesis; aldose reductase (clone 41), the rate-limiting enzyme in the polyol pathway central to the control of cellular flux of glucose; and CASH-alpha (clone ea2), a CASH-alpha cysteine protease with cytocidal effects having a critical role in all known programmed cell death processes (35). Quantification of the relative mRNA amounts of all three verified clones, expressed as multiples of increase for the estradiol-treated vs. untreated endothelial cells, demonstrated that estradiol upregulated expression of these three genes by 2.3-fold, 3.4-fold, and 4.2-fold, respectively. Two of five genes of interest were verified not to be significantly differentially expressed because they demonstrated less than twofold greater expression when normalized to GAPDH (clones 7b and 16).

Time and dose effects of estradiol on endothelial cell gene expression. Analysis of the estradiol treatment conditions that resulted in differential gene expression for the apparently upregulated genes indicated that over 50% occurred in response to 100 nM estradiol and after 24 h of incubation with this sex steroid. An additional group of apparently upregulated genes appeared in response to a dose of 1 nM estradiol, also at the 24-h time point. A few genes demonstrated apparent upregulated expression at all of the estradiol concentrations; however, this tended to also occur primarily at the 24-h time point. Interestingly, we did not observe evidence of apparent differential expression at the 4-h incubation time point for any of the estradiol concentrations used and therefore were not able to further characterize differentially displayed genes for this time point. Thus estradiol-induced upregulation of the apparently differentially expressed genes was relatively rapid, within 24 h after hormone treatment, and dose dependent, most consistently observed at the 100 nM estradiol concentration but detectable at estradiol concentrations as low as 1 nM.

The estradiol treatment conditions for the three genes verified to be significantly differentially expressed are detailed in Table 4. The gene similar to PAI-1 was upregulated after 24 h of treatment at 100 nM estradiol, and the gene for aldose reductase was upregulated after 24 h of treatment with estradiol at all study concentrations, with results verified at 100 nM estradiol. Similarly, the gene for CASH was also upregulated after 24 h of treatment with estradiol at 100 nM, although there was faint upregulation detectable as early as 0.5 h of treatment with estradiol at concentrations as low as 1 nM. Thus, for the three genes verified to be upregulated by estradiol, the time pattern of differential expression was similar and included temporal clustering to 24 h or less of incubation with this female sex steroid.

                              
View this table:
[in this window]
[in a new window]
  		Table 4.   Estradiol incubation treatment conditions for genes verified to be differentially expressed by semiquantitative RT-PCR in human aortic endothelial cells

The estradiol dose effect on gene expression for the three genes verified to be differentially expressed was consistent with differential gene expression at physiological concentrations. Although, in humans, physiological concentrations of estradiol are in the range of 1-10 nM, higher concentrations have been observed during pregnancy and 100 nM has been routinely used in cell culture systems. Therefore, our results have physiological relevance and relevance to standard in vitro experimental concentrations of estradiol.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We used mRNA differential display analysis to profile estradiol-regulated gene expression and to identify genes previously unknown to be regulated by estrogen in human aortic endothelial cells. Our study design differs from that of previous papers in that cells were studied in growth quiescence in which the effects of cell cycle were eliminated and the influence of dose and time of estradiol exposure could be elucidated. We show a time- and an estradiol dose-dependent differential upregulation of three genes previously not known to be regulated by estradiol. All three genes were clearly detectable within 24 h of estradiol stimulation at physiological concentrations; however, they encode a host of proteins with seemingly divergent functions.

The first gene identified to be upregulated by estradiol was CASH-alpha protein. CASH has been mapped to chromosome 7 of the human genome and is a novel CASH with death effector domains (DEDs) (3, 30). As such, it is of potential importance to atherogenesis, as apoptotic death of cells at the core of lesions is a characteristic of mature atherosclerotic plaques (35, 39). The caspases (e.g., CASP-8=FLICE1 and CASP-10=FLICE2) are members of a cysteine protease family that participate in apoptosis and play a critical role in all known programmed cell death processes (3). They are produced as inactive precursors that are activated by proteolysis on death induction. The precursor proteins contain unique NH2-terminal regions. Interactions of these "prodomains" with specific regulatory molecules allow differential activation of the various caspases by different death-inducing signals (1, 37). Caspases interact through their prodomains with MORT1/FADD, an adapter protein in the death signaling cascade (12). This interaction, required for the signaling to death, involves a protein-binding motif called the DED or the MORT1 motif. Recently, two spliced variants of the novel protein CASH were cloned from mouse liver mRNA by RT-PCR (30). The spliced variants differ at their COOH-termini but contain two shared NH2-terminal DEDs that can bind to MORT1/FADD. The longer COOH-terminal spliced variant (CASH-alpha ) has marked cytocidal effects, whereas the shorter COOH-terminal spliced variant (CASH-beta ) strongly inhibits cytotoxicity induction. In this way, CASH can trigger as well as inhibit signaling for death. Both spliced variants interact with MORT1/FADD and regulate apoptosis in in vitro systems. These studies suggest that the upregulation of CASH-alpha protein by estradiol may be associated with apoptotic cell death. Although estrogen has been reported to function as a survival factor (6, 34), there are accumulating data in other vascular systems for estrogen- and glucocorticoid-stimulated apoptosis (25, 50) suggesting estrogen may have a dual role in the regulation of apoptosis.

The second gene upregulated by estradiol was aldose reductase, the rate-limiting enzyme regulating increased flux of glucose through the polyol pathway. Located on chromosome 7 and 3 of the human and murine genome, respectively, aldose reductase is involved in acceleration of the polyol pathway under hyperglycemia, one of the mechanisms implicated in the pathogenesis of diabetic vascular complications. Induction of aldose reductase has been demonstrated in cultured human microvascular endothelial cells by advanced glycation end products (18). Furthermore, aldose reductase was recently reported to be involved in vascular smooth muscle cell (VSMC) growth and lesion formation after arterial injury (4). Abnormal proliferation of VSMCs is an important feature of atherosclerosis, diabetes mellitus, restenosis, and hypertension. Because aldose reductase catalyzes the reduction of mitogenic aldehydes derived from lipid peroxidation, it is hypothesized that it might be a potential regulator of redox changes that accompany VSMC growth. In addition, it has recently been reported that nitric oxide (important in vascular tone and inflammatory responses under diabetic conditions) upregulates aldose reductase expression in VSMCs (23), providing a potential role for aldose reductase in vascular remodeling. Taken together, our data demonstrating upregulation of aldose reduction by estradiol indicate that estradiol may elicit activation of the polyol pathway. The upregulation of aldose reductase by estradiol in human vascular endothelial cells may in turn modulate aldose reductase-induced VSMC proliferation, cell death, and the ensuing vascular remodeling that occurs during inflammatory responses of the vasculature.

The band for clone 4 (PAI-1) was the third estradiol- upregulated gene product identified by our studies. However, in the case of PAI-1, our fragment matched intron e, a noncoding DNA sequence for PAI-1, and not an mRNA. The match was therefore not to the known mRNA of PAI-1, although our sequence did contain an open reading frame. Although this could be explained by possible genomic DNA contamination of the cDNA, it is also possible that we have identified an estradiol-regulated novel cDNA or an unknown spliced variant of PAI-1 mRNA with homology to a noncoding PAI-1 DNA intron. The PCR primers were designed to the gene sequence, and the FastA and NetBlast searches for the designed primers we used did not match a known mRNA; one primer was a 20/20 match to a human EST cDNA, and the other was a 16/16 non-full-length match to another human EST cDNA. Therefore, it is possible that the primers amplified a novel cDNA related to PAI-1. Further characterization of this differentially expressed product may be possible with future studies and allow for clarification of its possible significance. Should it be functionally related to PAI-1, this gene might be implicated in vascular diseases via procoagulant actions.

The concentration of estradiol (100 nM) at which a majority of the gene expression changes were observed was at or below that typically utilized in in vitro systems with this female sex steroid hormone. The observation that differential expression for some of the verified genes occurred only at this concentration of estradiol suggests that there may be concentration-sensitive differential gene expression by estradiol in endothelial cells. We attribute the observed effects of estradiol on differential gene expression in endothelial cell to direct actions of the hormone; however, we cannot exclude an indirect action by cellular metabolites such as estrone. The potential for indirect effects of estrogen has been demonstrated in other study systems (45, 47, 49). However, it is not clearly known whether endothelial cells have the enzymatic machinery to catabolize estradiol and whether such enzymes might be regulated by exposure to estrogen. Our studies lay the foundation for future work in this area.

A plausible mechanism by which estradiol could regulate gene expression in endothelial cells in our system is by estradiol-stimulated regulation of RNA transcription. However, in other systems, changes in gene expression may be a consequence of RNA breakdown (31). Therefore, it is possible that estradiol could affect both the transcriptional rate of the genes identified and/or the stability of their mRNAs. Furthermore, although endothelial cells are reported to express estrogen receptors, the primary focus of this paper was to screen for differentially expressed genes. We therefore did not specifically examine estrogen receptor activity or their related proteins. Present studies are in progress in the laboratory to examine the role of the estrogen receptor-alpha in vascular gene expression. Nonetheless, the data presented provide significant new information about vascular targets for hormone action and form the basis for additional future mechanistic work.

In this study, only genes whose sequences are present in GenBank were investigated, yielding information on estradiol regulation of known genes. It would be interesting as a next step to embark on the discovery of unknown genes regulated by estradiol. Novel genes may be represented by EST fragments that are in GenBank but have no known function or characterization. In addition, poor matches to GenBank nucleotide sequences also could be novel genes. In fact, some of the estradiol-regulated sequences found in this study corresponded to ESTs in the human genome database with unknown function or poor matches to known genes; perhaps some of these ESTs are to novel genes. Although those ESTs were not further characterized, cloning them would be of future interest.

We have demonstrated the utility of using differential DNA display as a molecular screen to profile genes that are expressed by estradiol in human aortic endothelial cells. This technique, although reliable in identifying differentially expressed gene products, does not permit a functional assessment of the differentially expressed genes identified. Investigation of the physiological activities of the genes identified and their potential regulators in vascular endothelial cells would also be of future interest.

In summary, our studies using RT-PCR differential mRNA display in human aortic endothelial cells demonstrate that a number of physiologically important known genes are differentially regulated by estradiol. This is the first study in which the association between estradiol and gene expression is revealed in aortic endothelial cells by using differential display. Estradiol induced the three upregulated genes with a similar time course and produced similar expression patterns. This regulatory effect is rapid, occurring within the first 24 h after hormone treatment, and dose dependent. Furthermore, dose-response studies established that the action of estradiol in human aortic endothelial cells was at physiologically relevant concentrations for all three of the estradiol upregulated genes identified. In addition, all of the differentially regulated genes identified and verified were upregulated by estradiol, yet they encode for cellular regulatory functions that are quite divergent, ranging from those involved in vascular smooth muscle cell growth and vascular remodeling, to those involved in apoptotic cell death and inflammatory responses of the vasculature. These results indicate that estradiol may affect a wide spectrum of endothelial regulatory functions and that estradiol-regulated changes in gene expression may have a role in processes not solely confined to the realm of atheroprotection. Our results have implications for better understanding the role of estradiol in the regulation of gene expression in the vasculature and provide new unanticipated clues to potential genetic targets for estrogen action in the vasculature. These results are relevant to understanding the changes that occur in the vasculature in the presence of estrogen and could provide a greater understanding of the molecular basis for estrogen action in the vasculature.


    ACKNOWLEDGEMENTS

This work was supported by a career development award to A. C. Villablanca from the National Institutes of Health (1-KO1-HL-04142-01) and National Institutes of Health Grant R01-HL-5567 and the Richard A. and Nora Eccles Harrison Endowed Chair in Diabetes Research to J. C. Rutledge.


    FOOTNOTES

Address for reprint requests and other correspondence: A. C. Villablanca, Cardiovascular Medicine, Women's Cardiovascular Health Program, Div. of Cardiovascular Medicine, Univ. of California, One Shields Ave., TB 172, Davis, CA 95616-8636 (E-mail: avillablanca{at}ucdavis.edu).

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

10.1152/japplphysiol.00374.2001

Received 20 April 2001; accepted in final form 26 October 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Altschul, SF, Gish W, Miller W, Myers EW, and Lipman DJ. Basic local alignment search tool. J Mol Biol 215: 403-410, 1990[ISI][Medline].

2.   Alvarez, RJ, Gips SJ, Moldovan N, Wilhide CC, Millikin EE, Hoang AT, Hruban RH, Silverman HS, Dang CV, and Goldschmidt-Clermont PJ. 17beta -Estradiol inhibits apoptosis of endothelial cells. Biochem Biophys Res Commun 197: 43-49, 1997.

3.   Ausubel, FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, and Struhl K. Current Protocols in Molecular Biology. New York: Greene Publishing Associates and Wiley-Interscience, 1990.

4.   Ball, RY, Stowers EC, Burton JH, Cary NR, Skepper JN, and Mitchinson MJ. Evidence that the death of macrophage foam cells contributes to the lipid core of atheroma. Atherosclerosis 114: 45-54, 1995[ISI][Medline].

5.   Bauer, D, Warthoe P, Rohde M, and Struss M. PCR Methods Manual Supplement. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1994.

6.   Bilsel, AS, Moini H, Tetik E, Aksungar F, Kaynak B, and Ozer A. 17beta -Estradiol modulates endothelin-1 expression and release in human endothelial cells. Cardiovasc Res 46: 579-584, 2000[Abstract/Free Full Text].

7.   Biswas, R, and Vonderhaar BK. Role of serum in the prolactin responsiveness of MCF-7 human breast cancer cells in long-term tissue culture. Cancer Res 47: 3509-3514, 1987[Abstract/Free Full Text].

8.   Boldin, MP, Goncharov TM, Goltsev YV, and Wallach D. Involvement of MACH, a novel MORT1/FADD-interacting protease, in Fas/APO-1- and TNF receptor-induced cell death. Cell 85: 803-815, 1996[ISI][Medline].

9.   Caulin-Glaser, T, Watson CA, Pardi R, and Bender JR. Effects of 17beta -estradiol on cytokine-induced endothelial cell adhesion molecule expression. J Clin Invest 98: 36-42, 1996[ISI][Medline].

10.   Chang, AS, Chang SM, Garcia RL, and Schilling WP. Concomitant and hormonally regulated expression of trp genes in bovine aortic endothelial cells. FEBS Lett 415: 335-340, 1997[ISI][Medline].

11.   Doldi, N, Bassan M, Fusi FM, and Ferrari A. In controlled ovarian hyperstimulation, steroid production, oocyte retrieval, and pregnancy rate correlate with gene expression of vascular endothelial growth factor. J Assist Reprod Genet 14: 589-592, 1997[ISI][Medline].

12.   Don, RH, Cox PT, Wainwright BJ, Baker K, and Mattick JS. "Touchdown" PCR to circumvent spurious priming during gene amplification. Nucleic Acids Res 19: 4008, 1991[Free Full Text].

13.   Duan, H, and Dixit VM. RAIDD is a new "death" adaptor molecule. Nature 385: 86-89, 1997[Medline].

14.   Dubey, RK, Jackson EK, Keller PJ, Imthurn B, and Rosselli M. Estradiol metabolites inhibit endothelin synthesis by an estrogen receptor-independent mechanism. Hypertension 37: 640-644, 2001[Abstract/Free Full Text].

15.   Farhat, MY, Abi-Younes S, and Ramwell PW. Non-genomic effects of estrogen and the vessel wall. Biochem Pharmacol 51: 571-576, 1996[ISI][Medline].

16.   Farhat, MY, Lavigne MC, and Ramwell PW. The vascular protective effects of estrogen. FASEB J 10: 615-624, 1996[Abstract].

17.   Godsland, IF, Wynn V, Crook D, and Miller NE. Sex, plasma, lipoproteins, and atherosclerosis: prevailing assumptions and outstanding questions. Am Heart J 114: 1467-1503, 1987[ISI][Medline].

18.   Goltsev, YV, Kovalenko AV, Arnold E, Varfolomeev EE, Brodianskii VM, and Wallach D. CASH, a novel caspase homologue with death effector domains. J Biol Chem 272: 19641-19644, 1997[Abstract/Free Full Text].

19.   Hayashi, K, Miyamoto A, Berisha B, Kosmann MR, Okuda K, and Schams D. Regulation of angiotensin II production and angiotensin receptors in microvascular endothelial cells from bovine corpus luteum. Biol Reprod 62: 162-167, 2000[Abstract/Free Full Text].

20.   Hishikawa, K, Nakaki T, Marumo T, Suzuki H, Kato R, and Saruta T. Up-regulation of nitric oxide synthase by estradiol in human aortic endothelial cells. FEBS Lett 360: 291-293, 1995[ISI][Medline].

21.   Innis, MA, Gelfand DH, Sninsky JJ, and White TJ. PCR Protocols. A Guide to Methods and Applications. San Diego, CA: Academic, 1990.

22.   Kleinert, H, Wallerath T, Euchenhofer C, Ihrig-Biedert I, Li H, and Förstermann U. Estrogens increase transcription of the human endothelial NO synthase gene: analysis of the transcription factors involved. Hypertension 31: 582-588, 1998[Abstract/Free Full Text].

23.   Konstadoulakis, MM, Kymionis GD, Karagiani M, Katergianakis V, Doundoulakis N, Pararas V, Koutselinis A, Sehas M, and Peveretos P. Evidence of apoptosis in human carotid atheroma. J Vasc Surg 27: 733-739, 1998[ISI][Medline].

24.   Kourembanas, S, Morita T, Liu Y, and Christou H. Mechanisms by which oxygen regulates gene expression and cell-cell interaction in the vasculature. Kidney Int 51: 438-443, 1997[ISI][Medline].

25.   Kumar, S. ICE-like proteases in apoptosis. Trends Biochem Sci 20: 198-202, 1995[ISI][Medline].

26.   Kushwaha, RS, and Hazzard WR. Exogenous estrogens attenuate dietary hypercholesterolemia and atherosclerosis. Metabolism 30: 359-366, 1981[ISI][Medline].

27.   Liang, P, and Pardee AB. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science 257: 967-971, 1992[Abstract/Free Full Text].

28.   Lippert, C, Seeger H, Mueck AO, and Lippert TH. The effects of A-ring and D-ring metabolites of estradiol on the proliferation of vascular endothelial cells. Life Sci 67: 1653-1658, 2000[ISI][Medline].

29.   MacRitchie, AN, Jun SS, Chen Z, German Z, Yuhanna IS, Sherman TS, and Shaul PW. Estrogen upregulates endothelial nitric oxide synthase gene expression in fetal pulmonary artery endothelium. Circ Res 81: 355-362, 1997[Abstract/Free Full Text].

30.   Mead, DA, Pey NK, Herrnstadt C, Marcil RA, and Smith LM. A universal method for the direct cloning of PCR amplified nucleic acid. Biotechnology 9: 657-663, 1991[Medline].

31.   Nakamura, N, Obayashi H, Fujii M, Yoshimori K, Ogata M, Hasegawa G, Shigeta H, Kitagawa Y, Yoshikawa T, Kondo M, Ohta M, Nishimura M, Nishinaka T, and Nishimura CY. Induction of aldose reductase in cultured human microvascular endothelial cells by advanced glycation end products. Free Radic Biol Med 29: 17-25, 2000[ISI][Medline].

32.   Nelson, K, Brannan J, and Kretz K. The fidelity of TaqPlus DNA polymerase in PCR. Strategies Mol Biol 8: 24-25, 1995.

33.   Neulen, J, Yan Z, Raczek S, Weindel K, Keck C, Weich HA, Marme D, and Breckwoldt M. Human chorionic gonadotropin-dependent expression of vascular endothelial growth factor/vascular permeability factor in human granulosa cells: importance in ovarian hyperstimulation syndrome. J Clin Endocrinol Metab 80: 1967-1971, 1995[Abstract].

34.   Pearson, WR. Rapid and sensitive sequence comparison with FASTP and FASTA. Methods Enzymol 183: 63-98, 1990[ISI][Medline].

35.   Pearson, WR, and Lipman DJ. Improved tools for biological sequence comparison. Proc Natl Acad Sci USA 85: 2444-2448, 1988[Abstract/Free Full Text].

36.   Piotrowicz, RS, Weber LA, Hickey E, and Levin EG. Accelerated growth and senescence of arterial endothelial cells expressing the small molecular weight heat-shock protein HSP27. FASEB J 9: 1079-1084, 1995[Abstract].

37.   Rozen, S, and Skaletsky H. Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol 132: 365-386, 2000[Medline].

38.   Ruef, J, Liu SQ, Bode C, Tocchi M, Srivastava S, Runge MS, and Bhatnagar A. Involvement of aldose reductase in vascular smooth muscle cell growth and lesion formation after arterial injury. Arterioscler Thromb Vasc Biol 20: 1745-1752, 2000[Abstract/Free Full Text].

39.   Sambrook, J, Fritsch E, and Maniatis T. Molecular Cloning: A Laboratory Manual (2nd ed.). Plainview, NY: Cold Spring Harbor Laboratory Press, 1989.

40.   Seo, HG, Nishinaka T, and Yabe-Nishimura C. Nitric oxide up-regulates aldose reductase expression in rat vascular smooth muscle cells: a potential role for aldose reductase in vascular remodeling. Mol Pharmacol 57: 709-717, 2000[Abstract/Free Full Text].

41.   Simoncini, T, De Caterina R, and Genazzani AR. Selective estrogen receptor modulators: different actions on vascular cell adhesion molecule-1 (VCAM-1) expression in human endothelial cells. J Clin Endocrinol Metab 84: 815-818, 1999[Abstract/Free Full Text].

42.   Simoncini, T, and Genazzani AR. Tibolone inhibits leukocyte adhesion molecule expression in human endothelial cells. Mol Cell Endocrinol 162: 87-94, 2000[ISI][Medline].

43.   Simoncini, T, Maffei S, Basta G, Barsacchi G, Genazzani AR, Liao JK, and De Caterina R. Estrogens and glucocorticoids inhibit endothelial vascular cell adhesion molecule-1 expression by different transcriptional mechanisms. Circ Res 87: 19-25, 2000[Abstract/Free Full Text].

44.   Spyridopoulos, I, Sullivan AB, Kearney M, Isner JM, and Losordo DW. Estrogen-receptor-mediated inhibition of human endothelial cell apoptosis. Estradiol as a survival factor. Circulation 95: 1505-1514, 1997[ISI][Medline].

45.   Tsukamoto, A, Kaneko Y, Yoshida T, Han K, Ichinose M, and Kimura S. 2-Methoxyestradiol, an endogenous metabolite of estrogen, enhances apoptosis and beta -galactosidase expression in vascular endothelial cells. Biochem Biophys Res Commun 248: 9-12, 1998[ISI][Medline].

46.   Villablanca, AC, and Hanley MR. 17beta -Estradiol stimulates substance P receptor gene expression. Mol Cell Endocrinol 135: 109-117, 1997[ISI][Medline].

47.   Wang, W, and Passaniti A. Extracellular matrix inhibits apoptosis and enhances endothelial cell differentiation by a Nfkappa B-dependent mechanism. J Cell Biochem 73: 321-331, 1999[ISI][Medline].

48.   Williams, JK, Adams MR, and Klopfenstein HS. Estrogen modulates responses of atherosclerotic coronary arteries. Circulation 81: 1680-1687, 1990[ISI][Medline].

49.   Yue, TL, Wang X, Louden CS, Gupta S, Pillarisetti K, Gu JL, Hart TK, Lysko PG, and Feuerstein GZ. 2-Methoxyestradiol, an endogenous estrogen metabolite, induces apoptosis in endothelial cells and inhibits angiogenesis: possible role for stress-activated protein kinase signaling pathway and Fas expression. Mol Pharmacol 51: 951-962, 1997[Abstract/Free Full Text].

50.   Zhu, YH, Vekemans S, and Cassiman JJ. Semiquantitative PCR of alpha 2 and alpha 4 integrin mRNA shows differential response to the transcriptional modulators TGF-beta 1 and TPA. Cell Mol Biol (Oxf) 43: 1221-1226, 1997.

J APPL PHYSIOL 92(3):1064-1073
8750-7587/02 $5.00 Copyright © 2002 the American Physiological Society


This article has been cited by other articles:


Home page
 Mol. Endocrinol.Home page
R. O'Lone, M. C. Frith, E. K. Karlsson, and U. Hansen
Genomic Targets of Nuclear Estrogen Receptors
Mol. Endocrinol., August 1, 2004; 18(8): 1859 - 1875.
[Abstract] [Full Text] [PDF]

Home page
 Physiol. GenomicsHome page
A. C. Villablanca, K. A. Lewis, D. Tham, and J. C. Rutledge
Differential regulation of gene expression by ovariectomy in mouse aorta
Physiol Genomics, February 6, 2003; 12(3): 175 - 185.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow All Versions of this Article:
92/3/1064    most recent
00374.2001v1
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
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 ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal