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Am J Physiol Regul Integr Comp Physiol 285: R754-R761, 2003. First published June 26, 2003; doi:10.1152/ajpregu.00540.2002
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LOCAL CONTROL OF CIRCULATION

Estrogen decreases biglycan mRNA expression in resistance blood vessels

Manoj C. Rodrigo, Douglas S. Martin, and Kathleen M. Eyster

University of South Dakota School of Medicine, Vermillion, South Dakota 57069

Submitted 5 September 2002 ; accepted in final form 23 June 2003


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
This study was designed to identify new gene targets of estrogen in the mesenteric arteries and to determine whether the soy phytoestrogens could mimic estrogen effects. Ovariectomized rats were treated with estradiol, genistein, or daidzein for 4 days. The mesenteric arteries were harvested, total RNA was extracted, mRNA was reverse transcribed in the presence of [33P]dCTP, and the labeled probes were hybridized with DNA microarrays. Analysis of the microarray data identified biglycan as a target of estrogenic regulation. Semiquantitative RT-PCR was used to confirm and quantitate the decrease in biglycan gene expression in response to estrogen (-37%), genistein (-15%), and daidzein (-10%). Treatment with the pure estrogen receptor antagonist ICI-182,780 reversed the inhibition of biglycan gene expression. The decrease in biglycan gene expression in response to estrogens was paralleled with a decrease in biglycan protein expression. Biglycan protein was localized to the media of the mesenteric arteries by immunohistochemistry. Collectively, these data suggest that biglycan is a vascular protein regulated at the genomic level by estrogens.

DNA microarray; genistein; daidzein; mesenteric arteries

THE DIFFERENCE IN THE OCCURRENCE of cardiovascular disease between men and premenopausal women is remarkable, and this difference is purported to be due, at least in part, to the presence of estrogen in premenopausal women. Estrogen has been reported to provide vasculoprotective effects through a variety of mechanisms. For example, estrogen can directly affect the vessel wall by decreasing mitosis, thereby decreasing intimal hyperplasia (44-46). Estrogen can affect the function of blood vessels by regulating the expression of enzymes involved in vasodilatory pathways such as nitric oxide synthase (52), and estrogen can cause direct vasodilation by regulating ion channels in the vessel wall (54, 55). Estrogen has also been shown to reduce atherogenesis (34). In contrast, a recent clinical study reported that estrogen replacement therapy with premarin was found to have a deleterious effect on cardiovascular disease (56). Thus there remains considerable controversy regarding the effects of estrogen on the vasculature. One approach to clarify the effects of estrogen on the vasculature is to assess estrogen-regulated gene expression in blood vessels.

Estrogenic substances can be obtained from plant as well as animal sources. Soybeans and soy-derived food products are rich in the isoflavones genistein and daidzein (4). They bind and activate intracellular estrogen receptors and are therefore known as phytoestrogens (25, 49). In addition to its estrogenic effects, genistein is a potent tyrosine kinase inhibitor (2, 23). Daidzein, in contrast, is devoid of tyrosine kinase-inhibitory effects (2). Many health benefits have been claimed for these soy-derived compounds, from anticancer effects (5) to cardiovascular protective effects (15, 50). The putative health benefits of soy compounds could result from their estrogenic effects or from the tyrosine kinase-inhibitory effects.

The first goal of the current study was to identify new targets of estrogen in the vasculature, specifically the mesenteric arteries, through application of DNA microarray technology. The second goal of this project was to determine whether the soy phytoestrogens could mimic the effects of estrogen on gene expression in blood vessels. The initial microarray analyses identified biglycan as an estrogen-responsive gene in the mesenteric arteries. Biglycan is an extracellular matrix protein that belongs to the small proteoglycan family of proteins (19). Matrix proteoglycans such as biglycan are important in modulating the vessel wall structural integrity (24) and would be expected to influence biophysical properties such as compliance. Abnormal expression of some of these proteoglycans in the vasculature has been identified in the development and progression of cardiovascular pathologies (8, 31). Given the potential importance of biglycan in regulating vascular function, this gene was chosen for followup studies using semiquantitative RT-PCR to confirm and quantitate estrogen-regulated biglycan mRNA expression. Western blot and immunohistochemical approaches were used to demonstrate estrogen-responsive protein expression and localization.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Treatments. Immature female Sprague-Dawley rats (Taconic Farms, Germantown, NY) were bilaterally ovariectomized at 4 wk of age and fed a casein-based, soy-free diet (ICN Biomedicals, Aurora, OH) to avoid potential contamination with soy phytoestrogens in the diet. Treatments were initiated 2 wk after the surgery. The rats were given daily intraperitoneal injections of 10% dimethyl sulfoxide (vehicle control), 0.15 mg/kg estradiol benzoate (41), 1 mg/kg genistein (7), or 1 mg/kg daidzein for 4 days. The dose of estradiol chosen has been shown to regulate the expression of the beta isoform of the estrogen receptor in the mesenteric arteries (41). Genistein at 1 mg · kg-1 · day-1 lowered blood pressure in the borderline hypertensive rat model (7). A separate cohort of animals was given the same control, estrogen, or phytoestrogen treatments plus 2 mg · kg-1 · day-1 of the pure estrogen receptor antagonist ICI-182,780 (Tocris Cookson, Ellisville, MO) (6) in the same injection volume as hormone alone. Another group of animals received 2 mg · kg-1 · day-1 of tyrphostin-25 (Sigma, St. Louis, MO) (42), a specific tyrosine kinase inhibitor. This animal protocol was approved by the Institutional Animal Care and Use Committee and conforms to the American Physiological Society's "Guiding Principles for Research Involving Animals and Human Beings" (3).

Tissue isolation and RNA extraction. On the fourth treatment day, the rats were euthanized. The mesenteric arcade was dissected free and placed in RNAlater (Ambion, Austin, TX). The mesenteric vessels were used because they contribute significantly to the total vascular resistance, they can be isolated rapidly to preserve RNA integrity, and they have been implicated in cardiovascular disease states. The mesenteric arteries were dissected free from the mesenteric veins and adipose, nervous, and connective tissue while submerged under RNAlater (41). Total RNA was isolated from the mesenteric arteries with TRI reagent (Molecular Research Center, Cincinnati, OH). The samples were treated with DNase (Promega, Madison, WI), and the amount of total RNA in each sample was quantified with a spectrophotometer. The purity of the isolated RNA was evaluated by agarose gel electrophoresis.

Protein extraction. The organic phase from the TRI reagent (Molecular Research Center) RNA extraction protocol was saved for total protein extraction (41). To recover the protein, 0.3 ml of 100% ethanol was added to the organic phase. The sample was centrifuged (2,000 g for 5 min at 4°) to remove precipitated DNA. The proteins in the supernatant were precipitated with isopropanol. After centrifugation (12,000 g for 10 min at 4°), the protein pellet was washed three times in a 0.3 M guanidine hydrochloride solution. The protein pellet was solubilized in 0.2 ml of 1% sodium dodecyl sulfate (SDS) by incubating the samples at 60°C for 5 min and sonicating twice for 10 s. The bicinchoninic acid protein assay (Pierce, Rockford, IL) was used to measure the protein content of the samples.

DNA microarrays. The Rat Genes GeneFilters (Release I) were obtained from ResGen (Huntsville, AL). Label preparation and hybridization were carried out per company instructions. Total mesenteric artery RNA (1 µg) was reverse transcribed to cDNA in the presence of [33P]dCTP. The 33P-labeled cDNA was purified, the hybridization reaction was carried out, and the GeneFilters were washed as described (12). The GeneFilters were incubated with film (Amersham, Piscataway, NJ), and a Fuji Supercooled CCD camera was used to obtain a digital image of the exposed film. The digital image was imported into Pathways software (ResGen) for analysis. The density of each differentially expressed gene was normalized to the density of the housekeeping gene cyclophilin C (53).

RT-PCR. For multiplex semiquantitative RT-PCR, 2 µg of total mesenteric artery RNA was primed with 5 µg of random primers [poly d(N)6] at 70°C for 5 min, reverse transcribed at 40°C for 1 h, and incubated at 95°C for 5 min. The 18S rRNA primer pair from Ambion (Austin, TX) was used as an internal control for the multiplex PCR reactions. The mixture for multiplex semiquantitative PCR consisted of 4 µl reverse transcribed template cDNA, 2 µl each of 10 µM gene specific primers for biglycan, 1 µl of 18S rRNA, and 0.5 µl of [33P]dCTP, and Ready-to-go RT-PCR bead (Amersham Pharmacia Biotech, Piscataway, NJ). The sequence of the sense primer for biglycan was 5'-GAGACAACTGACCTCATAAGC-3', and the sequence of the antisense primer was 5'-ACACACACACACACACCATC-3'. The PCR reactions were carried out in a thermocycler (Perkin-Elmer Geneamp 2400) for 27 cycles. The appropriate number of cycles was determined by performing a linear amplification curve and choosing a cycle number within the linear amplification range (Fig. 1). Cycling parameters were set at 95°C for 30 s, 50°C for 30 s, and 72°C for 1 min. PCR products were separated on a 6% acrylamide gel, dried, and autoradiographed. The optical density of the bands was assessed by a densitometer. The results of the multiplex semiquantitative PCR are expressed as a percentage of internal control signal produced in each lane by the 18S rRNA after background subtraction. The specificity of the amplified product was evaluated by using a nested primer technique before evaluating biglycan gene expression. The sequences of the nested primers for biglycan were sense 5'-TGACCTCATAAGCAGCACTC-3' and antisense 5'-ACACACACACACCATCTTCTC-3'.



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  		Fig. 1. Linear range of amplification for biglycan. A sample was removed from the PCR tube every 2 cycles while the reaction was in progress. The amplified products were separated on a denaturing gel, vacuum dried, and autoradiographed for densitometry. The 27th cycle was chosen for remaining PCR experiments since the linear range of amplification for biglycan extended to 29 cycles. Top: a representative autoradiograph of the PCR curve. Bottom: the abundance of the biglycan PCR target product as a function of the number of PCR cycles.

 

Western blot protocol. Seventy-five micrograms of protein isolated from the mesenteric arteries was electrophoresed on 10% SDS polyacrylamide gels. The proteins were transferred to polyvinylidine difluoride membranes, and Western blots were run using a standard protocol (11). The antibody against biglycan was LF06 rabbit polyclonal donated by Dr. L. Fisher, Bone and Matrix Biology Department, National Institutes of Health, Bethesda, MD, and was used at a dilution of 1:1,500. The secondary antibody was horseradish peroxidase-labeled goat anti-rabbit (1:1,000). The protein bands were visualized by an enhanced chemiluminescence reaction (Amersham Pharmacia Biotech) and exposed to X-ray film. Bands on the X-ray film were quantitated by a densitometer, and the data are expressed as relative densitometric units after background subtraction.

Immunohistochemistry. The avidin-biotin complex (Vector Laboratories, Burlingame, CA) was used to enhance the immunohistochemical detection of biglycan protein (9). Mesenteric arterial tissue was cryoembedded in OCT reagent (Tissue-Tek, Torrance, CA), and 7-µm sections were mounted on positive-coated slides (Daigger, Vernon Hills, IL). The primary antibody against biglycan (LF06) was used at 1:1,000. The secondary antibody was biotinylated goat anti-rabbit at 1:1,000. The immunohistochemical signal was developed with diaminobenzidine.

Data analysis. Biglycan gene expression data are expressed as a percentage of the 18S rRNA internal control. The data from the experiments containing multiple treatment groups were subjected to a one-way ANOVA followed by post hoc comparison. Dunnett's test was used to compare the different treatments to a single control. One-way ANOVA followed by Student's t-test was used for selected pairwise comparisons in the estrogen receptor blockade experiments. An unpaired t-test was used to make comparisons between control and tyrphostin-25 treatment. The Western blot data from control and treated samples were also compared by an unpaired t-test. All data are expressed as means ± SE, and P < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 DISCLOSURES
 REFERENCES
 
Effect of estrogen, genistein, and daidzein on biglycan gene expression. DNA microarray experiments were performed twice and were used as an initial step to qualitatively identify potential target genes for experiments using semiquantitative RT-PCR. Analysis of the DNA microarray data identified a decrease in the expression of biglycan in mesenteric arterial samples from animals treated with estrogen (data not shown). Followup studies were then initiated to confirm and quantitate the estrogenic regulation of this gene. A nested primer set was used to validate the standard biglycan primer set. Product from a preliminary RT-PCR study using the standard biglycan primers was used as template for a PCR experiment using the nested set of biglycan primers. The presence of PCR product from the nested PCR reaction of the predicted size for that nested primer set validated the standard primer set for biglycan (data not shown). The DNA microarray data also suggested that the genes for endothelin converting enzyme 1, aldehyde dehydrogenase, {alpha}1-adrenergic receptor, fibroblast growth factor receptor activating protein-1, sodium chloride betaline/GABA transporter, megalin, and Sprague-Dawley protein kinase C receptor were regulated by estrogen in the mesenteric arteries (data not shown). However, confirmatory studies have been performed only for endothelin converting enzyme 1 (40). We are currently analyzing the estrogen sensitivity of the remaining genes.

Multiplex amplification by RT-PCR followed by resolution of the products on denaturing gels indicated two distinct bands at ~500 bp and 400 bp. The 500-bp band was identified as the 18S rRNA internal control band and was the correct molecular size of the product described by the manufacturer (Ambion). The 400-bp product was the predicted size for the biglycan PCR product. The data from the semiquantitative RT-PCR experiments indicated a significant decrease in biglycan gene expression in response to estrogen treatment compared with control (control: 60.1 ± 4.6; estrogen: 37.7 ± 5.5; P = 0.015). All RT-PCR data are expressed as percentage of the 18S rRNA internal control (Fig. 2). There was also a trend toward reduced biglycan gene expression in the genistein-treated (genistein: 51.3 ± 4.4, P = 0.18) but not the daidzein-treated groups (daidzein: 54.3 ± 4.4, P = 0.37) (Fig. 2).



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  		Fig. 2. Modulation of biglycan gene expression by estrogen and phytoestrogens in mesenteric arteries. Top: a representative autoradiograph of the multiplex RT-PCR products of control (Ctl, n = 9), estrogen (E2, n = 10)-, genistein (Gen, n = 10)-, and daidzein (Daid, n = 9)-treated mesenteric arterial tissue. The Std lane contains DNA molecular weight markers. Each of the other lanes contains the 18S rRNA internal control (~500-bp band) and the band specific for biglycan (~400-bp band). Bottom: summary data for biglycan gene expression as a percentage of the 18S rRNA internal control. Data are expressed as means ± SE. The inhibition of biglycan expression by estrogen was statistically significant (P = 0.015).

 

Effect of estrogen receptor blockade. Estrogen receptor blockade did not, of itself, produce a marked change in biglycan gene expression (Fig. 3A). Treatment with ICI-182,780 reversed the estrogen-mediated decrease in biglycan gene expression (control: 51.6 ± 3.7; estrogen: 36.8 ± 4.1, P = 0.03; ICI-182,780 + control: 56.4 ± 3.7; ICI-182,780 + estrogen: 51.9 ± 4.3, P = 0.44). The trend toward decreased biglycan mRNA expression in response to genistein was reproducible in that biglycan gene expression was again reduced slightly in the genistein-treated animals. Despite the reduction of 15% in biglycan gene expression in response to genistein treatment, the comparison of control vs. genistein did not reach significance. Nevertheless, treatment with ICI-182,780 abolished the trend toward a decrease in biglycan gene expression response to genistein (control: 55.6 ± 8.7; genistein: 39.6 ± 5.3, P = 0.14; ICI-182,780 + control: 70.6 ± 7.1; ICI-182,780 + genistein: 61.5 ± 6.0, P = 0.35) (Fig. 3B), suggesting that this effect was a real phenomenon. Similarly, daidzein also produced a trend toward reduced biglycan gene expression (10% decrease). This effect was mediated by an estrogen receptor mechanism since it was reversed by ICI-182,780 (control: 50.3 ± 3.1; daidzein: 41.4 ± 4.8, P = 0.17; ICI-182,780 + control: 52.7 ± 3.9; ICI-182,780 + daidzein: 55.3 ± 3.4, P = 0.63) (Fig. 3C). It is interesting to note that the pattern, if not the magnitude, of biglycan gene response was similar among the three estrogenic substances tested. In contrast to the effect of genistein, treatment with the tyrosine kinase inhibitor tyrphostin-25 did not inhibit biglycan gene expression; in fact, there was a tendency for biglycan expression to increase in response to tyrphostin-25 (control: 46.0 ± 1.8; tyrphostin-25: 55.8 ± 4.7; P = 0.11) (Fig. 4).



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  		Fig. 3. Effects of treatments [estrogen (A), genistein (B), and daidzein (C)] on biglycan mRNA expression in the presence and absence of the pure estrogen receptor antagonist ICI-182,780. The top of each panel shows a representative autoradiograph of the multiplex RT-PCR products of control (n = 5), treatment (n = 6), control + ICI 182,780 (n = 6), and treatment + ICI 182,780-treated (n = 6) mesenteric arterial tissue, and DNA molecular weight markers (Std). Each lane contains the 18S rRNA internal control (~500-bp band) and the band specific for biglycan (~400-bp band). The bottom of each panel shows summary data for biglycan mRNA expression as a percentage of the 18S rRNA internal control. Data are expressed as means ± SE. The inhibition of biglycan gene expression by estrogen was statistically significant (P = 0.03).

 


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  		Fig. 4. Modulation of biglycan gene expression in response to tyrphostin-25 in mesenteric arteries. Top: a representative autoradiograph of the multiplex RT-PCR products of control (n = 5) and tyrphostin-25 (Tyr, n = 6)-treated mesenteric arterial tissue, and DNA molecular weight markers (Std). Each lane contains the 18S rRNA internal control (~500-bp band) and the band specific for biglycan (~400-bp band). Bottom: summary data for biglycan gene expression as a percentage of the 18S rRNA internal control. Data are expressed as means ± SE.

 

Effect of treatment on biglycan protein expression. Biglycan protein expression was qualitatively similar to biglycan mRNA expression in that there was a trend to decreasing biglycan protein expression in response to estrogen, genistein, and daidzein treatment (P = 0.09). Biglycan protein expression was decreased 30% in response to estrogen, 32% in response to genistein, and 37% in response to daidzein treatment compared with control (control: 59.0 ± 7.0; estrogen: 41.5 ± 4.5; genistein: 40.0 ± 1.0; daidzein: 37.0 ± 4.0) (Fig. 5).



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  		Fig. 5. Western blot of biglycan protein expression in the mesenteric arteries in response to estrogen and phytoestrogen treatment. Top:a representative Western blot of control and estrogen-, genistein-, and daidzein-treated mesenteric arteries probed for biglycan protein. Bottom: summary data for Western blots quantitated by densitometric analysis. Data are expressed as means ± SE; n = 2 in each case.

 

Biglycan protein was localized in the veins and arteries of the mesentery by immunohistochemistry with very little staining present in the adipose or the mesenteric nerves (Fig. 6, left). The biglycan protein was specifically localized in the media of the superior mesenteric artery with the absence of staining in the intima and in adventitial fibroblasts (Fig. 6, right).



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  		Fig. 6. Immunohistochemical localization of biglycan in the mesenteric arcade and the superior mesenteric artery. The localization of biglycan was restricted to the veins and arteries in the mesentery. The surrounding adipose tissue and the mesenteric nerve trunk show minimal biglycan protein expression. Microscope magnification was set at x40 (left) and x200 (right). Positive staining (brown color) for biglycan was observed in the media/intima of the superior mesenteric arterial wall (right). Morphology: superior mesenteric artery (MA), superior mesenteric vein (MV), adipose tissue (AD), and the mesenteric nerve trunk (MNT), tunica intima (TI), tunica media (TM), the tunica adventitia (TA), and the lumen (L).

 


   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
DISCLOSURES
 REFERENCES
 
DNA microarrays were used in this project to identify new gene targets of estrogen action in the mesenteric arteries. Analysis of the microarray data suggested that among the genes affected by estrogen treatment, biglycan gene expression was decreased in the mesenteric arteries in response to estrogen. Semiquantitative RT-PCR was used to verify the finding from the microarray study and to quantitate the decrease in biglycan gene expression in response to estrogen. The data reported here validate the power of DNA microarray technology to discover new gene targets of estrogen in the vasculature.

Biglycan is an extracellular matrix protein that belongs to the small proteoglycan family of proteins (19). Matrix proteoglycans such as versican, decorin, and biglycan are important in modulating the structural integrity of the blood vessel wall (24). If the content of a structural protein, such as biglycan, is increased in the blood vessel wall, it will decrease the compliance of that vessel and will create a stiffer vessel. Abnormal expression of biglycan has been identified in the development and progression of cardiovascular pathologies (8, 31). Biglycan, in particular, has been shown to bind extracellular proteins such as growth factors (10, 20) and large macromolecules such as low-density lipoproteins (14, 35, 36, 43) in the subendothelium of the vessel wall. Increased concentrations of biglycan have been found in arterial plaques in experimental animal models (31, 32, 57) as well as in human vessel biopsies (18). Moreover, the application of mechanical deformation or shear forces on vascular smooth muscle cells has been shown to upregulate biglycan mRNA expression (26). Transforming growth factor-{beta} (TGF-{beta}) treatment also upregulates biglycan expression (21, 29, 32). Finally, ANG II, a humoral factor important in cardiovascular disease, has recently been shown to enhance biglycan mRNA expression and protein synthesis (14). Collectively, these data suggest that biglycan is an important vascular proteoglycan that is regulated by both physical (e.g., shear) and chemical (TGF-{beta}) factors that may play a role in vascular remodeling and cardiovascular disease.

There has been relatively little previous work examining the effects of estrogen on biglycan gene expression. Biglycan gene expression was significantly attenuated in pregnant rabbit articular cartilage compared with nonpregnant rabbits (17), suggesting in vivo modulation of biglycan in an animal model with high circulating estrogen (as well as progesterone) levels. Conversely, a clinical study evaluating extracellular matrix protein expression in paraurethral tissue in response to estrogen replacement reported no significant change in biglycan expression (13). The differential regulation of biglycan in these studies may be due to the different types of tissues examined or species differences. The present study extends previous work by demonstrating that estrogens are also biglycan gene regulatory molecules in rat vascular smooth muscle.

Previous studies showed that rats consuming a soy diet had plasma genistein levels of ~1-1.5 µM (30). Direct intravenous injection of genistein at a dose of 1.5 mg/kg resulted in plasma concentrations of ~19 µM at 90 min after injection (39). However, extrapolation to the current study is difficult because intraperitoneal injections were used in this study and only once per day. The plasma concentrations of genistein were not measured because at the time of the study, measurement of plasma levels required relatively large amounts of blood that could only be obtained at death. In addition, since the half-life of genistein is ~5-6 h in the rat, the majority of the dose would have been cleared at death. Therefore it is difficult to predict the actual concentration to which the rats were exposed. Nevertheless, it might be predicted to be in the low micromolar range at its peak.

The phytoestrogens genistein and daidzein showed an interesting trend toward mimicking the effect of estrogen on biglycan expression. The inhibition of biglycan gene expression by estrogen, and the inhibitory trend by genistein and daidzein, was reversed by concurrent treatment with ICI-182,780. This ICI compound inhibits the classical genomic estrogen receptors (ER) (22, 48). The observation that ICI-182,780 reversed the effect of estrogen, genistein, and daidzein on biglycan gene expression suggests that the effects were mediated through the classical ER. The pattern, if not the magnitude, of biglycan gene expression was similar between estrogen and the phytoestrogens. It may be that the smaller effects of the phytoestrogen simply reflect an overall lower affinity at estrogen receptors in general compared with estrogen. Alternatively, because differential affinities for estrogen subtypes have been described (33), the phytoestrogens may exert competing influences at ER-{alpha} and ER-{beta}. This latter case opens the possibility to development of phytoestrogen analogs that are selective for estrogen receptor subtypes, and this could allow selective targeting of effects.

Because genistein is a potent tyrosine kinase inhibitor, it was necessary to investigate whether the effect on biglycan gene expression was mediated by a tyrosine kinase-inhibitory mechanism. Tyrphostin-25, another tyrosine kinase inhibitor that has been shown to affect the vasculature (27, 28), was used to evaluate the possible modulation of biglycan by a tyrosine kinase-inhibitory mechanism in the mesenteric arteries. Tyrphostin-25 did not inhibit biglycan gene expression; in fact, there was a trend toward an increase in biglycan expression in response to tyrphostin-25. These data support the concept that the regulation of biglycan gene expression does not occur through a tyrosine kinase-mediated mechanism. This idea was further supported by the fact that genistein and daidzein exhibited a similar trend in the regulation of biglycan expression although only genistein inhibits tyrosine kinase activity.

No classical estrogen response element has been identified in the human biglycan gene promoter sequence (16, 47). However, the estrogen/receptor complex can interact with other transcription factor binding sites, such as the AP-1 site, to modulate the transcription of genes without classical estrogen response elements (38). The biglycan gene contains an AP-1 site (51). ER-{alpha} induces gene expression at the AP-1 site, whereas ER-{beta} represses gene transcription at the same site (38). Since ER-{beta} represses gene transcription at the AP-1 site, we can speculate that the decrease in biglycan gene expression may be mediated through ER-{beta}. This is also consistent with the ability of the phytoestrogens to mimick estrogen effects because these agents are thought to have preferential agonist activity at ER-{beta} (23, 25).

The cellular content of a given RNA does not always correlate directly with the cellular content of its cognate protein; thus it was important to examine the protein expression of biglycan as well as the mRNA expression. Estrogen and phytoestrogen treatments were associated with a trend toward a reduction in protein expression in mesenteric blood vessels. These data indicate that the effect of estrogens on biglycan gene expression was, indeed, translated into a functional result at the protein level. Moreover, our immunohistochemical data show that biglycan protein is localized to the media of the mesenteric arteries, indicating that this proteoglycan is expressed in an appropriate location to contribute to subendothelial retention of lipoproteins, a hallmark of atherogenesis (35-37). Therefore, estrogenic suppression of biglycan protein expression may be of beneficial effect to the vascular wall by reducing retention of lipoproteins. This is consistent with previous observations that estrogen (34) and phytoestrogen (1) treatments reduce atherogenesis.

In summary, through the use of DNA microarray technology, we have identified biglycan as a possible new gene target for estrogenic regulation in the vasculature, and we have confirmed the responsiveness of biglycan gene and protein expression to estrogen by semiquantitative RT-PCR and by Western blot. We also demonstrated that the phytoestrogens genistein and daidzein produced a similar pattern of response albeit of lesser magnitude. In all cases these responses were abolished by treatment with the estrogen receptor antagonist ICI-182,780. These data provide the first direct demonstration of estrogenic modulation of biglycan expression in vascular smooth muscle. Further studies are needed to elucidate the physiological effects of estrogenic regulation of the biglycan gene and its cognate protein in the blood vessels.

Perspectives

The difference in cardiovascular disease between men and premenopausal women is remarkable, and this difference is purported to be due, at least in part, to the presence of estrogen in the premenopausal women. These data present an estrogen-regulated gene that may play a cardioprotective role. Biglycan may be involved in both function and remodeling of blood vessels. This dual mode of action may exert synergistic effects in blood vessel physiology. Because estrogen and phytoestrogens reduced expression of biglycan, these substances may also exert both structural and functional effects that interact with other known mechanisms (e.g., upregulation of nitric oxide synthesis) that contribute to cardiovascular health.


   DISCLOSURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 DISCLOSURES
REFERENCES
 
This work was supported in part by the South Dakota Soybean Research and Promotion Council (K. M. Eyster and D. S. Martin), National Science Foundation Grant NSF-EPSCoR 9720642 (M. C. Rodrigo), National Heart, Lung, and Blood Institute (NHLBI) Grant HL-69886 (K. M. Eyster), and American Heart Association Grant 0140157N and NHLBI Grant HL-63053 (D. S. Martin).


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. M. Eyster, USDSM, 414 E. Clark St., Vermillion, SD 57069 (E-mail: keyster{at}usd.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.


   REFERENCES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 

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