Estradiol Suppresses MCP-1 Expression In Vivo

Implications for Atherosclerosis

From the Department of Obstetrics and Gynecology (S.P., R.S., G.C., L.N.), the Department of Molecular and Medical Pharmacology (R.S., G.C.), and the Department of Internal Medicine (M.N.), UCLA School of Medicine, University of California, Los Angeles, and the Department of Pathobiology and Program in Nutritional Sciences, University of Washington, Seattle (M.E.R.).

Correspondence to Lauren Nathan, MD, Department of Obstetrics and Gynecology, 27-139 CHS, UCLA School of Medicine, 10833 LeConte Ave, Los Angeles, CA 90095-1740.

	Abstract

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Abstract—The mechanisms by which 17ß-estradiol retards atherogenesis are not known. The adhesion of monocytes to endothelial cells followed by the migration of monocytes into the artery wall are key cellular events that occur throughout the entire atherogenic process and may be responsive to estradiol. Monocyte chemoattractant protein-1 (MCP-1), a chemokine that is expressed in atherosclerotic lesions, is thought to play a major role in stimulating the migration of blood monocytes into developing atherosclerotic lesions. We therefore assessed the effects of estradiol in vivo on MCP-1 protein and mRNA expression in the descending thoracic aorta of rabbits fed a cholesterol-enriched (0.5%) diet for 6 weeks and in animals fed normal chow. MCP-1 protein was quantified by Western blot analysis and monocyte chemotaxis bioassay, and reverse transcription–polymerase chain reaction was used to ascertain the level of MCP-1 mRNA expression. We observed that in both ovary-intact and ovariectomized (OVX) animals, MCP-1 protein and mRNA expression were significantly increased by 6 weeks in animals fed a high-cholesterol diet. The cholesterol-induced increase in MCP-1 protein and mRNA expression was significantly attenuated in OVX rabbits supplemented with estradiol pellets (1.5- and 10.0-mg 60-day-release pellets), which yielded a range of estradiol concentrations encompassing the physiological levels. MCP-1 protein and mRNA expression were increased in normocholesterolemic OVX rabbits compared with normocholesterolemic ovary-intact animals, and this increase was prevented in OVX animals supplemented with estradiol pellets. Our observations indicate that both basal and hypercholesterolemia-induced increases in MCP-1 protein are modulated by physiological concentrations of estradiol.

Key Words: monocyte chemoattractant protein-1 • atherosclerosis • estrogen • estradiol

	Introduction

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The mechanisms by which estradiol protects against cardiovascular morbidity and mortality are not known. The adhesion of monocytes to endothelial cells^{1 2} followed by subendothelial migration are essential elements of an inflammatory response and occur continuously throughout the entire atherogenic process. We have recently presented preliminary data that estradiol supplementation to ovariectomized (OVX) rabbits fed a cholesterol-enriched (0.5%) diet inhibits both the adhesion of monocytes to endothelial cells and their subsequent subendothelial migration in vivo when compared with animals not receiving estrogen supplementation.³ It is therefore possible that estradiol inhibits the development of atherosclerotic lesions by regulating this inflammatory component of the atherogenic process. One chemokine that has been demonstrated to stimulate the migration of monocytes through an arterial endothelial monolayer is monocyte chemoattractant protein-1 (MCP-1).⁴ MCP-1 is a monomeric polypeptide with an estimated molecular mass of 12 kDa.^{5 6} Unlike other chemoattractants, MCP-1 is relatively specific for monocytes and lymphocytes. Polymorphonuclear leukocytes lack MCP-1 receptors and therefore do not respond to MCP-1.⁷ This chemokine is expressed in atherosclerotic lesions of both animals^{8 9} and humans,^{8 10} especially in areas of lesions enriched in macrophages. Studies by Cushing and Fogelman¹¹ have further suggested that differentiating monocytes produce MCP-1, which serves to amplify their own recruitment into the lesions. Estradiol inhibits JE/MCP-1 mRNA expression in murine macrophages induced by lipopolysaccharide¹² and talc,¹³ as well as in platelet-derived growth factor–induced murine fibroblasts.¹⁴ The inhibitory effect of estradiol on MCP-1 mRNA expression led to the speculation that the antiatherogenic effects of estradiol may be mediated by the prevention of macrophage accumulation in the atherosclerotic area.¹² Estradiol in addition inhibited the migration of human monocytes stimulated with either MCP-1¹⁵ or minimally oxidized LDL¹⁶ in vitro.

We therefore assessed whether feeding a cholesterol-enriched diet to rabbits increases the production of MCP-1 in the thoracic aorta and whether supplementation with estradiol can inhibit this production. The rabbit was chosen as the animal model because it is in persistent estrus¹⁷ ; therefore, the animals are under the constant influence of estradiol with no compounding influence of progesterone. Furthermore, previous studies¹⁸ of the time course of lesion development in hypercholesterolemic rabbits have provided important information as to when monocyte adhesion first occurs at lesion-prone sites and thus have enabled us to focus the current investigation on this inflammatory process and the role of estrogen in inhibiting MCP-1 expression.

	Methods

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Animals
New Zealand White rabbits weighing 3 to 3.5 kg were used (Irish Farms). All animals were initially fed regular chow for 2 weeks and then were divided into various groups as outlined under experimental protocols.

Ovariectomy was performed in some animals under anesthesia with halothane, following the regulations of the Animal Research Committee of the University of California. Under strictly aseptic conditions, a midline incision was made and both ovaries were removed. The abdominal incisions were closed in layers with 3-0 Dexon sutures. All animals received prophylactic antibiotics (Kefzol, 500 mg IM). At the time of surgery, animals were implanted subcutaneously with 60-day-release 17ß-estradiol pellets (1.5- or 10-mg pellets) or placebo pellets (Innovative Research of America). Animals were fed normal chow for 1 week after surgery, after which they were changed to a high-cholesterol (0.5% wt/wt normal chow) diet (Purina Mills, Inc) or continued on normal chow for 6 weeks.

Experimental Protocols
Protocol 1: MCP-1 Protein Expression in Ovary-Intact and OVX Cholesterol-Fed Rabbits
This protocol was designed to elucidate the effect of cholesterol feeding and the modulating role of endogenous estradiol production on MCP-1 expression. Animals were divided into the following 3 groups of 5 animals each: (1) ovary-intact animals fed normal chow; (2) ovary-intact animals fed a 0.5% cholesterol diet for 6 weeks; and (3) OVX animals fed a 0.5% cholesterol diet for 6 weeks. At the end of the feeding period the animals were euthanized, and the aortic tissue was dissected out, snap-frozen in LN₂, and stored for Western blot analysis for MCP-1 protein as described below.

Protocol 2: Effect of Ovariectomy and Estradiol Supplementation on MCP-1 mRNA and Protein Expression in Animals Fed a Cholesterol-Enriched Diet
This second protocol was designed to elucidate the modulating role of exogenous estradiol on MCP-1 production in cholesterol-fed animals. The animals were divided into the following 3 groups of 5 animals each: (1) OVX animals implanted with placebo pellets and fed a 0.5% cholesterol diet for 6 weeks; (2) OVX animals implanted with estradiol pellets (1.5 mg) and fed a 0.5% cholesterol diet for 6 weeks; and (3) OVX animals implanted with estradiol pellets (10 mg) and fed a 0.5% cholesterol diet for 6 weeks. At the end of the feeding period the animals were euthanized, and the aortic tissue was dissected out and stored for analysis of MCP-1 by Western blotting and bioassay. These tissues were also analyzed for MCP-1 mRNA by reverse transcription–polymerase chain reaction (RT-PCR).

Protocol 3: Effect of Ovariectomy and Estradiol Supplementation on Basal MCP-1 mRNA and Protein Expression in Animals Fed Normal Chow
The third protocol was designed to elucidate the modulating role of endogenous estradiol on MCP-1 expression in animals fed normal chow. Animals were divided into 4 groups of 5 animals each as follows: (1) ovary-intact animals fed normal chow for 6 weeks; (2) OVX animals implanted with placebo pellets and fed a normal diet for 6 weeks; (3) OVX animals implanted with 1.5-mg estradiol pellets and fed normal chow for 6 weeks; and (4) OVX animals implanted with 10-mg estradiol pellets and fed normal chow for 6 weeks. At the end of the feeding period the animals were euthanized, and the aortic tissue was dissected out and stored for analysis of MCP-1 protein and mRNA by Western blotting and RT-PCR, respectively.

Collection of Tissues
Animals were anesthetized with pentobarbital (50 mg/kg IV) and were euthanized by exsanguination. A section of aorta that extended from the bifurcation of the subclavian artery to the origin of the renal arteries was removed from each animal. Care was taken to protect the endothelial lining. The vessels, which were kept on ice, were then cleared of adhering adventitial fat and connective tissue and quickly snap-frozen in LN₂. RNA and protein were extracted from these tissues to ascertain the level of MCP-1 mRNA and protein by RT-PCR and Western blotting, respectively. Measurements were made on pooled samples of both the protein and mRNA from all 5 animals in each group and individually from 3 animals in each of the groups under protocol 2.

RT-PCR
The RT-PCR technique was used to ascertain levels of MCP-1 mRNA in rabbit descending thoracic aortas after having undergone various treatments. Tri-reagent (Molecular Research Center) was used to homogenize thoracic aortas and to extract total RNA according to the manufacturer's instructions. RT for MCP-1 was performed with 3 µg of the total RNA sample, 50 U of Moloney murine leukemia virus reverse transcriptase, and 100 pmol of oligo-dT. The reaction was run at 42°C for 20 minutes. The resulting cDNA samples were PCR-amplified with a GeneAmp RNA PCR kit (Perkin-Elmer) and 100 ng each of rabbit MCP-1 sense and antisense primers in a 100-µL reaction mixture. The final reaction mixture was initially heat-denatured at 94°C for 5 minutes followed by PCR amplification of MCP-1 cDNA for 40 cycles each of denaturation (94°C for 1 minute), primer annealing (60°C for 1 minute), and extension (72°C for 1.5 minutes). This procedure was followed by a final extension at 72°C for 5 minutes. A 500-ng aliquot of total RNA was reverse-transcribed under similar conditions followed by PCR amplification of GAPDH cDNA for 30 cycles each of denaturation (94° for 30 seconds), primer annealing (55°C for 30 seconds), and extension (72°C for 1 minute). Initially, the quantity of total RNA for RT-PCR and the number of cycles were determined to ensure that PCR amplification of target cDNAs (MCP-1 and GAPDH) remained in the exponential range and that saturation had not occurred. Primers specific for GAPDH were used as a positive control for RT-PCR. Control samples analyzed without reverse transcriptase were free of any amplification product (data not shown).

The following primers were used (Custom Primers, GIBCO-BRL): MCP-1, (rabbit spleen) 5'-GTCTCTGCAACGCTTC- TGTGCCTG-3' (sense) and 5'-CAATGAAGTAGTAGTAGAGG- GTGT-3' (antisense)¹⁹ ; GAPDH, (rat brain) 5'-GTGAAGGTCGGT- GTCAACCGGATTT-3' (sense) and 5'-CACAGTCTTCTGAGTG- GCAGTGAT-3' (antisense).²⁰

Each final PCR product sample (30 µL) was electrophoresed on a 1.5% agarose gel and visualized by ethidium bromide staining under UV light. The amplified DNA fragments obtained were of the appropriate base-pair sizes: 369 bp (MCP-1) and 558 bp (GAPDH). The relative intensities of the bands were quantified by densitometric analysis (Personal Densitometer SI, Molecular Dynamics). PCR products were cloned into TA vector (pCR 2.1, Invitrogen), and their identities were confirmed by DNA sequencing (data not shown).

Western Blotting
The tissues were homogenized in 50 mmol/L triethanolamine hydrochloride (pH 7.4) containing 0.1 mmol/L EGTA, 0.1 mmol/L EDTA, 0.5 mmol/L DTT, 1 µmol/L pepstatin A, and 2 µmol/L leupeptin at 4°C with the aid of a tissue grinder fitted with a Teflon pestle. The protein concentrations were determined by using Bio-Rad protein reagent (Bio-Rad Laboratories) and BSA as the standard. Protein samples (50 µg per lane) were separated on 15% SDS polyacrylamide gels and electrophoretically transferred to a Hybond polyvinylidine difluoride (PVDF) membrane (Amersham Life Science, Inc) by using a semidry transfer system (Pharmacia Biotech). Equal loading in each lane was confirmed by staining the PVDF membrane with Ponceau S solution (Sigma Chemical Co). The nonspecific binding on the PVDF membrane was blocked by incubation with 10% (wt/vol) nonfat dry milk and 0.1% Tween-20 in PBS (wash buffer) for 2 hours at room temperature. The membrane was incubated with an anti-human MCP-1 mouse monoclonal antibody (R&D System) at 1:500 dilution in PBS containing 1% nonfat milk and 0.1% Tween-20 for 1 hour at room temperature. The membrane was washed 3 times for 15 minutes each with wash buffer (PBS containing 0.1% Tween-20) and incubated for 1 hour at room temperature with a donkey anti-mouse horseradish peroxidase–conjugated secondary antibody at 1:2500 dilution. The membranes were washed 3 times with wash buffer, after which they were incubated for 2 minutes at room temperature in chemiluminescence reaction detection reagents (ECL Western blotting, Amersham). The membranes were then exposed to autoradiography film (Hyperfilm-ECL, Amersham). The relative intensities of the bands were quantified by densitometric analysis.

Monocyte Chemotaxis Bioassay
The protein samples from aortic extracts were assayed for monocyte chemotactic activity by using a standard Neuroprobe chamber as described.²¹ The extracts were diluted in Tyrode's buffer at 1:20, 1:40, 1:80, and 1:160 dilutions. Triplicate wells of the base of the chamber were filled with the sample. Positive (fMet-Leu-Phe) and negative controls were also run in separate chambers. The 5-µm-pore-diameter polycarbonate membrane (Nucleopore) was placed on the top and the chamber was tightened. The chamber was prewarmed at 37°C. Aliquots of a suspension of human monocytes containing 200 000 monocytes per milliliter were then added to the upper wells. The chamber was incubated at 37°C for 60 minutes. The membrane was then removed. Adherent monocytes on the top were eliminated and the membrane was stained with 0.1% crystal violet. The numbers of transmigrated monocytes in 9 fields were then counted under high magnification (x400). Data are presented as mean±SD of the counted monocytes in triplicate wells. Specificity of the bioassay for MCP-1 in aortic extracts was tested by performing the assays after preincubation with anti-human MCP-1 antibodies (Antigenix America).

Radioimmunoassay (RIA) for Estradiol
The plasma concentrations of estradiol (E₂) were measured by specific RIAs as previously described.²² Known amounts (400 cpm) of [³H]E₂ were added to 0.6 to 0.8 mL of plasma. Each plasma sample was then extracted with diethyl ether and the extract evaporated to dryness. The dried plasma extracts were dissolved in isooctane and then applied to a Celite column for chromatographic separation of E₂ as described by Brenner and coworkers.²³ Steroid fractions collected from the column were dried and reconstituted with assay buffer for RIA. The results showed that peaks of radioactive and immunoreactive estrogens coincided in the Celite column fractions.¹⁷

Assay for Lipids
Plasma total cholesterol (TC) was measured at baseline and at the time of sacrifice in each animal. All samples were analyzed in triplicate with enzymatic colorimetric assays as described previously.^{24 25 26} The assays were performed in the Molecular Biology Institute Lipid and Lipoprotein Laboratory, UCLA, Los Angeles, Calif. This laboratory is certified by the Centers for Disease Control and Prevention, National Heart, Lung, and Blood Institute (National Institutes of Health, Bethesda, Md) Lipid Standardization Program and meets their criteria for precision and accuracy.

	Results

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Protocol 1: MCP-1 Protein Expression in Ovary-Intact and OVX Cholesterol-Fed Rabbits
The mean E₂ and TC levels of the animals in the 3 groups are shown in Table 1. Western blot analysis of pooled samples from each group, as well as the arbitrary densitometric units (ADUs), are shown in Figure 1A and 1B, respectively. In ovary-intact animals fed a cholesterol-enriched diet, MCP-1 expression increased by 60%, whereas in the OVX animals fed a cholesterol-enriched diet, MCP-1 expression increased by 260%.

View larger version (29K): [in this window] [in a new window]   Figure 1. A, Effect of high-cholesterol diet and ovariectomy on MCP-1 protein expression. Ovary-intact rabbits were fed normal chow for 6 weeks (lane 1), fed a high-cholesterol diet for 6 weeks (lane-2), or OVX and fed a high-cholesterol diet for 6 weeks (lane 3). Western blotting was performed with 50 µg of protein from homogenates obtained from descending thoracic aortas as described in Methods. Approximate molecular weight for MCP-1 protein as detected by Western blot was 12 kDa. Western blotting was performed with pooled (n=5) tissues from the same 3 groups. Nonspecific band corresponding to 66 kDa was also observed. B, Densitometric scan of Western blot in Figure 1A. * and ** signify significant difference (P<0.05) from lane 1 and from lane 2, respectively.

Protocol 2: Effect of Ovariectomy and Estradiol Supplementation on MCP-1 mRNA Expression and Protein Production in Animals Fed a Cholesterol-Enriched Diet
The mean E₂ and TC levels in the animals in the 3 groups are shown in Table 1. These results indicated that ovariectomy reduced endogenous circulating E₂ concentrations, whereas supplementation with 1.5- or 10-mg estradiol pellets led to a dose-related increase in the circulating concentrations of E₂ that encompassed the physiological range. The Western blot analysis of MCP-1 protein expression of pooled samples from the 3 groups is shown in Figure 2A, and the ADUs of Western blots performed on each of the 3 groups is shown in Figure 2B. Estradiol at both the lower and higher dose reduced MCP-1 expression by 20% and 47%, respectively. To assess the range of MCP-1 expression in individual animals in the OVX and estradiol-supplemented (10-mg pellet) groups, we performed a Western blot analysis of MCP-1 for each individual animal, ie, 3 from the OVX group and 3 from the estradiol-supplemented group. All samples were run on the same gel (Figure 3A). The mean ADUs from the 2 groups, with their SEs, are shown in Figure 3B. Estradiol supplementation significantly suppressed MCP-1 protein by 37%. The results of the bioassay for MCP-1 protein from these 2 groups of animals are summarized in Table 2. When the aortic extracts of OVX and placebo-treated animals were tested for their ability to cause transmigration of monocytes, a concentration-dependent effect was observed. This activity was significantly attenuated in the presence of anti-human MCP-1 antibodies. The ability of aortic extracts from OVX and estradiol-treated animals was significantly less when compared with those obtained from OVX and placebo-treated animals. Positive and negative controls were 30±2.5 and 1.3±0.4, respectively. The bioassay results paralleled the data obtained by Western blot analysis of MCP-1.

View larger version (34K): [in this window] [in a new window]   Figure 2. A, Effect of 17ß-estradiol supplementation on MCP-1 protein expression in OVX rabbits fed a high-cholesterol diet for 6 weeks. Rabbits were OVX, given placebo, and fed a high-cholesterol diet (lane 1); OVX, supplemented with 17ß-estradiol pellets (1.5 mg), and fed a high-cholesterol diet (lane 2); or OVX, supplemented with 17ß-estradiol pellets (10 mg), and fed a high-cholesterol diet (lane 3). Western blotting was performed with 50 µg of protein from homogenates obtained from descending thoracic aortas as described in Methods. Western blotting was performed with pooled (n=5) samples from the same 3 groups. Approximate molecular weight for MCP-1 protein was 12 kDa. Nonspecific band corresponding to 66 kDa was also observed. B, Densitometric scan of Western blot in Figure 2A. *Signifies significant difference (P<0.05) from lane 1.

View larger version (47K): [in this window] [in a new window]   Figure 3. A, Effect of 17ß-estradiol supplementation on MCP-1 protein expression in OVX rabbits fed a high-cholesterol diet for 6 weeks. Rabbits were OVX, supplemented with placebo, and fed a high-cholesterol diet (lanes 1 through 3) or OVX, supplemented with 17ß-estradiol (10 mg), and fed a high-cholesterol diet (lanes 4 through 6). Western blotting was performed with all samples (n=3) run individually on the same gel. B, Mean of densitometric analyses from Western blots in Figure 3A. *Signifies significant difference (P<0.05) from high cholesterol–fed and placebo pellet–implanted animals.

The pooled mRNA expression paralleled the protein expression (Figure 4A). In the OVX groups, MCP-1 mRNA expression showed a 4.5-fold increase when compared with GAPDH. However, in the animals supplemented with the 1.5- and 10-mg pellets, no rise in MCP-1 mRNA was seen, and expression was 2.8-fold and 1.4-fold of that of GAPDH, the latter being similar to that observed in ovary-intact animals.

View larger version (50K): [in this window] [in a new window]   Figure 4. A, Effect of 17ß-estradiol on MCP-1 mRNA expression in OVX rabbits fed a high-cholesterol diet for 6 weeks. Rabbits were OVX and fed high-cholesterol diet (lane 1); OVX, supplemented with 17ß-estradiol (1.5 mg), and fed a high-cholesterol diet (lane 2); or OVX, supplemented with 17ß-estradiol (10 mg), and fed a high-cholesterol diet (lane 3). Total RNA was extracted from descending thoracic aortas by using Tri-reagent, and RT-PCR was performed with 3 µg of total RNA taken from pooled samples of each group with the use of rabbit MCP-1–specific primers (top) and 500 ng of total cellular RNA with the use of GAPDH-specific primers (bottom) as described in Methods. Each final PCR product sample (30 µL) was loaded on 1.5% agarose gel, electrophoresed, and visualized under UV light after ethidium bromide staining. Base pair (bp) size markers (x174/HaeIII) are shown at left of each panel. Arrows indicate expected sizes of PCR product; 369 bp for MCP-1 and 558 bp for GAPDH. B, Densitometric scan of RT-PCR product of pooled samples in Figure 4A. *Signifies significant difference (P<0.05) from lane 1.

Protocol 3: Effect of Ovariectomy and Estradiol Supplementation on Basal MCP-1 mRNA Expression and Protein Production in Animals Fed Normal Chow
The mean E₂ and TC levels are shown in Table 1. The Western blot analysis of pooled samples for MCP-1 expression from each of the 4 groups is shown in Figure 5A. The ADUs of Western blot analysis performed on each of the 4 groups are shown in Figure 5B. Compared with ovary-intact rabbits, ovariectomy increased MCP-1 expression by 2.3-fold. Estradiol supplementation at both the lower and higher dose attenuated the increase in MCP-1 protein expression by 34% and 51%, respectively. The values for pooled mRNA expression paralleled those for protein expression (Figure 6A). In Figure 6B are shown the ADUs of mRNA from each of the groups. In OVX animals fed normal chow, the increase in MCP-1 mRNA expression was 2.6-fold that of GAPDH. In OVX rabbits that were also implanted with 1.5- or 10-mg estradiol pellets, MCP-1 mRNA expression was considerably less and had increased by only 2-fold or 1.2-fold, respectively, relative to GAPDH (Figure 6B).

View larger version (39K): [in this window] [in a new window]   Figure 5. A, Effects of ovariectomy and 17ß-estradiol supplementation on MCP-1 protein expression in rabbits fed normal diet for 6 weeks. Rabbits were ovary-intact and fed normal chow (lane 1); OVX, supplemented with placebo pellets, and fed normal chow (lane 2); OVX, supplemented with 17ß-estradiol (1.5 mg), and fed normal chow (lane 3); or OVX, supplemented with 17ß-estradiol (10 mg), and fed normal chow (lane 4). Western blotting was done with 50 µg protein obtained from descending thoracic aorta tissue homogenates as described in Methods. Western blotting was performed with pooled samples (n=5) from the same groups. B, Densitometric scan of Western blot in Figure 5A. *Signifies significant difference from lane 1. **Signifies significant difference from lane 2.

View larger version (56K): [in this window] [in a new window]   Figure 6. A, Effects of ovariectomy and 17ß-estradiol supplementation on MCP-1 mRNA expression in rabbits fed a normal diet for 6 weeks. Rabbits were ovary-intact and fed normal chow (lane 1); OVX, supplemented with placebo, and fed normal chow (lane 2); OVX, supplemented with 17ß-estradiol (1.5 mg), and fed normal chow (lane 3); or OVX, supplemented with 17ß-estradiol (10 mg), and fed normal chow (lane 4). RT-PCR was performed as described in legend to Figure 4 and Methods by using pooled total RNA from descending thoracic aortas from animals in each group. Top panel depicts MCP-1, and bottom, GAPDH bands. B, Densitometric scan of RT-PCR product in Figure 6A. *Signifies significant difference (P<0.05) from lane 1. **Signifies significant difference (P<0.05) from lane 2.

	Discussion

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In concurrent studies, we have recently presented preliminary evidence that estrogen inhibits both monocyte adhesion and transendothelial migration in hypercholesterolemic rabbits.³ We further demonstrated that the estrogen-mediated reduction in monocyte adhesion is probably due to an inhibition of the expression of vascular cell adhesion molecule-1 (VCAM-1). However, it is not clear from these studies whether the associated reduction in the number of monocytes within the subendothelial space is simply a function of a reduction in adhesion or is also due to an estrogen-induced inhibition of the expression of monocyte-specific chemokines such as MCP-1. Thus, the primary objective of this study was to assess the role of estradiol in modulating both basal and hypercholesterolemia-induced MCP-1 protein production and mRNA expression in the thoracic aortas of rabbits in vivo. In animals fed normal chow, MCP-1 mRNA expression was apparently undetectable, because a visible band for MCP-1 appeared only after 3 µg of total cellular RNA was used for 40 cycles of PCR amplification and visualization on 1.5% agarose gel after ethidium bromide staining. Feeding the animals a cholesterol-enriched diet led to an 3-fold increase in MCP-1 levels by 6 weeks. This time interval was selected on the basis of our previous quantification of the actual number of monocytes both adherent to the endothelium and located immediately beneath the endothelium in the thoracic aorta in vivo at 4 weeks in animals fed a cholesterol-enriched diet.^{3 18} The modulating role of estradiol on MCP-1 expression was assessed after feeding animals a cholesterol-enriched diet for 6 weeks to ensure that the effects of estradiol on MCP-1 expression were manifested for a longer duration of time.

Estradiol at physiological levels significantly reduced, in a dose-dependent fashion, both MCP-1 gene and protein expression in animals fed a cholesterol-enriched diet to values observed in ovary-intact animals fed normal chow. This decrease in the expression of MCP-1 was also confirmed in a monocyte chemotactic bioassay. These results indicate that estradiol most likely exerts its regulatory effect on MCP-1 at the level of transcription. However, a direct inhibitory action of estradiol on MCP-1 production in macrophages has also been demonstrated by other investigators.^{12 13 14 16}

Potential mechanisms by which estradiol may inhibit MCP-1 expression must also be considered. For example, NO inhibits MCP-1 gene expression²⁷ and estradiol increases NO production in endothelial cells.^{28 29 30 31} Thus, it is possible that estradiol indirectly inhibits the expression of MCP-1 by increasing NO production. In human umbilical vein endothelial cells, basal MCP-1 is increased after inhibition of NO synthesis, whereas exogenous NO inhibits MCP-1 expression.²⁷ In the same study, it was shown that inhibition of NO synthesis activated proteins capable of binding to oligonucleotides containing the consensus sequence for a nuclear factor-B binding site, suggesting a molecular link between an oxidant-sensitive transcriptional regulatory mechanism and NO synthesis in human umbilical vein endothelial cells . It is possible that estradiol, acting either directly or through NO, may also modulate other transcription factors such as SP-1 and AP-1. AP-1 has recently been characterized as an antioxidant-responsive transcription factor.^{32 33} Estradiol-induced decreases in MCP-1 mRNA expression^{12 14} as well as increases in NO synthase expression³⁴ have been assumed to be mediated in part by estrogen receptor (ER), because tamoxifen, an ER receptor antagonist, attenuates these effects. However, it is possible that part of the effect of estradiol on the vessel wall may be mediated by the newly discovered ERß, as it has recently been demonstrated that after carotid arterial injury in a mouse model in which the ER gene is disrupted, estradiol inhibited vascular smooth muscle cell proliferation.³⁵ Further studies on the type of receptor on which estradiol acts to retard atherogenesis need to be conducted.

Estradiol also acts as an antioxidant in vivo,³⁶ and in its absence, an increase in lipoprotein oxidation in the artery wall could account for the increase in MCP-1 expression observed in the OVX animals. This scenario is also possible in the animals fed normal chow, as rabbit chow contains significant amounts of linoleic acid, which makes normal rabbit LDL especially susceptible to oxidation.³⁷ Furthermore, there is evidence that circulating estradiol can also modulate the release of cytokines in vivo. Cytokines such as tumor necrosis factor- (TNF-) are known to modulate MCP-1 expression.^{38 39} Physiological concentrations of estradiol have a marked inhibitory effect on the production of TNF- by human peripheral blood mononuclear cells from postmenopausal women.⁴⁰ Similarly, estrogen withdrawal is associated with an increased potential for human bone marrow cells to release various cytokines, including TNF-, interleukin-1, and interleukin-6.⁴¹ Therefore, removal of the endogenous source of estradiol by ovariectomy may, by itself, lead to endothelial cell activation by the release of cytokines, thereby resulting in increased MCP-1 expression in vascular tissue.

In summary, our results demonstrate for the first time that estradiol at physiological concentrations inhibits MCP-1 expression in vivo. This finding therefore may be one potential mechanism by which estradiol retards atherogenesis.

	Acknowledgments

 
This work was supported in part by the American Federation for Aging Research (AFAR), Inc (L.N.); the Hartford Foundation (L.N.); the National Heart, Lung, and Blood Institute grants HL-46843 and HD-31467 (National Institutes of Health, Bethesda, Md) (G.C.); and the Laubisch Fund for Cardiovascular Research (L.N., G.C.). We wish to thank Janis Cuevas for her technical expertise in the animal preparations.

Received July 11, 1997; accepted April 10, 1998.

	References

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