Upregulation of Estrogen Receptor- Expression in Rabbit Cardiac Allograft

From the Departments of Surgery, Physiology and Biophysics (P.W.R.) and Lombardi Cancer Center (M.B.M., A.S.), Georgetown University Medical Center, Washington, DC.

Correspondence to Marie L. Foegh, MD, DSc, Georgetown University Medical Center, 4000 Reservoir Rd, NW, Washington DC 20007. E-mail mfoegh{at}aol.com

	Abstract

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Abstract—Estrogen receptor (ER) expression has been detected in different tissues, and estradiol-17ß treatment protects against experimental transplant arteriosclerosis. In this study, ER- expression in the rabbit hearts and attached aortas before and after cardiac-aorta allograft transplantation was examined. Ten male New Zealand White rabbits were transplanted with cardiac-aorta allografts from male Dutch Belted rabbits. This transplant arteriosclerosis model uses a 0.5% cholesterol diet and immunosuppression with cyclosporin A (10 mg · kg^-1 · d^-1) until euthanatization 42 days later. The cardiac grafts with the attached aorta were harvested. Strong staining of ER- protein was shown in the coronary arteries of the cardiac allografts by immunohistochemistry with the use of a mouse anti-human ER- monoclonal antibody (ID5). In contrast, both the nongrafted hearts of the recipients and donor hearts expressed only weak staining. RNase protection assay with the use of a ³²P–labeled ER- antisense riboprobe (pOR 300) proved that the basal expression of ER- mRNA is similar in the nongrafted aorta of both recipients and donors. A marked increase of ER- mRNA was observed in the allograft aorta compared with the nongrafted aorta (289±69%, P<0.02) by reverse transcription and polymerase chain reaction. The DNA sequence analysis confirmed that the polymerase chain reaction–amplified fragment corresponded to ER-. This is the first observation of ER- upregulation in the allograft vasculature and may relate to the allograft cardiovascular protective effects of estrogen.

Key Words: cardiac allograft • artery, coronary • estrogen receptor • gene expression

	Introduction

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Transplant arteriosclerosis compromises the long-term success of organ transplantation.^{1 2} We discovered earlier that estradiol-17ß treatment protects against myointimal hyperplasia in aorta and cardiac transplantation models in rats and rabbits.^{3 4 5} More recently, (1) abolition of class II major histocompatibility complex antigen^{5 6} and (2) inhibition of both insulin-like growth factor-I (IGF-I) and IGF-I receptor expression in coronary arteries and aorta of cardiac allografts, were evident after estradiol-17ß treatment.^{7 8 9} These inhibitory effects were associated with abrogation of proliferation of cultured primary smooth muscle cells as well as allograft aorta explants ex vivo.^{7 8 10}

Estrogen has diverse effects on various organs, tissues, and cells.^{11 12 13 14 15 16 17 18} These effects are mediated through the estrogen receptors (ER) that belong to the super family of ligand-activated nuclear transcription factors. In vascular tissue, ER- expression has been demonstrated in coronary arteries,^{12 13 14} aorta, and venous smooth muscle cells (SMC),^{15 16 17 18} and endothelial cells.¹² In this study, we examined whether ER- is expressed in the cardiac allograft and attached aortas, whether the expression of this receptor is altered after transplantation, and whether altered ER- expression occurs at the mRNA level.

	Materials and Methods

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Rabbit Cardiac Transplantation
Ten male New Zealand White (NZW) rabbits received cardiac-aorta allografts from 10 male Dutch Belted (DB) rabbits through the use of end-to-side aorta anastomosis to the carotid artery and pulmonary artery anastomosis to the external jugular vein, as described previously.⁷ Four recipient and 4 donor strains served as nongraft controls. All of the animals were fed a 0.5% cholesterol diet for 1 week before transplantation and until killed. Four recipient and 4 donor strains on a regular diet served as additional controls for nongraft controls. The transplant recipients were immunosuppressed with cyclosporin A (10 mg · kg^-1 · d^-1, Sandoz) for graft survival from the day of transplantation until death at 6 weeks after transplantation.

Harvest of Hearts and Aortas
At the time of death, both native and allograft hearts from the recipients (n=10) as well as the hearts from nongraft control of donors (n=4) and recipients (n=4) on a 0.5% cholesterol diet for 1 week were pressure perfused with tissue fixative (Histochoice, Amresco).⁵ The native hearts from both donors and recipients on a regular diet (n=4) were also harvested and served as additional controls. Slides (5 µm) were prepared from transversally sectioned hearts embedded in paraffin for immunohistochemistry staining. The attached aortas of the hearts were isolated, snap-frozen in liquid nitrogen, and stored at -70°C until mRNA analysis. Four aortas from the nongraft recipient strain were also harvested for primary SMC cultures, as described earlier,⁷ and these cells exhibited characteristic positive SMC -actin immunostaining.

Immunohistochemistry for Identification of ER-
The presence of ER- in the coronary artery was detected by immunohistochemical staining with the use of Histostain SP kit (Zymed), as described previously.⁷ ERID5 (Immunotech Inc), a mouse anti-human ER monoclonal antibody that recognizes the A/B domain of ER-,¹⁹ was used as a primary antibody at a dilution of 1:50 (IgG concentration of 2.8 µg/mL). Three negative controls were applied. They consisted of (1) the use of a preimmune mouse IgG (Sigma) at 5 µg/mL as a primary antibody, (2) omission of a primary antibody, and (3) the use of an irrelevant monoclonal antibody (anti-human interferon gamma [IFN-], Genzyme). ER- expression was semiquantified by comparison, in a double-blind manner, for the intensity of positive-staining under the light microscope. The staining was graded (1) none (same as negative control), (2) weak (light orange color), (3) moderate (orange color), and (4) extensive (brownish color).

RNase Protection Assay (RPA)
Total cellular RNA of the aorta and of the aorta SMC was extracted using TRIzol reagent (Life Technologies), and the total RNA-concentration was measured spectrophotometrically. An RPA II kit (Ambion) was used in accordance with the protocol of the manufacturer. Two plasmids, pOR 300 (containing a 288-bp segment of exon 1 of human ER- DNA)¹² and pT7 18S (80 bp of human 18S, Ambion) were used to generate ³²P-labeled antisense RNA probes. Forty micrograms of total RNA from samples; an ER-positive human breast cancer cell line, MCF-7; and human saphenous vein SMC (HSVSMC) from a male volunteer (gift of Dr P. Libby, Brigham & Women's Hospital, Boston, Mass) were hybridized with probes. The protected mRNA bands were analyzed on a 5% polyacrylamide gel containing 8 mol/L urea and quantified by the use of the National Institute of Health image 1.6 software.

Reverse Transcription–Polymerase Chain Reaction (RT-PCR)
Because of the small size of the allograft aorta, only a limited amount of RNA can be obtained, which precludes the use of RPA to quantitate the mRNA in the allografts. Thus the expression of ER- mRNA in the allograft aorta and the control samples were semiquantified by highly sensitive RT-PCR. Two micrograms of total RNA from the samples was used for reverse transcription. Two microliters of the first strand cDNA for the vascular samples and 1 µL for MCF-7 cells were amplified for ER- and 1 µL for GAPDH, which served as an internal control. The primers for ER- (5', 762 to 779 and 3', 1040 to 1023 nucleotides of rat ER- cDNA)¹⁵ and GAPDH (5', 228 to 253 and 3', 964 to 939 nucleotides of human GAPDH cDNA) were used for amplification with an RNA PCR kit (Perkin Elmer). In the presence of ³²P dCTP (0.5 µCi/100 µL), the amplification was done according to the standard protocol for 35 cycles of 1 minute at 95°C, 1 minute at 55°C, and 1 minute at 72°C after initial denature at 95°C for 4 minutes. The final cycle was followed by 3 minutes of extension. The amplification products were size-fractionated by electrophoresis in a 1% agarose gel and visualized by ethidium-bromide staining. The DNA fragments, which correspond to the predicted size for ER- (279 bp) and GAPDH (738 bp), were isolated. Incorporation of ³²P into the fragments was measured in a scintillation counter.

Statistics
The semiquantification of ER- mRNA by RT-PCR and RNase protection assay was normalized from at least 3 analyses and corrected by GAPDH for the same sample. Results are presented as mean±SEM. Statistical analyses were performed using a paired Student t test. The value of P<0.05 was considered significant.

	Results

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Enhanced Expression of ER- Protein in Allograft Vessels
Extensive staining of the ER- was observed by immunohistochemistry predominantly in the myointima and the media of the coronary arteries of the cardiac allografts (Figure 1A), and moderate diffused staining was seen in the media of the coronary arteries of the recipient hearts after transplantation (Figure 1B). Only weak staining was evident in the medium of the coronary arteries of the donor (Figure 1C) and the recipient nongraft hearts (Figure 1D). Weak diffused staining was shown in the coronary arteries of both control donor and recipient hearts on a regular diet (Figure 1E and 1F). ER-–positive staining was also shown in the infiltrating monocytes in the myocardium of the cardiac allografts (Figure 1A). No staining was shown in the native and cardiac allografts when preimmune mouse IgG was used as a primary antibody or when either the secondary antibody only or an irrelevant monoclonal antibody (anti-human INF-) was used (data not shown).

View larger version (165K): [in this window] [in a new window]   Figure 1. Expression of ER- in coronary arteries of native hearts and cardiac allografts by immunohistochemistry with the use of a mouse anti-human ER- monoclonal antibody (ERID5, final dilution of 1:50). A, Extensive staining for ER- in the entire myointimal and medial area of an allograft coronary artery. B, Moderate staining of a coronary artery in the native heart (x400). C, Weak staining in a coronary artery of the donor heart before transplantation. D, Weak staining in a coronary artery of the recipient heart before transplantation. E and F, Weak staining in coronary aorta of the donor and recipient hearts before transplantation on a regular diet (x600). Magnification of the sections is x200 unless stated otherwise.

ER- mRNA Expression in Vascular Wall and SMC of Native Vessels
RNase protection assay was used to measure the steady-state levels of ER- mRNA in the rabbit aortas, cultured rabbit aorta SMC, and the controls. Weak protected bands were observed in the nongraft aorta from both recipient (NZW, Figure 2, lanes 1 and 2) and donor (DB, Figure 2, lanes 3 and 4) rabbits, the recipient aortic SMC (Figure 2, lane 6) as well as the HSVSMC (positive control, Figure 2, lane 7). Nongraft aorta of NZW and DB rabbits showed no significant difference in the band intensity (in absorbency units, 118±7 versus 104±6, P>0.05). An intense band consistent with the size of the ER- riboprobe was shown in the positive control, MCF-7 (288 bp, Figure 2, lane 5). The bands shown in the aorta and SMC were slightly smaller than those of the MCF-7 cell (280 bp). The intensity of the band in the MCF-7 cell was 21-fold greater than the rabbit aortas and SMC (710±60 versus 34±3.7, P<0.02) and 6-fold stronger than that of HSVSMC (710±60 versus 120.4±14, P<0.02).

View larger version (78K): [in this window] [in a new window]   Figure 2. An autoradiograph of ER- mRNA expression in the aorta of both donor and recipient rabbits before transplantation and also in recipient aorta SMC, as detected by RNase protection assay. Forty micrograms of total RNA of the samples was used in every reaction. A strong protected band of ER- mRNA expression was seen in MCF-7 cells (288bp, lane 5). Weak mRNA fragments were consistently shown in the rabbit aorta from recipients (NZW, lanes 1 and 2) and donors (DB, lanes 3 and 4), recipient aortic SMC (lane 6), and HSVSMC (lane 7). Human 18S riboprobe (80 bp) was used as loading control (lower panel). The figure represents 3 similar results.

Increased Expression of ER- mRNA in the Allograft Vessels
In the RT-PCR, 35 cycles were chosen to ensure the linear range of specific amplifications from the earlier study (data not shown). Agarose gel electrophoresis of the amplified products showed a single band approximately 279 bp (Figure 3A), which was confirmed by sequence analysis to correspond to the C-terminal of the A/B domain through the DNA-binding domain of ER-. The sequence of this DNA fragment is 89% identical to human ER- (M12674) (unpublished data). The strong ER- mRNA expression was observed in the allograft aortas (Figure 3, lane 4) compared with the nongraft aortas (Figure 3, lanes 1, 2, and 3). When measured by incorporation of ³²P, the expression of ER- mRNA in the allograft aortas was increased 289±69% versus that of the donor nongraft aorta (100±10.2%, P<0.02). There was no significant difference in the ER- mRNA expression among the recipient aorta–harvested pretransplantation (PRE TX) (100±18%, Figure 3, NZW-PRE TX) and posttransplantation (POST TX) (121±23%, P>0.05, NZW-POST TX), the aorta SMC (123±14%, lane 5, NZW SMC), and the HSVSMC (132±19%, lane 7; all P>0.05). The ER- mRNA expression in the MCF-7 cell line was 6-fold higher than rabbit SMC (596±56% versus 123±14%, P<0.002). The quantitative data for the ER- RT-PCR are shown in Figure 3B and summarized in the Table.

View larger version (38K): [in this window] [in a new window]   Figure 3. Expression of ER- mRNA in rabbit aorta and aorta SMC by RT-PCR. A, RT-PCR product resulted in the 1% agarose gel displaying a single band of ER- (279 bp) and loading control, GAPDH (738 bp). NZW-PRE TX, indicates recipient aorta harvested before transplantation; NZW-POST TX, recipient aorta harvested after transplantion; DB-PRE TX, donor aorta harvested before transplantation; DB-ALLOGRAFT, allograft aorta; and NZW SMC, aortic SMC derived from recipient aorta before transplantation. MCF-7 cells are 1/2 volume of the template DNA as the rest samples. B, Semiquantification of ER- mRNA by measuring 32P incorporation into the amplified ER- DNA after correction by GAPDH. The data represents 4 experiments and expresses as percentage change of mean±SEM of count per minute (CPM) compared with donor aorta before transplantation.

View this table: [in this window] [in a new window]   Table 1. ER- mRNA Expression in Various Tissues by RT-PCR

	Discussion

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In this study, we report for the first time that the expression of ER- protein and ER- mRNA are upregulated in male rabbit allograft vessels compared with the nongraft vessels of the donors and recipients. The expression of ER- in coronary arteries of the rabbit hearts was detected by immunohistochemistry with the use of a mouse anti-human ER antibody because a specific anti-rabbit ER- antibody is not available. The epitope of this antibody is in the N terminal of the A/B domain of ER-, which has very low homology compared with that of ER-ß (16.5%),²⁰ and it does not crossreact with the androgen, glucocorticoid, or progesterone receptors.¹⁹ This antibody is routinely used to screen human breast and uterus carcinoma at 1:100 dilution. We chose to use a higher antibody concentration (1:50 dilution) because of the lesser number of ER- expressed in the vasculature, as shown by RPA in this study and because of reduced sensitivity of the antibody caused by the sequence differences between human and rabbit ER-. No staining was observed in the negative controls when double the amount of preimmune mouse IgG was used as the primary antibody, in the secondary antibody only condition, or when an irrelevant monoclonal antibody was used. We believe that the positive staining detected in the coronary arteries is ER- protein. The moderate-to-weak expression of ER- in the nongraft hearts of donor and recipient versus the extensive expression in the cardiac allografts suggests that basal expression of ER- in the rabbit vasculature can be upregulated in the allograft vasculature.

The localization of ER- protein exclusively in the myointimal and medial layers of the coronary arteries indicates that the cells expressing ER- are endothelial cells, vascular SMC, and infiltrating monocytes.^{5 21} The ER- staining in the nongraft arteries seems to be diffuse, which has often been seen in the vasculature in the recipient and donor heart with or without a cholesterol diet, instead of typical nuclear localization of ER- in isolated aorta SMC (data not shown).¹⁸

Our next question was whether the upregulation of ER- protein occurred at the mRNA level after transplantation. Because we have always found that the aorta of the cardiac allograft exhibits histological changes (ie, expression of growth factors and mitogenic response) similar to the coronary arteries, we omitted the immunostaining of the aorta of the cardiac allograft and used these tissues for demonstrating and semiquantificating the expression of mRNA of ER-. The steady-state levels of ER- mRNA in the nongraft aortas of the donor and recipient strains were similar when measured by either RT-PCR or RPA. However, the expression of ER- mRNA was found to be significantly upregulated (3-fold) in the allograft aorta compared with the nongraft aortas when semiquantitative RT-PCR was used. This supports our finding of upregulated expression of ER- protein in the allograft vasculature. By the protection assay, the amount of the mRNA in the aorta and SMC was substantially less (21 times) than the ER-–positive control, MCF-7 cells; this also has been reported by others.^{11 17} The protected fragment of ER- mRNA in the vascular and SMC was slightly smaller (280 bp) than the size of MCF-7 cell (288 bp). Because the RNA probe (pOR 300) corresponds to the A/B domain of the human ER-, these ER mRNA fragments are probably not ER-ß but occur because of alternative promoter usage or splice variants of ER-.¹¹

The mechanisms involved in upregulation of ER- protein and gene expression in the allograft remain unknown. Overexpression of some growth factors and their receptors has been found in the allograft vessels.^{6 7 8 22 23 24 25} We speculate that the overexpressed ER- may relate to the phenotypic changes of the vascular cells during the progression of the transplant arteriosclerosis. We also noticed no significant difference in the ER- expression in the coronary arteries of both donor and recipient hearts with 1 week on a 0.5% cholesterol diet or on a regular diet. However, the coronary arteries of transplant recipient hearts, which were maintained on a 0.5% cholesterol diet and immunosuppressant for 6 weeks after transplantation, showed a moderate increase of ER- expression when compared with the hearts with 1 week on a 0.5% cholesterol diet or regular diet. The mRNA expression of the ER- in the aorta of recipients 6 weeks after transplantation did not show a significant increase compared with the aorta of recipients before the transplantation. The mechanisms of this moderate increase should be investigated further.

The function of upregulated ER- and mRNA in the allograft vasculature is also unknown. We observed that the allografts have a significant proliferative response to growth factor stimulation compared with the nongraft vessels. With estrogen treatment, abolition of this proliferative response was seen in the allografts, but only partial inhibition was seen in the nongraft vessels.^{7 8} The correlation of the abolition of proliferative response and overexpression of ER- in the allografts implies that the inhibition of proliferation for transplant arteriosclerosis by estrogen may relate to the upregulation of ER- expression in the allograft vessels. In an ER-–knockout mouse model, 17ß-estradiol was shown to markedly inhibit aorta medial proliferation to the same degree in ER-–deficient and wild-type mice.¹⁶ ER-ß expression was demonstrated in the ER-–knockout mouse aorta, which suggests that the antiproliferative effect of estradiol in a nonallograft model can occur in the absence of ER- and possibly through ER-ß. The formation of the heterodimers of ER- and ER-ß,²⁶ in addition to homodimers of ER- or ER-ß, allows for diverse and tissue-specific (activation and inhibitory) regulation by estrogen ligand through ER- and ER-ß when complexed with either ERE or an AP-1 site.^{26 27} We speculate that rabbit vasculature expresses ER-ß in addition to ER-. Whether ER-ß expression is also altered after transplantation and/or directly mediates inhibition of cell proliferation is under investigation.

	Acknowledgments

 
This work was supported by National Institute of Health grant R01-HL-56119. We thank Dr Peter Libby for the gift of HSVSMC and Drs Nevin M. Katz, Teruaki Kodama, and Yining Wang for their assistance.

Received May 20, 1998; accepted July 31, 1998.

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