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(Circulation. 1996;94:3355-3361.)
© 1996 American Heart Association, Inc.

Articles

Inhibition of Transplant Coronary Arteriosclerosis in Rabbits by Chronic Estradiol Treatment Is Associated With Abolition of MHC Class II Antigen Expression

Hong Lou, MD; Teruaki Kodama, MD; Ye Jun Zhao, MD; Peter Maurice, MS; Yi Ning Wang, MD; Nevin Katz, MD; Marie L. Foegh, MD, DSc

Georgetown University Medical Center, Washington, DC.

Correspondence to Marie L. Foegh, MD, DSc, Georgetown University Medical Center, 4000 Reservoir Rd NW, Building D, Room 398, Washington DC 20007. E-mail mfoegh@aol.com.

	Abstract

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Background Accelerated coronary arteriosclerosis is a major complication in long-term survivors of cardiac transplantation. Estrogen prevents transplant arteriosclerosis in experimental cardiac and aortic allografts and may act by an immune mechanism.

Methods and Results New Zealand White rabbits immunosuppressed with cyclosporine were recipients of cardiac allografts from Dutch Belted rabbits. The recipients received either estradiol or placebo daily until they were killed 6 weeks later. Histological cross sections of the cardiac allograft were used for quantification of major histocompatibility complex (MHC) class II antigen expression, T lymphocytes, and macrophages by immunohistochemistry using monoclonal antibodies. MHC class II antigen expression was not detectable in allograft coronary arteries from any of the estradiol-treated recipients, whereas this antigen expression was present in the allograft coronary arteries from all the placebo-treated recipients. Macrophage and lymphocyte infiltration of the allograft coronary artery myointima was significantly less frequent in the estradiol-treated group. Rejection was moderate but slightly less in the estradiol-treated group. These findings were associated with a 60% decrease in allograft coronary artery myointimal thickening (determined by morphometry) in the estradiol-treated compared with the placebo-treated group.

Conclusions Estradiol treatment of cardiac allograft recipients abolishes MHC class II antigen expression in the coronary arteries and decreases macrophage infiltration in all three layers of the vessel wall, whereas T-lymphocyte infiltration is decreased only in the myointima. These findings are associated with estradiol inhibition of myointimal proliferation. Thus, estradiol treatment may have a beneficial effect on graft arteriosclerosis through immune mechanisms.

Key Words: arteries • transplantation • arteriosclerosis • rejection • antigens

	Introduction

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Coronary allograft arteriosclerosis in recipients of cardiac transplants has emerged as the major cause of death or retransplantation after the first 3 months. The incidence of cardiac transplant arteriosclerosis approaches 80% at 5 years.¹ It is characterized by tubular and concentric myointimal proliferation throughout the entire coronary artery tree. In patients, the cellular infiltrate of the proliferative lesions consists of smooth muscle cells (SMCs), macrophages, and T lymphocytes.^{2 3 4 5 6} The pathogenesis is poorly understood, but chronic immunological events are believed to play a significant role. The vascular wall of the graft is one of the targets of these ongoing allogeneic immune responses. Here, we present immunological data related to the protective role of estrogen.

The protective role of estrogen against cardiovascular disease in women is well documented. Epidemiological data indicate that the incidence of cardiovascular disease is lower in premenopausal women than in men and that in postmenopausal women, estrogen replacement therapy reduces coronary disease by 50%.^{7 8 9} Experimentally, we find that transplantation of hearts of female syngeneic rats to male recipients is associated with less myointimal hyperplasia.¹⁰ Furthermore, our previous studies using male donors and recipients have shown that estrogen treatment protects against transplant arteriosclerosis in cardiac and aortic allografts in a dose-dependent manner.^{11 12}

Here, we used the immunosuppressed male rabbit cardiac heterotopic transplant model, in which the histological appearance of the vascular lesions is comparable to that seen in cardiac transplant patients. Using immunocytochemistry, we examined the inhibitory effects of chronic estradiol treatment on late immune activity, determined by measurement of vascular infiltration of macrophages and lymphocytes as well as vascular major histocompatibility complex (MHC) class II antigen expression.

	Methods

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Transplantation Procedure
The cardiac recipients were male New Zealand White rabbits weighing 3.0 to 3.5 kg, and the donors were male Dutch Belted rabbits weighing 1.8 to 2.3 kg (Hazelton Research Products Inc, Denver, Pa). The animals were housed separately at constant room temperature and with a 12-hour light cycle in a facility approved by the American Association for Accreditation of Laboratory Animal Care. The rabbits were fed a 0.5% cholesterol diet for 7 days before transplantation, and recipients continued this diet until they were killed 6 weeks later. The recipients received 10 mg·kg^-1·d^-1 cyclosporin A (Sandoz) as an immunosuppressant until they were killed. One dose of 0.5 mL IM of Bicillin (Wyeth Laboratories, Inc) was given before surgery to prevent infection.

Donors and recipients were prepared for sterile surgery and anesthetized with 80 mg ketamine and 20 mg xylazine IM. The donors were heparinized (330 U/kg) via ear-vein catheter before surgery; the hearts were excised and immediately placed in an iced, heparinized (10 U/mL) saline bath. Heterotopic cardiac transplantation was performed on the right side with end-to-side anastomosis between the aorta and carotid artery and between the pulmonary artery and external jugular vein.

The recipients were randomly assigned to two groups as follows: the estradiol-treated group (n=8) received 100 µg·kg^-1·d^-1 estradiol cypionate IM (Depo-Estradiol, Upjohn); the placebo-treated group (n=9) received an equal volume of placebo. Rabbits were killed at 6 weeks after transplantation, at which time blood was drawn for determination of serum levels of estradiol.

The cardiac allografts were isolated and pressure-perfused at 80 mm Hg for 30 minutes with saline and later with formalin tissue fixative (Histochoice, Amresco). Five serial cross sections of each cardiac allograft were embedded in paraffin. Sections 3 µm thick were cut from each block for immunohistochemical analysis and stained with either hematoxylin-eosin or an elastin stain for histological and morphometrical evaluation.

Rejection Grading
Hematoxylin-eosin–stained cross sections were used for the histological grading of rejection. Rejection was graded from 0 to 4 according to the guidelines of the International Society for Heart and Lung Transplantation: briefly, absence of inflammatory cells, myocytolysis, and fibrosis, 0; no rejection, scattered perivascular and endocardial mononuclear cell infiltration, 1; mild rejection, prominent mononuclear cell infiltration with early focal myocytolysis, 2; moderate rejection, prominent myocytolysis and interstitial hemorrhage, 3; and severe rejection, fibrosis with severe hemorrhage, 4.

Immunohistochemistry
Immunohistochemistry was performed with Histostain SP Kit (Zymed) according to the manufacturer's protocol. Briefly, the paraffin-embedded sections were dewaxed in xylenes and rehydrated in serial concentrations of ethanol. Endogenous peroxidase activity was eliminated by exposure to 3% hydrogen peroxide/methanol and followed by incubation in a blocking solution to reduce the background. The sections were exposed to primary antibody for 1 hour, to biotin-labeled secondary antibody for 30 minutes, and then to a streptavidin-peroxidase reaction system. Lymphocytes were identified by a mouse IgG1 (clone LII-135) that detects cell-surface glycoprotein determinants present on rabbit thymocytes and peripheral T cells. MHC class II antigen expression was determined with the monoclonal antibody 2C4 (IgG2a), which binds to a framework determinant of the -chain of rabbit MHC-II antigen (HLA-DR) (both were gifts from Dr Peter Libby, Brigham and Women's Hospital, Boston, Mass). Anti–rabbit macrophage monoclonal antibody RAM 11 (IgG1) was a gift from Dr Allen Gown (University of Washington, Seattle, Wash). Anti–SMC -actin antibody 1A4 (IgG2a) was obtained from Sigma Chemical Co.

Semiquantification of antigen expression was evaluated under the light microscope at x200 or x400 magnification by two different methods of counting the immunostaining within the intima, media, and adventitia, in a blinded manner. One evaluation method was based on the frequency and the other on the degree of positive staining. The frequency of positive staining was quantified by counting of the number of stained and unstained coronary arteries in cross sections of the cardiac allograft; the frequency was expressed as the percentage of positively stained coronary arteries over the total number (15 to 20 cross sections of coronary arteries were evaluated from each graft). The degree of positive immunostaining was graded on a scale of 0 to 4, as follows: 0, negative (same as negative control); 1, weak staining (with single scattered red staining); 2, moderate (patched staining in <25% of the area); 3, extensive staining (25% but <50% of the area was positively stained); and 4, severe staining (>50% positive staining, and color of the staining stronger than 3). The first five most positively stained vessels in each section were evaluated for grading, and 4 to 7 graft sections were averaged in each group.

Morphometric Analysis
The degree of intimal thickening was determined by computerized morphometric analysis (The Morphometer, Woods Hole Educational Associates).¹² Five horizontal sections from each allograft were used for morphometry. Fifteen to 20 cross sections of coronary arteries were evaluated for each graft. The intimal thickness was expressed as the area of myointima over the total vessel areax100%.

Measurement of Serum Estradiol
The blood obtained at the time animals were killed was centrifuged, and serum was separated and stored at -70°C until analysis. Serum estradiol was purified by extraction and chromatography and determined by radioimmunoassay (Hazleton).

Statistics and Significance
All the morphological measurements and scoring were done by two investigators blinded to the group allocations. In the event of disagreement, the sections were reexamined, and a consensus was reached. Comparisons between estradiol-treated and placebo-treated groups were done by ANOVA and unpaired Student's t test, with a value of P<.05 considered significant. Unless otherwise noted, all values were expressed as mean±SEM.

	Results

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Rejection Grading
There was a significant difference in rejection grading between the two groups. The rejection grading of the cardiac allografts from the placebo- and estradiol-treated recipients was 2.72±0.12 and 2.08±0.27, respectively (P<.05; Table).

View this table: [in this window] [in a new window]   Table 1. Rejection Grading in Estradiol- and Placebo-Treated Groups

Immunohistochemistry: Quantification of MHC Class II Antigen Expression, T Lymphocytes, and Macrophages in Graft Coronary Arteries
Frequency of Immunostaining
No MHC class II antigen expression was detected in coronary arteries from any of the allografts from the 17ß-estradiol–treated recipients, whereas all the allografts from the placebo-treated recipients showed MHC class II antigen expression. The frequency of MHC class II antigen–positive cross sections of coronary arteries was 29.5±1.0% in the grafts from the placebo-treated animals (Fig 1). There was no difference in the frequency of T lymphocyte–positive cells between the two groups (12.2±1.0% and 11.3±1.8% for the placebo- and estradiol-treated groups, respectively). The coronary arteries from the placebo-treated group showed 49.7±7.0% positive staining with the anti-macrophage antibody, whereas only 16.1±4.0% positive staining was seen in coronary arteries from the estradiol-treated group (P<.02).

View larger version (16K): [in this window] [in a new window]   Figure 1. Frequency of positive staining in coronary arteries expressed as percentage of positive-stained vessels over total number of vessels examined for rabbit major histocompatibility complex class II antigen (MHC II), T lymphocytes (T-Cell), and macrophages in coronary arteries from placebo-treated group (open bars) and estradiol-treated group (hatched bars).

Degree of Immunostaining
The estradiol-treated group showed no positive staining for MHC class II antigen expression. In the placebo-treated group, the MHC class II antigen expression was abundant and located primarily in the myointima (44%) and adventitia (50%) of the coronary arteries of the cardiac allograft. (Figs 2A, 2B, and 3).

View larger version (831K): [in this window] [in a new window]   Figure 2. Facing page. Immunohistochemistry staining with Histo SP kits. Rabbit major histocompatibility complex class II antigen staining in placebo-treated group (A) and estradiol-treated group (B); rabbit macrophage staining in placebo-treated group (C) and estradiol-treated group (D); T-lymphocyte staining in placebo-treated group (E) and estradiol-treated group (F); smooth muscle cell -actin staining in placebo-treated group (G) and estradiol-treated group (H). Magnification x200 for D, E, G, and H; x400 for A, B, C, and F.

View larger version (20K): [in this window] [in a new window]   Figure 3. Grading on a scale of 0 to 4 of immunohistochemistry staining of major histocompatibility complex class II (MHC-II) antigen, T cells, and macrophages in intima, media, and adventitia of rabbit coronary arteries from cardiac allograft. Placebo-treated group, open bars; estradiol-treated group, hatched bars.

Estradiol treatment significantly reduced immunopositive staining of macrophages in all three layers of the arterial wall. The myointima of the coronary arteries showed macrophage-positive staining grades of 1.49±0.17 and 0.26±0.04 (P<.005) for the placebo- and estradiol-treated groups, respectively. In the medial layer of the coronary arteries, the degrees of positive immunostaining were 0.47±0.05 and 0.03±0.01 for the placebo- and estradiol-treated groups, respectively (P<.02). In the adventitia of the coronary arteries, the degrees of positive immunostaining were 1.20±0.07 and 0.49±0.08 for the placebo- and estradiol-treated groups, respectively (P>.05) (Figs 2C, 2D, and 3).

Estradiol treatment also significantly reduced the degree of positive immunostaining for T lymphocytes in the myointima and media of the graft coronary arteries but not in the adventitia. The grades of positive-stained T lymphocytes in the myointima were 0.38±0.04 and 0.05±0.02 for the placebo- and estradiol-treated groups, respectively (P<.02). In the medial layer, the estradiol-treated group showed no positive staining, whereas the placebo-treated group showed a grade of 0.09±0.03. In the adventitia, the staining grades of T lymphocytes were 0.47±0.02 and 0.35±0.1 in the placebo- and estradiol-treated groups, respectively (Figs 2E, 2F, and 3).

The degrees of positive immunostaining for vascular SMC -actin were not significantly different between the placebo- and estradiol-treated groups in the myointima and adventitia (Fig 4). In the myointima, the grades were 1.67±0.08 and 1.20±0.32 in the placebo- and the estradiol-treated groups, respectively. In the adventitia, the -actin staining was weak in both groups (both 0.6±0.1). The medial layer of the graft coronary arteries exhibited a significantly (P<.05) higher degree of positive -actin staining in the estradiol-treated group (3.60±0.13) than in the placebo-treated group (2.73±0.11). Moreover, the SMC staining had an even appearance in the medial layer of the coronary arteries in the estradiol-treated group, whereas it had a disrupted and irregular appearance in the placebo-treated group (Fig 2G and 2H).

View larger version (19K): [in this window] [in a new window]   Figure 4. Intensity grading of immunohistochemistry staining for smooth muscle cell (SMC) -actin in rabbit coronary arteries in cardiac graft. An increased SMC -actin staining was seen in the intimal area in the placebo-treated group (open bars), but less staining was seen in the estradiol-treated group (hatched bars). There was disrupted and reduced SMC -actin staining in medial area in the placebo-treated group.

Morphometric Analysis
Estradiol treatment inhibited myointimal thickening in the cardiac allograft. Myointimal thickening of the graft coronary arteries was decreased from 44.3±3.6% in the placebo-treated group to 17.9±1.5% in the estradiol-treated group (P<.002; Fig 5).

View larger version (16K): [in this window] [in a new window]   Figure 5. Intimal thickening of coronary arteries from cardiac allograft in placebo-treated group (open bar) and from chronic estradiol-treated recipients (hatched bar). Intimal thickening is expressed as a percentage of myointimal area over total vessel area (=area within external elastic lamina). The estradiol treatment significantly reduced coronary artery transplant arteriosclerosis.

Serum Estradiol
Serum estradiol levels at the time the animals were killed were increased in the estradiol-treated animals to 433±126 pg/mL compared with 34±17 pg/mL in the placebo-treated group (P<.05; Table).

	Discussion

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We found that a significant reduction of coronary artery transplant arteriosclerosis induced by estradiol treatment is associated with (1) abolition of vascular MHC class II antigen expression, (2) a reduction in both lymphocyte and macrophage infiltration of the vascular wall, (3) a slight reduction in rejection grade, and (4) preservation of SMC -actin staining in the medial layer of the coronary arteries.

Transplant arteriosclerosis is a characteristic of all transplanted organs; several features suggest that immune activity is an important element in the development of the disease.^{13 14 15 16} Thus, the incidence of graft arteriosclerosis is lower in patients on higher doses of cyclosporin A than in patients on lower doses. Moreover, arteriosclerosis is readily obtained by transplantation across strains in animal models and use of suboptimal immunosuppression. Inbreeding decreases the development of cardiac graft vascular disease. Finally, analysis of a continuous series of rejecting allografts has demonstrated that a distinct inflammatory stage precedes that of SMC accumulation in the intima, suggesting that mononuclear cells play an important role in the development of transplant arteriosclerosis.^{2 5}

Male animals were used in this study to obtain the highest degree of transplant arteriosclerosis. The male sex was associated with the most extensive graft myointimal hyperplasia in the rat syngeneic aorta graft model in a previous study.¹⁰ In patients, the highest degree of coronary transplant arteriosclerosis was in male recipients of grafts from women; there was also more transplant arteriosclerosis in male recipients of grafts from men than in female recipients of grafts from men.¹⁷

The finding that the cardiac allograft from the estradiol-treated recipients showed less cell-mediated rejection than the controls is important, because estrogen is thought to increase immune activity by activating macrophages and by promoting the differentiation and maturation of lymphocytes, resulting in increased immunoglobulin production and release of cytokines.^{18 19} Nuclear estrogen receptors have been found in lymphoid cells.^{20 21} Estrogen increases the activity of the interferon- promoter in lymphoid cells and interferon- mRNA expression in concanavalin A–treated murine spleen cells.²¹ Adult mice are protected from genital herpes virus at estrus and metestrus, as well as after estradiol treatment,²² suggesting an estrogen-induced increase in immune activity. Collectively, these observations indicate that estrogen increases the immune response. However, our present study in rabbits shows that chronic estradiol treatment in the presence of cyclosporin A immunosuppression does not increase cell-mediated immune responses determined by rejection grading of the cardiac graft. To the contrary, a slight but statistically significant decrease in rejection grading was seen in the estradiol-treated group. Furthermore, the estradiol treatment significantly reduced immunoactivity in the myointima of the cardiac allograft coronary arteries. Thus, chronic estradiol treatment in prevention of transplant arteriosclerosis does not increase the host systemic immune response.

The expression of MHC class II antigen in the graft coronary arteries is a sign of immune activation of the allograft. Endothelial cells and SMCs normally do not present detectable levels of MHC class II antigen but will express this protein after immunological activation, particularly by interferon-, which allows these cells to act as antigen-presenting cells eliciting allogeneic responses.^{6 16} The MHC antigens are the main signals through which the blood vessel of the donor organ might be recognized as foreign by host helper (CD4⁺) T lymphocytes, which could make graft vascular cells a target for immune system–mediated injury. Cytokines and growth factors secreted during this process promote SMC proliferation and migration, resulting in accelerated arteriosclerosis.^{2 3 4 5 6} We found that MHC class II antigen was not detectable in any of the coronary arteries of the estradiol-treated recipients. This was associated with a low degree of coronary artery myointimal hyperplasia. In contrast, MHC class II antigen expression was prevalent in the myointima and adventitia of the control graft coronary arteries, in which there was a high degree of transplant arteriosclerosis. This suggests that one of the mechanisms of estrogen prevention of transplant arteriosclerosis is prevention of MHC class II antigen expression. Such a mechanism is supported by other investigators who found similar estrogen effects in other experimental systems. For example, estradiol treatment decreases MHC class I and class II antigen expression on guinea pig blood mononuclear cells, including killer cells,²³ and estradiol protects human breast carcinoma cells against interleukin-1–induced MHC class II expression in vitro.²⁴ The mechanism whereby estradiol modulates MHC class II antigen expression is not clear. Some investigators find pharmacological doses of estradiol to inhibit Ca²⁺ influx and Ca²⁺ release induced by thromboxane A₂ in porcine coronary artery.²⁵ Estradiol showed an effect similar to that of calcium channel blockers, which have been reported to play a role in inhibiting transplant arteriosclerosis.²⁶ We observed that MHC class II antigen expression was absent in the intimal area of the coronary arteries from estradiol-treated rabbits despite the presence of macrophages and lymphocytes in this area. Macrophages and some T lymphocytes constitutively express MHC class II antigen; thus, the estradiol-induced abolition of MHC class II antigen expression is most likely a direct effect of estradiol on class II antigen expression. The estrogen-induced abrogation of graft-vessel MHC class II antigen expression may also relate to reduced adhesion molecule expression, thus preventing macrophage and T-lymphocyte accumulation and adhesion to the vascular wall. A recent study in mouse cardiac allograft vasculopathy did indeed show an increased expression of both intracellular and vascular adhesion molecules.²⁷

Monocytes/macrophages and T lymphocytes are the principal effectors of cell-mediated immunity. In the allograft, immune injury of endothelial cells increases expression of adhesion molecules for monocytes/macrophages and T-lymphocyte adhesion, causing activation of the cytokine cascades^{2 3 4 5 6 28} and release of growth factors such as insulin-like growth factor I,^{29 30} platelet-derived growth factor,^{31 32} epidermal growth factor, basic fibroblast growth factor, and transforming growth factor-ß.^{28 33} These growth factors are also expressed in SMCs as well as endothelium and act as autocrine and/or paracrine growth regulators to accelerate SMC proliferation and thus arteriosclerosis.^{28 29 34} Although all the recipient rabbits received cyclosporin A daily as an immunosuppressant for the duration of this study, 50% of the vessels in the placebo-treated group showed positive staining for macrophages in the coronary artery. In contrast, only 16% of the vessels showed positive staining for macrophages in the estradiol-treated group. In the present study, we find macrophages and T lymphocytes in the graft arterial wall but to a much lesser extent in graft coronary arteries of the estrogen-treated recipients than in the placebo-treated group. The observation of the substantial inhibition of transplant arteriosclerosis and the total absence of MHC class II antigen expression in the coronary arteries of all estradiol-treated rabbits despite the presence of macrophages and T lymphocytes and its presence in grafts from all the placebo-treated recipients suggest that MHC class II antigen may serve as an important mechanism in transplant arteriosclerosis.

A significant reduction of the staining intensity of T lymphocytes was observed in graft coronary arteries of the estradiol-treated recipients, although there was no significant difference in the frequency of T-lymphocyte staining between the estradiol-treated recipients and the placebo-treated recipients. The decrease in intensity of staining of T lymphocytes seen in the graft coronary arteries from the estradiol-treated group may be related to the decrease in MHC class II antigen expression or may be due to a direct inhibitory effect of estrogen on activation of T lymphocytes. Estrogen receptors are found in human suppressor/cytotoxic T cells (CD8⁺) but not in helper T cells (CD4⁺)¹⁹ ; thus, the estradiol-induced increase in the activity of T helper cells must be an indirect effect associated with a reduced activity of suppressor T cells.³⁵ The antibody used in the study presented here is a mouse IgG1, which detects a 120-kD cell-surface glycoprotein determinant present on rabbit thymocytes and peripheral T cells. The monoclonal antibody does not discriminate among T cells carrying CD4, CD8, or CD3 markers. Thus, we cannot determine which subset of T lymphocytes is affected by the estrogen treatment.

Estrogen in vitro at high pharmacological doses inhibits lymphocyte proliferation.³⁶ We found that estrogen inhibits proliferation of rabbit SMCs in vitro.³⁷ Estradiol also inhibits cell proliferation of porcine coronary artery explants.³⁸ In vivo, we also find estradiol to inhibit myointimal hyperplasia after balloon injury, with inhibition of DNA synthesis in the injured arterial wall,³⁹ and to inhibit cell proliferation in aorta explants from the rabbit heterotopically transplanted cardiac grafts ex vivo.⁴⁰ This suggests that estradiol inhibition of SMC proliferation by nonimmune mechanisms may contribute to the observed inhibition of myointimal thickening. We also noted less SMC -actin–positive staining in the media of graft coronary arteries from placebo-treated rabbits than in those from estradiol-treated rabbits, suggesting more phenotypic changes of SMCs from the contractile to the secretory type. The latter cells express increased amounts of ß-isoform and -isoform actin rather than -actin,^{41 42} so that a reduced and disrupted -actin staining will be observed in vessels with severe transplant arteriosclerosis, as seen in the medial layer of coronary arteries from the placebo-treated group in the experiment reported here. This is in accordance with previous findings in rabbit aorta transplants, in which secretory SMCs were seen in the control group but not in the estradiol-treated group, as determined by transmission electron microscopy.¹²

Hyperlipidemia has been implicated in a few studies as a contributing factor in the development of transplant arteriosclerosis.^{43 44} Lipoprotein concentration has been reduced by estrogen replacement therapy in postmenopausal women.^{45 46} However, we found no significant effect on total serum cholesterol 6 weeks after administration of estradiol in two models of cholesterol-fed rabbits, a cardiac transplant model¹¹ and an aorta transplant model,¹² in which estradiol inhibited transplant arteriosclerosis. Other investigators found no effect of estrogen on serum cholesterol levels in cholesterol-fed rabbits after 3 weeks of treatment.⁴⁷

We do not rule out an effect of estradiol on lipoprotein metabolism's causing some of the inhibition of transplant arteriosclerosis in the present study, but as demonstrated in our previous studies, the benefit of estradiol in preventing transplant arteriosclerosis is not directly dependent on lowering total serum cholesterol levels.^{11 12} In primates as well, it has been shown that only some of the antiatherosclerotic effect can be attributed to the lipid-lowering effect.⁴⁸ In the study presented here, the apparent evidence of reduced graft allogenicity through the abolition of MHC class II antigen expression in all the estradiol-treated recipients compared with an abundant expression in all the cardiac grafts from the placebo-treated group suggests this to be an important and novel mechanism of estrogen effects.

In conclusion, this study provides evidence that chronic estradiol treatment does not increase cell-mediated rejection in heterotopic cardiac allografts from recipients immunosuppressed with cyclosporin A. Furthermore, chronic estradiol treatment abolished MHC class II antigen expression in all the graft coronary arteries; the graft coronary arteries from all the placebo-treated recipients expressed MHC class II antigen. Infiltration and proliferation of macrophages and T lymphocytes in the intima and media were suppressed. Finally, these estradiol effects were associated with a substantial inhibition of coronary artery transplant arteriosclerosis. These findings suggest that estradiol may reduce transplant arteriosclerosis by attenuating the alloimmune response by abolishing MHC class II antigen expression and decreasing macrophage and T-lymphocyte presence in the coronary arteries of cardiac allografts.

	Acknowledgments

 
This study was supported by NIH grant 40069 and the Schering Foundation, Berlin, Germany. We are grateful to Nikheel Kolatkar for his technical assistance and Cecile Heatley for manuscript preparation.

Received May 23, 1996; revision received July 24, 1996; accepted July 31, 1996.

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