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(Circulation. 1996;94:2096-2102.)
© 1996 American Heart Association, Inc.

Articles

Increased Immunoreactive Endothelin-1 in Human Transplant Coronary Artery Disease

Stefano Ravalli, MD; Matthias Szabolcs, MD; Arline Albala, MA; Robert E. Michler, MD; Paul J. Cannon, MD

the Departments of Medicine, Division of Cardiology (S.R., A.A., P.J.C.); Pathology (M.S.); and Surgery, Division of Cardiothoracic Surgery (R.E.M.), Columbia University College of Physicians and Surgeons, New York, NY.

Correspondence to Paul J. Cannon, MD, Division of Cardiology, Columbia University College of Physicians and Surgeons, 630 W 168th St, New York, NY 10032.

	Abstract

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Background The pathogenesis of transplant coronary artery disease (TCAD) is unknown, but it is thought to derive from an interaction between immune and nonimmune factors, leading to smooth muscle cell proliferation and accumulation in the expanded neointima. Endothelin-1 (ET-1), a potent vasoconstrictor with mitogenic properties for vascular smooth muscle cells, has recently been demonstrated in native vessel atherosclerosis. The present study used immunohistochemistry to investigate the role of ET-1 in TCAD.

Methods and Results ET-1 immunoreactivity and cellular localization were assessed in human coronary arteries with TCAD (n=13) and in normal coronary arteries (n=10) with single- and double-label immunohistochemistry. The intensity of immunostaining was determined by a semiquantitative method. Diffuse and intense ET-1 immunoreactivity was found in 11 of 13 patients with TCAD (85%), mainly in myointimal cells and, in lesser amounts, in macrophages and endothelial cells. In contrast, normal coronary arteries had only faint immunostaining localized to the endothelial layer. Mean semiquantitative grade was significantly higher in TCAD than in normal arteries (1.8 versus 0.7; P<.05). ET-1 was more frequently present in lipid-rich, atheromatous lesions than in lipid-poor, proliferative ones. Intimal neovessels consistently immunostained for ET-1.

Conclusions Immunoreactivity for ET-1 is significantly increased in TCAD, possibly as a result of stimulatory cytokines and growth factors that are upregulated in the posttransplant state. The results suggest a role for this mitogenic peptide in the pathogenesis of graft arteriosclerosis.

Key Words: arteriosclerosis • transplantation • endothelin

	Introduction

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The success of cardiac transplantation as treatment for intractable heart failure has been clearly demonstrated. Long-term survival has been limited, however, by the development of a proliferative vasculopathy in the allograft coronary arteries. This vasculopathy, called graft arteriosclerosis or TCAD, is the leading cause of death after the first year after transplantation, is found in 40% of patients at 5 years after transplantation, and is associated with the development of myocardial infarction, ventricular failure, malignant arrhythmias, and sudden death.¹

The pathogenesis of TCAD is incompletely understood but probably multifactorial. A chronic immune reaction against alloantigens on graft endothelial cells is thought to initiate the disease process.² Migration and proliferation of SMCs in the neointima, infiltration by lymphocytes and macrophages, and uptake of oxidized lipids by macrophages and SMCs to form "foam cells" occur to various extents in different patients. Risk factors for the development of TCAD include multiple episodes of rejection,³ cytomegalovirus infection,⁴ the development of anti–donor HLA antibodies,⁵ the release of donor soluble HLA antigens,⁶ dyslipidemia,⁷ and increased serum lipoprotein(a).⁸ The importance of dyslipidemia has been highlighted by recent studies in which hypercholesterolemia increased the extent of lesions in a rabbit transplant model⁹ and by the clinical report that administration of lipid-lowering drugs to cardiac transplant recipients improved 1-year survival and the degree of TCAD.¹⁰

Knowledge concerning cellular growth factors that induce SMC proliferation and might contribute to the development of TCAD is limited. Allogeneic lymphocytes have been shown to induce aortic endothelial cells to increase mRNA synthesis for the SMC mitogens PDGF A chain, basic FGF, and TGF-ß.¹¹ TNF- is expressed by medial SMCs during acute rejection in rabbit allografts.¹² PDGF and acidic FGF mRNA and proteins have been found in intramyocardial arterioles of human cardiac allografts.¹³

ET-1 is a 21-amino-acid vasoconstrictor polypeptide that was originally isolated from supernatants of cultured porcine endothelial cells¹⁴ but has more recently been found also in neurons, astrocytes, hepatocytes, and renal cells.¹⁵ Human ET-1 is produced from a single gene first as a 212-amino-acid polypeptide precursor, preproendothelin, that undergoes cleavage to form a 38-amino-acid product, proendothelin ("big endothelin"), which is in turn cleaved by the endothelin-converting enzyme to form the final product.¹⁶ ET-1 interacts with specific receptors, labeled ET_A and ET_B, to initiate a variety of physiological responses. Not only is ET-1 the most potent vasoconstrictor yet discovered, but it also enhances the vasoconstrictor effect of substances such as norepinephrine, serotonin, and angiotensin II.¹⁷ ET-1 has been shown to stimulate the proliferation of vascular SMCs and fibroblasts.¹⁸

ET-1 immunostaining has recently been observed in human atherosclerotic arteries, localized in endothelial cells, macrophages, and intimal SMCs.^{19 20} The objective of the present study was to investigate whether ET-1 immunoreactivity is present in the coronary arterial lesions of patients with TCAD.

	Methods

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Patient Characteristics
Coronary arteries were obtained from the explanted allografts of 13 patients (10 male, 3 female) undergoing retransplantation for severe TCAD. Histologically normal coronary arteries were obtained for comparison from the native hearts of 10 patients undergoing transplantation for idiopathic dilated cardiomyopathy. Cyclosporine, corticosteroids, and azathioprine were used in combination for immunosuppression. Hypertension was defined as a systolic blood pressure >140 mm Hg and/or a diastolic blood pressure >95 mm Hg or by the need for antihypertensive medications. Serum cholesterol levels were measured at the time of routine endomyocardial biopsies and reported as a mean, for each patient, of samples taken at 3, 6, 9, 12, and 24 months after transplantation.

Tissue Preparation
Coronary arteries were fixed in 10% buffered formalin, embedded in paraffin, sectioned at 4-µm intervals, and stained with hematoxylin-eosin. Selected sections were stained with Masson's trichrome for collagen and Verhoeff–van Gieson's stain for elastic tissue.

Immunohistochemistry
The source of each antibody used and the optimal working dilutions are summarized in Table 1. The monoclonal anti–ET-1 antibody (mouse IgG1; Biodesign International) recognizes human ET-1 with a 10% cross-reactivity with ET-3. HAM-56 (Dako Corp) reacts with fixed tissue macrophages and a subpopulation of endothelial cells, particularly those lining capillaries and small blood vessels.²¹ It was used to identify macrophages. A rabbit anti–human von Willebrand factor antibody (Dako Corp) was used as a specific endothelial cell marker.²² SMCs were identified with monoclonal antibodies directed against -smooth muscle actin²³ (BioGenex) and vimentin²⁴ (Dako).

View this table: [in this window] [in a new window]   Table 1. Antibodies Used for Immunohistochemistry

Sections were deparaffinized with xylene, rehydrated in sequential alcohol baths, and then washed in PBS. Endogenous peroxidase was inactivated with 3% hydrogen peroxide in ethanol for 30 minutes, and nonspecific antibody binding was suppressed with 20% nonimmune serum in PBS for 30 minutes. Sections were incubated in a humidified chamber overnight at 4°C with the ET-1 antibody. With intervening washes in PBS, sections were then incubated for 30 minutes at room temperature with a 1:100 dilution of a biotinylated horse anti-mouse IgG (Vector Laboratories) and with the avidin-biotin-immunoperoxidase complex (ABC Elite, Vector). Peroxidase activity was visualized with diaminobenzidine (Sigma Chemical Co). Sections were washed in tap water, counterstained with Mayer's hematoxylin, dehydrated in sequential alcohols and xylene, and mounted with coverslips. Normal human and pig coronary arteries, which constitutively express ET-1 mRNA and peptide, were used as positive controls. As negative controls, the immunostaining was performed after the anti–ET-1 antibody was adsorbed overnight with the same concentration of synthetic ET-1 peptide (Peninsula Laboratories) or after the primary antibody was substituted with nonimmune serum.

Double-Label Immunohistochemistry
To identify the cell types immunoreactive for ET-1, double-labeling with ET-1– and cell-specific antibodies was performed in the following manner. Sections were first stained for ET-1 as described above with diaminobenzidine as a chromogen to yield a brown reaction product. After a washing in PBS, sections were then incubated with -actin, von Willebrand factor, or HAM-56 antibodies for 1 hour at room temperature, followed by application of the biotinylated secondary antibody. Finally, sections were incubated with the alkaline phosphatase/anti–alkaline phosphatase complex (ABC-AP, Vector). The reaction was visualized with Vector Red (Vector) or BCIP/NBP (BioGenex), yielding a red or blue reaction product, respectively.

Histological/Immunohistochemical Analysis
Sections were independently examined by two observers for the presence of ET-1 immunoreactivity and its distribution in the vessel wall. The intensity of endothelin staining was graded numerically on a scale from 0 to 3, as follows: 0, no staining; 1, weak; 2, moderate; and 3, intense. Coronary arteries with TCAD were classified as atheromatous if they had at least three of the following histological features: (1) eccentric plaque, (2) disrupted internal elastic lamina, (3) large lipid deposits ("cholesterol clefts"), and (4) calcifications. Conversely, lesions were defined as proliferative if they consisted of a concentric accumulation of myointimal cells, with a mostly intact internal elastic lamina, without lipid deposits and calcifications. Intimal neovessels were defined as vascular spaces lined by endothelial cells and/or containing red blood cells in the area between the vessel lumen and the internal elastic lamina. Allograft rejection was graded according to the formulation of the ISHLT.²⁵

Statistical Analysis
All data are reported as mean±SEM. Student's t test was used for statistical comparison of continuous variables and ² for discrete variables. A value of P<.05 was considered statistically significant.

	Results

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Patients
Patient characteristics are summarized in Table 2. Allograft survival ranged from 15 to 103 months (mean, 51.5±7.7) from transplantation. Eleven of 13 patients (84%) were hypertensive. Mean serum cholesterol was 266±11 mg/dL. Three of 13 patients (23.1%) had no cellular rejection (ISHLT grade 0) in the explanted heart. Mild rejection was present in the remaining 10 patients (76.9%), divided as follows: grade 1A in 4, grade 1B in 4, and grade 2 in 2.

View this table: [in this window] [in a new window]   Table 2. Characteristics of the Patient Population

Normal Coronary Arteries
Normal coronary arteries had immunostaining for endothelin in 7 of 10 patients (70%). The degree of staining was always very light (Fig 1A), as demonstrated by a mean semiquantitative grade of 0.7. The staining was localized to von Willebrand factor–positive endothelial cells lining the vessel intimas and occasionally to SMCs of the tunica media.

View larger version (312K): [in this window] [in a new window]   Figure 1. Sections of a normal coronary artery (A) and of a coronary artery with TCAD (B) immunostained with the anti–ET-1 antibody. Diaminobenzidine was used as a chromogen in the immunoperoxidase reaction, yielding a brown reaction product. The normal artery has only faint immunoreactivity localized to endothelial cells and medial SMCs. In contrast, the artery with graft arteriosclerosis has diffuse and intense immunostaining throughout the vessel wall. Hematoxylin counterstain, x25.

Transplant Coronary Artery Disease
A total of 36 coronary artery sections were analyzed (mean, 2.8 per patient). Diffuse and intense endothelin immunoreactivity was present in 11 of 13 patients (85%) with TCAD (Fig 1B). The immunoreactivity was usually confined to the cytoplasm of individual cells in the expanded neointima (Fig 2), but in lesions with a large amount of necrotic material, areas of diffuse extracellular staining could occasionally be observed (not shown). The staining was mostly localized to neointimal spindle-shaped cells (Fig 3A) and to endothelial cells lining epicardial coronary arteries. Lipid-laden foam cells were also found to contain ET-1 immunoreactivity, although less consistently than myointimal cells (Fig 3A). All endothelin immunoreactivity was abolished when the primary antibody was applied after being preadsorbed with synthetic ET-1 peptide (Fig 3B) or when it was replaced with nonimmune serum, confirming the specificity of the immunostaining. The mean semiquantitative immunostaining grade was significantly higher in coronary arteries with graft arteriosclerosis than in normal coronary arteries (1.8 versus 0.7, P<.05).

View larger version (147K): [in this window] [in a new window]   Figure 2. Representative section of a coronary artery with graft arteriosclerosis double-labeled with the anti–ET-1 antibody (brown) and the von Willebrand factor antibody (blue), which was used to identify endothelial cells. Spindle cells accumulating in the neointima show a very intense cytoplasmic immunoreactivity for ET-1. Magnification x100.

View larger version (311K): [in this window] [in a new window]   Figure 3. Representative section of a coronary artery with graft arteriosclerosis immunostained for ET-1. Positive cells are dark brown. Several spindle cells and occasional foam cells immunostained for ET-1 are noted (A). All immunoreactivity was abolished when the primary antibody was preadsorbed with synthetic ET-1 peptide at similar concentration (B), confirming the specificity of the immunostaining. Hematoxylin counterstain. A, Magnification x400; B, magnification x100.

Specific antibodies were used to identify the origin of cells immunoreactive for endothelin. A double-label technique was used that allowed us to stain the same tissue section first for ET-1, then for cell-specific markers. The spindle-shaped cells, which represented the majority of ET-1–positive neointimal cells, were of smooth muscle origin, as demonstrated by their positivity for the anti–-actin antibody (Fig 4A). ET-1–positive foam cells were usually reactive for the macrophage marker HAM-56 (Fig 4B), although occasional actin- and vimentin-positive foam cells were also noted (not shown).

View larger version (298K): [in this window] [in a new window]   Figure 4. Representative sections of two coronary arteries with TCAD double-labeled with the ET-1 antibody (brown) and a smooth muscle actin (blue, A) or a macrophage (red, B) marker. In A, note several ET-1–positive neointimal spindle cells also stained with the anti–-actin antibody, indicating their SMC origin. In B, only a few of the numerous ET-1–positive foam cells were recognized by the specific macrophage marker HAM-56. Magnification x400.

Intimal neovessels, ie, vascular spaces located in the thickness of the atherosclerotic lesions, were present in 5 of 13 patients with TCAD. These neovessels were consistently and intensely immunostained by the ET-1 antibody (Fig 5). Intramyocardial arterioles, on the other hand, were usually negative (not shown).

View larger version (137K): [in this window] [in a new window]   Figure 5. Representative section of a coronary artery with graft arteriosclerosis stained for ET-1 (brown) and double-labeled with the von Willebrand factor antibody (red) to identify plaque neovessels. Three such neovessels are present in the field, all intensely immunostained for ET-1. Magnification x100.

Coronary arteries with graft arteriosclerosis were classified as atheromatous in 8 of 13 patients (61%) and proliferative in 5 of 13 (39%) on the basis of preestablished criteria that included lesion focality (ie, eccentric versus diffuse), the presence or absence of extracellular lipid deposits in the expanded intima, the integrity or disruption of the internal elastic lamina, and the presence or absence of calcifications. Foam cells and an intense neointimal lymphocytic infiltrate were present in both types of lesion. Intraplaque hemorrhage was not common, occurring in 3 of 13 cases (23%). Representative examples of both types of graft arteriosclerosis are shown in Fig 6. ET-1 immunoreactivity was more frequently present in cases of TCAD with the atheromatous features (8 of 8, 100%) than in those with the proliferative features (3 of 5, 60%), (P=.05). In either type, ET-1 immunostaining was found predominantly in the -actin–positive myointimal cells. The semiquantitative grade, ie, the intensity of ET-1 immunostaining, was not significantly different between the two groups. A trend toward an association between serum cholesterol levels and tissue ET-1 immunoreactivity was present, but it did not reach statistical significance.

View larger version (315K): [in this window] [in a new window]   Figure 6. Representative sections of coronary arteries from two patients with advanced TCAD. Two histological forms are shown. The first (A) consists of a concentric proliferation of myointimal cells overlying an intact internal elastic lamina (in black with the elastin stain) without lipid deposits or calcifications. We refer to this as a proliferative lesion. The second type (B), indicated as atheromatous, is indistinguishable from the complex lesions of native vessel atherosclerosis. Intracellular and extracellular lipid deposits are present. Internal elastic lamina fragmentation is also apparent. A, Verhoeff–van Gieson stain, x25; B, hematoxylin-eosin, x12.5.

	Discussion

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In the present study, we used immunohistochemistry to detect the expression of ET-1, a potent vasoconstrictor with mitogenic properties for SMCs, in human coronary arteries affected by TCAD. This disease is a frequent and clinically important complication of cardiac transplantation, representing the leading cause of death after the first year after transplantation.¹ We found that tissue ET-1 immunoreactivity was significantly increased in coronary arteries with TCAD compared with normal vessels. The most common cell types immunostaining for ET-1 were the neointimal SMCs and the endothelial cells lining the epicardial coronary arteries. Macrophages were also found to contain ET-1 immunoreactivity, but less frequently than SMCs. The intensity of ET-1 staining, as determined by a semiquantitative method, was significantly higher in TCAD than in normal coronary arteries. To the best of our knowledge, this study is the first to assess ET-1 immunoreactivity and its localization to specific cell types in the human lesions of TCAD. The results confirm and extend, in human tissue, work recently presented by Watschinger and collaborators²⁶ in a rat heterotopic cardiac transplant model. These authors demonstrated marked upregulation of ET-1 mRNA and protein in coronary arteries of allografts with acute and chronic rejection. Upregulation was not seen in the isograft controls or in the recipient native hearts and spleens, suggesting that the process was specific to the transplanted allograft. Interestingly, the major cell type expressing ET-1 in the chronically rejecting rat allografts was the neointimal macrophage, with a smaller number of ET-1–positive myointimal cells, whereas in the human lesions in our study, ET-1 immunoreactivity was predominantly localized in neointimal SMCs.

In native-vessel atherosclerosis, several authors have shown that, in addition to endothelial cells, both macrophages and intimal SMCs immunostain for ET-1.^{19 20} Tissue ET-1 immunoreactivity could conceivably represent circulating ET-1 tightly bound to its receptors or internalized by target cells,²⁷ rather than active local production. On the other hand, the ability of macrophages and SMCs to synthesize endothelin has been firmly established by several authors. Resink and Hahn and coworkers^{28 29} demonstrated the induction of ET-1 mRNA expression and synthesis of ET-1 peptide in cultured human and rat vascular SMCs by several growth factors and cytokines. Ehrenreich et al,³⁰ using a combination of radioimmunoassay, high-performance liquid chromatography, and immunocytochemistry, found ET-1 peptide in cultured and tissue human macrophages and in peripheral blood monocytes. Winkles and coworkers³¹ showed that ET-1 mRNA expression is elevated in atherosclerotic lesions compared with normal aorta.

As previously reported,³² we observed two histological forms of TCAD. One is characterized by large lipid deposits ("cholesterol clefts"), plaque eccentricity, fragmentation of the internal elastic lamina, and frequent calcifications and thus closely resembles native atherosclerosis. The cell population in these lesions consists of a mixture of SMCs, macrophages, and lymphocytes. The second type consists of a concentric accumulation of SMCs, with an intact internal elastic lamina and without lipids and calcium deposits. In the present study, ET-1 immunoreactivity was more frequently observed in the atheromatous than in the proliferative lesions. In both types, ET-1 was found predominantly in -actin–positive myointimal cells. Taken together, these findings suggest that the lesions of TCAD are not homogeneous and that each histological type may be induced by separate pathogenetic factors.

The expression of endothelin is regulated, mainly at the transcriptional level, by several cytokines, growth factors, and vasoactive substances. PDGF, interleukin-1, TNF-, and TGF-ß are potent stimulators of ET-1 mRNA production in cultured vascular endothelial cells^{29 33} and SMCs.^{28 29} Angiotensin II, epinephrine, arginine-vasopressin, and ET-1 itself increase the expression of ET mRNA in vascular endothelial cells^{14 29} and SMCs.²⁸ Atrial and brain natriuretic peptides, on the other hand, inhibit the angiotensin II–induced secretion of ET-1 in endothelial cells.³⁴ Thrombin, released in the setting of endothelial damage and platelet aggregation, is another potent stimulator of ET production.^{14 29 33 35} Interestingly, nitric oxide, which is also released from endothelial cells during stimulation with thrombin, inhibits ET-1 production via a cGMP-dependent pathway.³⁵ Additional factors may contribute to ET-1 upregulation in transplant patients. Cyclosporine elevates serum ET-1 levels in patients with solid-organ transplants,³⁶ induces a dose-dependent increase of endothelin synthesis in human endothelial cells in vitro,³⁷ and impairs endothelium-dependent vasorelaxation in dog coronary arteries.³⁸ Hypercholesterolemia, commonly seen in transplant patients as a result of corticosteroid treatment, elevates plasma and tissue ET-1 levels.³⁹ A tendency for patients with the highest cholesterol levels to have more intense tissue immunoreactivity was noted in this study, although it did not reach statistical significance. Oxidized LDL but not native LDL stimulates the expression of ET mRNA and protein from human and porcine vascular endothelial cells.⁴⁰ This effect is potentiated by thrombin and is probably mediated by the endothelial scavenger receptor. We have previously shown⁴¹ that 15-lipoxygenase, an enzyme involved in the cellular oxidation of LDL, is overexpressed in TCAD, suggesting the presence of oxidized LDL in these lesions. Upregulation of ET-1 synthesis by lipids might explain how hypercholesterolemia, interacting with the immune response, markedly augments graft arteriosclerosis in the experimental animal.^{9 42}

In the posttransplantation state, several mechanisms might therefore participate in the upregulation of ET-1 production. The immune response may play a prominent role in this process by producing an inflammatory infiltrate in the cardiac allograft. Activated macrophages not only release cytokines⁴³ that upregulate ET-1 production but can also synthesize the peptide themselves. Endothelial injury caused by reperfusion ischemia, viruses, and cyclosporine causes platelet adhesion and, in turn, thrombin, TGF-ß, and PDGF release. Cyclosporine, as discussed above, provides a protracted stimulus to ET-1 production. Finally, elevated serum cholesterol and triglycerides induced by immunosuppressive drugs create a favorable milieu for the development of atherosclerotic lesions.^{9 42}

The association of ET-1 immunoreactivity with intimal neovessels in cardiac allografts observed in this study is in agreement with the findings of others in native-vessel atherosclerosis.²⁰ Intense ET-1 immunostaining has also been reported in the neovessel-rich areas of previous biopsy sites in transplanted human hearts.⁴⁴ The role of these microvessels is still unclear, but the presence of intraplaque vascular channels releasing vasoconstrictor and mitogenic substances may be important in the phenomena of vascular remodeling, plaque growth, and rupture.

Regardless of the overall histological architecture (ie, atheromatous or proliferative), the proliferation and accumulation of SMCs in the neointima represent a major component of the pathogenesis of graft arteriosclerosis. ET-1 may play an important role in this process. In addition to its effects in producing vasoconstriction and enhancing the action of other vasoconstrictors,¹⁷ ET-1 causes vascular SMCs and fibroblast proliferation.^{16 18} This mitogenic effect can be direct^{18 45} or can derive from a synergistic action with known SMC mitogenic factors, such as PDGF.⁴⁶ ET-1 also induces quiescent vascular SMCs to produce mRNA for the mitogens PDGF and TGF-ß²⁹ and stimulates the expression of the proto-oncogenes c-fos and c-myc.^{18 47} These mitogenic properties are mostly mediated by the ET_A receptor⁴⁸ and are therefore potentially inhibited by the use of specific ET-1 receptor antagonists. In this regard, selective blockade of endothelin receptors has been shown to decrease the extent of atherosclerosis in the hypercholesterolemic hamster⁴⁹ and to reduce neointimal hyperplasia in the rat carotid artery after balloon angioplasty.⁵⁰ Treatment of experimental animals with specific ET-1 receptor antagonists may contribute to understanding whether this peptide plays an active role in the pathogenesis of graft arteriosclerosis.

Limitations of the Study
In the present study, immunohistochemistry was the only technique used to detect the presence of ET-1 in coronary arteries with TCAD. The human tissue we used had been fixed in formalin for histological sections and was therefore not suitable for more sophisticated molecular studies. Nonetheless, a previous study in rats has shown increased ET-1 mRNA production in TCAD by means of reverse transcription polymerase chain reaction.²⁶ Another limitation lies in the small sample analyzed, which reflects the very limited number of donor hearts available to retransplant patients with TCAD. For this reason, we have analyzed approximately three tissue sections per patient, thereby decreasing the likelihood of sample bias.

	Selected Abbreviations and Acronyms

 

ET-1 = endothelin-1 FGF = fibroblast growth factor ISHLT = International Society for Heart and Lung Transplantation PDGF = platelet-derived growth factor SMC = smooth muscle cell TCAD = transplant coronary artery disease TGF = transforming growth factor TNF = tumor necrosis factor

	Acknowledgments

 
This work was supported in part by National Heart, Lung, and Blood Institute grants HL-21006 and HL-54764.

Received May 2, 1996; accepted June 3, 1996.

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