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(Circulation. 1995;92:2594-2604.)
© 1995 American Heart Association, Inc.

Articles

Cytomegalovirus Antigen Expression, Endothelial Cell Proliferation, and Intimal Thickening in Rat Cardiac Allografts After Cytomegalovirus Infection

Karl Lemström, MD; Petri Koskinen, MD, PhD; Leena Krogerus, MD; Mat Daemen, MD, PhD; Cathrien Bruggeman, PhD; Pekka Häyry, MD, PhD

From the Transplantation Laboratory (K.L., P.K., L.K., P.H.), University of Helsinki (Finland), and the Departments of Medical Microbiology (C.B.) and Pathology (M.D.), University of Limburg, Maastricht, the Netherlands.

Correspondence to Dr Karl Lemström, Transplantation Laboratory, PO Box 21 (Haartmaninkatu 3), University of Helsinki, FIN-00014 Helsinki, Finland.

	Abstract

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Background Cardiac allograft arteriosclerosis is the primary cause of late death in heart transplant recipients. Clinical studies have suggested that humoral and cellular immune response, hyperlipidemia, and cytomegalovirus (CMV) infection may amplify the disease. In this study, the role of CMV infection in the development of rat cardiac allograft arteriosclerosis is investigated.

Methods and Results Heterotopic rat cardiac allografts were performed from the DA to the WF rat strains. To prevent rejection, the recipients received triple-drug (cyclosporine A 20 mg · kg^-1 · d^-1, azathioprine 2 mg · kg^-1 · d^-1, and methylprednisolone 0.5 mg · kg^-1 · d^-1) immunosuppression postoperatively. Recipient rats were infected intraperitoneally (n=21) with 10⁵ plaque-forming units of rat CMV (RCMV) 1 day after transplantation or were left uninfected and used as controls (n=18). The grafts were removed 7 and 14 days and 1 and 3 months after transplantation. In 42% (9 of 21) of cardiac allografts in RCMV-infected rats, an intramural, mononuclear cell inflammation of small intramyocardial arterioles was observed compared with none in uninfected rats (P=.005). Acute RCMV infection was associated with an early perivascular inflammatory cell response of helper T (W3/25), cytotoxic T (OX8), and NK (3.2.3) cells, macrophages (OX42), and major histocompatibility complex class II expression around small intramyocardial arterioles and capillaries. No upregulation of interleukin-2 receptor expression was seen. In arteries and small intramyocardial arterioles, RCMV infection was associated with a significant endothelial cell proliferation and a clear increase in intimal thickening. Significant endothelial cell proliferation was also observed in the capillaries after RCMV infection. Immunohistochemistry revealed specific focal RCMV early and late antigen expression in epicardial and interstitial ED1–immunoreactive mononuclear cell infiltrates and around small arterioles of RCMV-infected cardiac allografts. Occasionally, media cells of stenosed small intramyocardial arterioles also showed strong focal RCMV antigen expression. In addition, infectious RCMV could be recovered by plaque assay in cardiac allografts expressing RCMV antigens.

Conclusions These results demonstrate a productive RCMV infection in cardiac allograft structures and suggest that RCMV infection accelerates cardiac allograft arteriosclerosis, particularly in small intramyocardial arterioles mediated by inflammatory responses in the vascular wall and perivascular space.

Key Words: viruses • rejection • arteriosclerosis • transplantation • cyclosporine

	Introduction

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Persistent low-grade vascular inflammation, SMC proliferation, and intimal thickening are characteristic features of cardiac allograft arteriosclerosis, which is the primary cause of death and retransplantation among long-surviving heart transplant recipients.¹ In coronary angiography, the approximate incidences of cardiac allograft arteriosclerosis are 10%, 20%, and 50% at 1, 2, and 5 years after transplantation, respectively.^{2 3 4} When more sensitive techniques, such as intracoronary ultrasound, are used, the incidences are even higher.⁵ Although several risk factors have been linked to cardiac allograft arteriosclerosis, the cause and mechanism of the disease remain unclear. No doubt, allograft arteriosclerotic changes are amplified by humoral and cellular immune response,^{4 6 7} hyperlipidemia,^{3 8} and viral infections.^{9 10 11}

CMV infection is an important cause of morbidity and mortality among cardiac allograft recipients.¹² Many clinical studies suggested an accelerating role of CMV infection in the generation of cardiac allograft arteriosclerosis.^{9 10 11} Endomyocardial biopsies have shown an association between CMV antigenemia and a mild inflammatory cell response in the vessel wall, with alterations in small intramyocardial arterioles leading to a narrowing of the vascular lumen of the graft.^{13 14} In an autopsy study of three heart allograft recipients with severe graft arteriosclerosis, CMV nucleic acids were recovered from the coronary arteries.¹⁵ Furthermore, a recent study demonstrated that CMV infection induces vascular wall changes resembling fatty streaks observed in the early stages of classic atherosclerosis.¹⁶

We demonstrated previously with nonimmunosuppressed rat aortic allografts that RCMV infection enhances SMC proliferation and intimal thickening in the allograft vascular wall.^{17 18 19 20} The purpose of the present study was to extend these observations to cardiac allografts in which acute rejection was prevented by oral high-dose triple-drug immunosuppression. In this study, we demonstrate a productive RCMV infection in cardiac allograft structures and show that RCMV infection enhances cardiac allograft arteriosclerosis in the rat.

	Methods

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Experimental Design
Cardiac allografts were transplanted from DA rat strain donors to WF rat strain recipients. Allograft recipients were infected intraperitoneally with 10⁵ PFU tissue culture–derived RCMV 1 day after transplantation or were left uninfected and used as controls. To minimize the effect of acute rejection episodes on the development of cardiac allograft arteriosclerosis, oral high-dose cyclosporine-based triple-drug (cyclosporine A 20 mg · kg^-1 · d^-1, azathioprine 2 mg · kg^-1 · d^-1, and methylprednisolone 0.5 mg · kg^-1 · d^-1) immunosuppression was used. The grafts were removed 7 and 14 days and 1 and 3 months after transplantation and processed for morphometry, autoradiography, and immunohistochemistry. There were 4, 5, 8, and 4 rats infected with RCMV that had their grafts removed after 7 and 14 days and 1 and 3 months, respectively, and 4, 3, 6, and 5 rats, respectively, that were left uninfected. For autoradiography, rats received 300 µCi IV [methyl-³H]thymidine 3 hours before euthanasia and graft removal. At graft removal, biopsies from salivary gland, liver, and spleen were taken for quantitative plaque assay to confirm a generalized RCMV infection in the recipient. The presence of RCMV early and late antigens in cardiac allografts was demonstrated by immunoperoxidase technique.

The strain combination DA-WF is mismatched for MHC and non-MHC antigens, and without immunosuppression these cardiac allografts develop an acute rejection 5 to 7 days after transplantation. We demonstrated previously that sufficient triple-drug immunosuppression inhibits endothelial cell proliferation and intimal thickening of long-surviving rat cardiac allografts dose-dependently and that rejection treatment with 20 mg · kg^-1 · d^-1 cyclosporine A inhibits almost all arteriosclerotic vascular wall changes in the cardiac allografts.²¹ In this study, we used triple-drug immunosuppression with 20 mg · kg^-1 · d^-1 cyclosporine A to minimize the effect of acute rejection on the generation of (background) allograft arteriosclerosis.

Rats
Inbred DA (AG-B4, RT1a) and WF (AG-B2, RT1u) rat strains were used as donors and recipients, respectively. The rats were purchased from the Laboratory Animal Center, University of Helsinki (Finland). They were 10 to 12 weeks of age and weighed 250 to 300 g. Water and rat chow (Altromin, Standard Dioet, Chr Petersen A/S) were administrated according to rat weight (see the discussion of the immunosuppressive regimen). The infected and uninfected rats were kept in separate colonies under similar conditions and given otherwise similar diets. The animals received humane care in compliance with the "Principles of Laboratory Animal Care" and the "Guide for the Care and Use of Laboratory Animals," prepared and formulated by the Institute of Laboratory Animal Resources and published by the NIH (NIH publication No. 86-23, revised 1985).

Cardiac Allografts
Intra-abdominal heterotopic cardiac allografts were transplanted with a modified technique of Ono and Lindsey.²² In short, the donor rats were anesthetized by use of ether. After perfusion of 200 IU heparin in 10 mL ice-cold PBS into the inferior vena cava, the venae cavae and pulmonary veins were ligated with 6-0 silk, and the pulmonary artery and aorta were cut 2 to 3 mm above their origin in the heart. Recipient rats were anesthetized with chloral hydrate 240 mg/kg IP and were given buprenorphine 0.25 mg/kg SC (Temgesic, Reckitt & Colman) for postoperative pain relief. A midline incision was made, the great abdominal vessels were dissected free from the surroundings, and the aorta and pulmonary artery were anastomosed with abdominal aorta and inferior vena cava in a running end-to-side fashion with 9-0 nylon suture. Total ischemic time was 45±15 minutes, during which the graft was kept in an ice bath of 4°C PBS for 15 minutes. The hearts were cooled during the procedure with frequent changes of saline-cooled gauze coverings. The grafts started beating vigorously after perfusion was established. Graft function was evaluated by abdominal palpation, and all the grafts were beating at graft removal.

Immunosuppressive Drugs
In a preliminary study, we determined the uptake of food and tap water in nontransplanted WF rats and observed that the rats drank 80 mL · kg^-1 · d^-1 water and ate 90 mL · kg^-1 · d^-1 rat chow. Thus, water and chow were administrated in 7-day intervals. We did not observe any difference in the uptake of water or chow between RCMV-infected and uninfected rats.

The rats received oral triple-drug immunosuppression for the whole observation time. The rats were perioperatively given cyclosporine A (Sandimmun; Sandoz Pharma AG) 15 mg/kg SC in the neck as a single dose. For subcutaneous injection, 50 mg/mL cyclosporine A infusion substance was dissolved in 200 mg/mL Intralipid (KabiVitrum) to a final concentration of 3 mg/mL. Thereafter, cyclosporine A 20 mg · kg^-1 · d^-1 was given postoperatively with regular rat chow using 100 mg/mL Sandimmun mixture. Methylprednisolone 0.5 mg · kg^-1 · d^-1 (Solu-Medrol 40 mg/mL, Upjohn sa) and azathioprine 2 mg · kg^-1 · d^-1 (Imuran, Wellcome) were administered with drinking water.

Rat Cytomegalovirus
RCMV infection in cardiac allograft recipients was established by inoculating the rats with 10⁵ PFU IP Maastricht strain RCMV^{17 23 24} 1 day after transplantation. The RCMV pool consisted of tissue culture–derived virus diluted in 1 mL PBS before inoculation. RCMV was passaged by infecting fibroblasts from 17-day-old DA rat embryos. The cells were cultured in flasks containing MEM (Flow Laboratories) supplemented with 200 mmol/L L-glutamine (Northumbria Biologicals Ltd), 10 000 IU/mL penicillin per 1000 µg/mL streptomycin solution (GIBCO Ltd), and 2% FCS (GIBCO) according to standard viral culture techniques.²⁵ The stocks of virus were stored at -70°C until use. Quantification of infectious virus was done by plaque assay with a modification of the method of Wentworth and French²⁶ as described previously.^{17 19}

Plaque Assays
At graft removal, tissue biopsies from salivary gland, liver, and spleen were obtained aseptically in minimal essential medium containing 2% FCS and stored at -70°C until quantification of infectious RCMV by plaque assay.²⁶ For this purpose, 10-fold dilutions of 10% organ homogenates (wt/vol) were inoculated on confluent rat embryo fibroblast monolayers. After 7 days, the number of plaques was monitored microscopically after methylene blue staining. The RCMV titers were calculated per 1 mL of 10% organ suspension, and the results are given as PFU (mean±SEM).

Demonstration of RCMV in Cardiac Allografts by Plaque Assay
A section of cardiac allograft was put into a cryotube (A/S Nunc) and stored in liquid nitrogen until use. Tissue section was homogenized in MEM containing 2% FCS in a Potter tube and centrifuged at 3000 rpm for 10 minutes. The rats were irradiated with 5 Gy; 4 to 6 hours later, they were injected intraperitoneally with the cell suspension derived from cardiac allografts expressing or not expressing RCMV antigens. After 26 days, the rats were euthanatized, and salivary gland was taken for plaque assay to demonstrate replicating virus in cardiac allografts.

Histological Evaluation
When removed, the grafts were immediately washed with ice-cold PBS, and three to four cross sections were fixed in 10% phosphate-buffered formalin overnight, routinely processed, and embedded in paraffin. Cross sections of cardiac allografts (4 µm thick) were stained with Mayer's hematoxylin-eosin for general evaluation, Masson's trichrome stain for fibrosis, Weigert–van Gieson stain for internal elastic lamina, and Unna-Pappenheim stain for pyroninophilic cells.

Slides from midsections of both donor and recipient hearts were examined by light microscopy, and the score was assigned by consensus of three observers in a blind review (K.L., P.K., and L.K., a fully trained cardiac transplant pathologist). Recipient native hearts served as internal controls. Changes in histological parameters were scored semiquantitatively from 0 to 3 (0=normal, 1=mild, 2=moderate, and 3=severe).

Vessel Definition
Vessels with large luminal diameters and well-defined SMC layers in the vascular wall were identified as arteries; vessels with small luminal diameters and thin SMC layers in the vascular wall were considered arterioles. To differentiate between intimal and medial layers, paraffin sections were stained with the Weigert–van Gieson procedure, which gives a black reaction in the internal elastic lamina. Vessels without recognizable SMC layers and with only one endothelial cell nucleus per vessel cross section were identified as capillaries; those with small luminal diameters and few endothelial cells were considered postcapillary venules. Vessels without clearly recognizable SMC layers and with larger luminal diameter were considered veins. Endothelial cell proliferation was scored mild when endothelial cells were closely packed in one layer, moderate in two layers, and severe in three layers. The intimal thickness of arteries and arterioles was scored mild when there was <25% occlusion, moderate with 25% to 50% occlusion, and severe with >50% occlusion in the vascular lumen.

Inflammation
Both mononuclear and polymorphonuclear leukocytes were investigated. Pyroninophilic cells were identified as enlarged, activated lymphocytes that stained positively by the Unna-Pappenheim procedure. Intramural inflammation was considered to be when inflammatory cells were detectable beneath the vascular endothelium in the SMC layer of the vessel wall. Perivascular inflammation was scored mild in groups consisting of 10 to 30 inflammatory cells per infiltrate, moderate with 30 to 50 inflammatory cells per infiltrate, and severe with >50 inflammatory cells per infiltrate or when fingerlike projections of inflammatory cells were identified between the myocytes. The structure of perivascular inflammation was scored by immunohistochemistry.

Autoradiography and Double-Staining Procedure
To demonstrate that the cells morphologically consistent with proliferating endothelial cells were endothelial cells, we performed double-staining with polyclonal antibody against von Willebrand factor (Dako A/S) and [methyl-³H]thymidine autoradiography from representative frozen cardiac allograft cross sections. All rats received 300 µCi IV [methyl-³H]thymidine ([³H]TdR, Amersham International plc) 3 hours before rat euthanasia and graft removal. First, frozen cardiac cross sections were stained with a polyclonal antibody against von Willebrand factor at a dilution of 1:20 by use of a two-layer indirect immunoperoxidase technique. After being washed in Tris buffer, the sections were incubated with goat anti-rabbit immunoglobulin (CALTAG Labs) at a dilution of 1:10 in Tris buffer with 50% rat normal sera. After the reaction was revealed by chromogen 3-amino-9-ethylcarbazole containing hydrogen peroxidase, the slides were left in Tris buffer, and emulsion film (Ilford) autoradiography was performed. The slides were sealed in light-tight boxes for 21 days at 4°C; then they were developed by the Kodak D 19 developer. The specimens were counterstained with hematoxylin, and coverslips were aqua-mounted (Aquamount, BDH Ltd).

Immunohistochemistry
A midsection of cardiac allografts was taken in optimal-cutting-temperature embedding (Tissue-Tek, Miles Inc), snap-frozen in liquid nitrogen, and stored at -70°C. Frozen sections were air-dried onto poly-D-lysine–coated slides, fixed in acetone at -20°C for 20 minutes, and stored at -20°C until use. Before immunostaining, the slides were refixed with chloroform and then air-dried. Cross sections (4 µm thick) were incubated with monoclonal antibodies with a three-layer indirect immunoperoxidase technique.²⁷ The primary antibodies were used at a dilution of 1:100 in Tris with 1% BSA. After a 30-minute incubation at room temperature, the sections were washed in Tris buffer and incubated for 30 minutes with peroxidase-conjugated rabbit anti-mouse immunoglobulin at a dilution of 1:10 in Tris buffer with 50% normal rat sera (Dako-Immunoglobulins A/S). After being washed in Tris buffer, the sections were incubated with goat anti-rabbit immunoglobulin at a dilution of 1:10 in Tris buffer with 50% normal rat sera (Caltag Labs). The reaction was revealed by chromogen 3-amino-9-ethylcarbazole containing hydrogen peroxidase. The specimens were counterstained with hematoxylin, and coverslips were aqua-mounted (Aquamount, BDH Ltd). No difference was observed regardless of whether incubation was with nonimmune rabbit sera for nonspecific reaction or methanol containing 1% H₂O₂ for endogenous peroxidase before staining. Primary antibody was omitted in the control rats; otherwise, the staining procedure was performed similarly, and the control rats did not show any nonspecific immunoreactivity.

To determine the structure of inflammation and immune activation of infiltrating leukocytes, the following monoclonal antibodies were used: W3/25 (Sera Laboratory), a mouse IgG1 monoclonal antibody to rat T helper cells (CD 4 equivalent); OX 8 (Sera Lab), a mouse IgG1 monoclonal antibody to rat T cells (nonhelper subset, Lyt-2/Lyt-3, CD8 equivalent); OX 42 (Sera Lab), a mouse IgG2a monoclonal antibody to rat macrophages (160-, 103-, or 96-kD polypeptide); 3.2.3, a monoclonal antibody to rat NK cells (a generous gift from Assistant Prof William H. Chambers, Pittsburgh (Pa) Cancer Institute); OX 6 (Sera Lab), a mouse IgG1 monoclonal antibody to rat MHC class II common determinant; and IL-2R (CD25), a monoclonal antibody to rat interleukin-2 receptor (a generous gift from Dr J. Kupiec-Weglinski, Harvard Medical School, Boston, Mass).

The structure of inflammation and the level of immune activation were assessed in inflammatory cells. The analysis was done semiquantitatively by scoring the cell staining from 0 to 3 (0=no visible staining, 1=few cells with faint staining, 2=moderate intensity with multifocal staining, and 3=intense diffuse staining).

Immunohistochemistry to Detect CMV Antigens
For the detection of RCMV antigens, 4-µm-thick formalin-fixed paraffin-embedded tissue sections of heart allografts were stained with a mixture of monoclonal antibodies against early (monoclonal 8) and late (monoclonal 35) antigens of RCMV.^{28 29} To differentiate between acute and chronic RCMV infection, RCMV-immunopositive tissue sections were stained separately with monoclonal antibodies against early and late RCMV antigens. For control rats, primary antibody was left out; otherwise, the staining procedure was performed similarly. Control rats did not show any nonspecific immunoreactivity.

The sections were applied onto Chromaluin (Merck 1036)-coated slides and deparaffinized, and endogenous peroxidase was blocked by methanol containing 0.6% H₂O₂. Before immunostaining, the sections were preincubated with 0.1% pepsin containing 0.1 mol/L HCl, washed, and preincubated with PBS containing 2% rat sera. Primary monoclonal antibodies were diluted in PBS with 0.1% BSA and 0.1% Tween 20. A second incubation was performed with biotinylated affinity-purified sheep anti-mouse IgG antibodies (Amersham Corp) diluted in PBS with 0.1% BSA and Tween 20. Thereafter, the tissue sections were incubated with streptavidin-biotin horseradish-peroxidase complex (Amersham) diluted in PBS with 0.1% BSA and 0.1% Tween 20. The reaction was revealed by chromogen 3,3'-diaminobenzidine tetrahydrocloride (Serva 18865) with 0.05 mol/L Tris-HCl and 0.1 mol/L imidazole (Sigma I-7125). The specimens were counterstained with hematoxylin, and coverslips were aqua-mounted with Entellan (Merck 7961).

Double-Staining Procedure to Identify Mononuclear Cells Containing RCMV
After incubation with 1.5% nonimmune horse serum (Vector Laboratories), acetone-fixed frozen sections of cardiac allografts were incubated with a mixture of monoclonal antibodies against RCMV early and late antigens for 1 hour. With intervening washes in PBS, the following steps were performed: biotinylated horse anti–mouse/anti-rat absorbed antibodies for 30 minutes; avidin-biotinylated horseradish complex (Vectastain Elite ABC Kit, Vector Laboratories) in PBS was applied for 30 minutes; and the reaction was revealed by 3,3'-diaminobenzidine (DAB Substrate Kit, Vector Laboratories) containing nickel chloride. Then the sections were incubated with 1.5% nonimmune horse serum, followed by an incubation with monoclonal antibodies against rat helper and cytotoxic T cells (as above) and monocytes/macrophages (ED1 [Serotec], an IgG1 antibody against rat monocytes and macrophages). With intervening washes in PBS, the same steps were performed as with the first primary antibody. The second reaction was revealed by chromogen 3-amino-9-ethylcarbazole containing hydrogen peroxidase. The specimens were counterstained with hematoxylin, and coverslips were aqua-mounted (Aquamount, BDH Ltd).

Statistical Analyses
All data are expressed as mean±SEM. A nonparametric test was chosen because of small sample sizes and an inability to determine whether the samples were normally distributed.³⁰ The Mann-Whitney U test, z-corrected for ties, was used to evaluate significance. Comparison of intramural inflammation between RCMV-infected and uninfected rats was done by ² test. All analyses were performed with the STATVIEW 512+ program (Brain Power Inc). Values of P<.05 were considered statistically significant.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Cardiac allografts were transplanted from DA donors to WF recipients. Allograft recipients were infected with 10⁵ PFU IP RCMV 1 day after transplantation or were left uninfected and used as controls. To minimize the effect of acute rejection on the development of cardiac allograft arteriosclerosis, oral triple-drug immunosuppression was applied. No vascular wall changes were observed in the native hearts of allograft recipients of either RCMV-infected or uninfected rats. The values of histological changes are expressed as mean±SEM.

RCMV Infection in Cardiac Allograft Recipients
The natural history of RCMV infection was described in detail previously.³¹ In the rat, acute infection with systemic virus dissemination occurs within 5 to 10 days if unmodified by immunosuppression. Shortly thereafter, the infection progresses to a chronic phase, and the virus can be recovered only from the salivary gland of the host. At 6 months after infection, infectious virions are no longer present in any tissue, and the rat is presumed to be latently infected.

In these immunosuppressed cardiac allograft recipients, hardly any infectious RCMV could be demonstrated in liver or spleen during the observation time. Small amounts (0.3±0.3x10⁴ PFU/mL) of infectious RCMV could be demonstrated from biopsies of salivary glands 14 days after transplantation; they contained large quantities (0.9±0.2x10⁴ PFU/mL) of infectious RCMV 1 month after transplantation. The amount of infectious RCMV in the biopsies of salivary glands increased to 2.7x10⁴ PFU/mL at 3 months.

Inflammation
Intramural Inflammation
In cardiac allografts of uninfected rats, no intramural inflammation was observed in the vascular wall of arterioles or arteries (Fig 1A). Intramural inflammation of mononuclear inflammatory cells was characteristic of allografts in RCMV-infected rats (P=.005), all of which (four of four) showed intramural inflammation of arterioles but not of arteries 7 days after transplantation (Fig 1B). Thereafter, intramural inflammation was occasionally found in the vascular wall of both arterioles and arteries in RCMV-infected rats (Table 1).

View larger version (79K): [in this window] [in a new window]   Figure 1. Photomicrographs showing intramyocardial arterioles after heart transplantation. A, Normal-looking small intramyocardial arteriole of an uninfected rat on day 7. B, Intramural inflammation in small intramyocardial arteriole of RCMV-infected rat on day 7. C, Moderate endothelial cell proliferation in an intramyocardial arteriole of RCMV-infected rat at 1 month. D, Stenosed small intramyocardial arteriole of RCMV-infected rat at 3 months (arrow indicates internal elastic lamina revealed by Weigert–van Gieson staining; Mayer's hematoxylin-eosin staining, x100). E indicates endothelial cell; S, smooth muscle cell; M, a cluster of mononuclear inflammatory cells.

View this table: [in this window] [in a new window]   Table 1. Intramural Inflammation in Cardiac Allografts in Noninfected (DAWF) and RCMV-Infected (DAWF+RCMV) Rats

Perivascular Inflammation
In uninfected rats, there was no perivascular inflammation around cardiac allograft arterioles or capillaries 7 days after transplantation; later, the intensity of perivascular inflammatory cell response was trace to mild (Table 2). Compared with cardiac allografts in uninfected rats, RCMV infection induced early moderate inflammatory cell response of helper T cells, cytotoxic T cells, NK cells, and macrophages, as well as immune activation of these cells in the form of MHC class II expression (Table 2) around both small intramyocardial arterioles and capillaries 7 days after transplantation. No upregulation of interleukin-2 receptor expression was seen in cardiac allografts in both uninfected and RCMV-infected rats. In light microscopy, most of the inflammatory cells were Unna-Pappenheim–positive pyroninophilic cells. Thereafter, there was no difference in the inflammatory response in cardiac allografts in RCMV-infected rats compared with uninfected rats.

View this table: [in this window] [in a new window]   Table 2. Structure of Inflammation and Level of Immune Activation in the Perivascular Space of Arterioles and Capillaries in Cardiac Allografts of Noninfected (DAWF) and RCMV-Infected (DAWF+RCMV) Rats

The intensity of perivascular inflammation around cardiac allograft arteries was trace to mild during the whole experiment in uninfected and RCMV-infected rats, and no statistical difference was found between these two groups (not shown). In the perivascular space of veins and venules, the intensity of perivascular inflammatory response was also trace to mild, and there was no difference between the uninfected and RCMV-infected rats (not shown).

Interstitial Inflammation
In the interstitium of cardiac allografts in uninfected rats, there was nonexistent to trace inflammatory response of mononuclear inflammatory cells during the observation time. Occasionally, pyroninophilic cells and neutrophils were seen. The interstitial inflammation was associated with mild edema and hemorrhage. RCMV infection induced mild to moderate focal interstitial inflammation of helper T cells, cytotoxic T cells, macrophages, and NK cells, in that order of magnitude, 7 days after transplantation (not shown). The inflammatory cell infiltrate consisted mostly of pyroninophilic cells as demonstrated by Unna-Pappenheim staining. Mild edema and hemorrhage were seen, but hardly any myocytolysis or necrosis was present in the cardiac allografts of RCMV-infected rats.

Arteriosclerotic Vascular Wall Alterations
Endothelial Cell Proliferation
Phenotype and proliferation of cells morphologically consistent with endothelial cells were demonstrated with a double-staining procedure against von Willebrand factor and [methyl-³H]thymidine autoradiography from representative frozen cardiac allograft cross sections, respectively. The cells morphologically consistent with endothelial cells showed positive immunoreactivity to the antibody against von Willebrand factor and were labeled with [methyl-³H]-thymidine (Fig 2), indicating that they are proliferating endothelial cells.

View larger version (0K): [in this window] [in a new window]   Figure 2. Photomicrograph showing autoradiography-immunohistochemistry double-staining procedure demonstrating the von Willebrand factor immunoreactive proliferating endothelial cells (E) in longitudinal section of small intramyocardial arteriole in cardiac allografts of RCMV-infected rat. Note proliferating mononuclear cells (M) in the perivascular space of arteriole (Mayer's hematoxylin counterstaining, x100).

In cardiac allograft arterioles and arteries in uninfected rats, hardly any endothelial cell proliferation was observed, reaching a score of 0.3±0.3 and 0.3±0.2 at 3 months after transplantation, respectively (Fig 3A and 3C). In cardiac allograft arterioles of RCMV-infected compared with uninfected rats, a clear increase in endothelial cell proliferation was found, peaking with a score of 1.4±0.4 (P=.02) at 3 months after transplantation (Figs 1C, 2, and 3A). The time pattern of endothelial cell proliferation was quite similar in cardiac allograft arteries and arterioles, but the difference compared with uninfected rats was less prominent (Fig 3C).

View larger version (28K): [in this window] [in a new window]   Figure 3. Graphs showing the effect of RCMV infection on the generation of rat cardiac allograft arteriosclerosis in intramyocardial arterioles (A, B) and epicardial arteries (C, D). Cardiac allograft recipients with oral triple-drug immunosuppression were infected intraperitoneally with 105 PFU of RCMV (DAWF+RCMV []) 1 day after transplantation or were left uninfected and used as controls (DAWF []). Grafts were removed 7 (n=4) and 14 (n=5) days and 1 (n=8) and 3 (n=4) months after transplantation. In the control group, allografts were removed at 7 (n=4) and 14 (n=3) days and 1 (n=6) and 3 (n=5) months after transplantation. Vascular wall changes were scored semiquantitatively from 0 to 3 (0=normal, 1=mild, 2=moderate, and 3=severe), and the score was assigned by the consensus of three observers in a blind review. No vascular wall changes were observed in the native hearts of allograft recipients in either RCMV-infected or uninfected rats. Values are expressed as mean±SEM. Significances were determined by a nonparametric Mann-Whitney U test. Note that the time is given in logarithmic scale to emphasize the early posttransplant period. DA and WF are rat strains.

In the capillaries of cardiac allografts in uninfected rats, there was only very mild endothelial cell proliferation, reaching 0.6±0.4 at 3 months. In the capillary and postcapillary endothelium of cardiac allografts in RCMV-infected compared with uninfected rats, a significantly more pronounced proliferative response was observed, reaching a score of 0.9±0.2 (P=.02) on day 14 and prevailing at 1 (1.1±0.3; P=.02) and 3 (0.9±0.3; P=NS) months after transplantation.

Intimal Thickening
In the cardiac allografts of uninfected rats, hardly any intimal thickening was observed in either arterioles or arteries (Figs 3B, 3D, and 4A). In RCMV-infected rats, a gradual increase in intimal thickening was seen in arterioles and arteries of cardiac allografts, reaching a score of 1.1±0.4 (P=.04) and 1.3±0.2 (P=.01), respectively, at 1 month after transplantation. Thereafter, intimal thickening reached a plateau level (Figs 1C, 3B, 3D, 4B, and 4C).

View larger version (111K): [in this window] [in a new window]   Figure 4. Photomicrographs showing epicardial arteries after heart transplantation. A, Intact cardiac allograft artery of uninfected rat at 3 months; B, moderate intimal thickening in cardiac allograft of RCMV-infected rat at 3 months; and C, severe intimal thickening compromising the vascular lumen in cardiac allograft of RCMV-infected rat at 3 months (Wiegert–van Gieson staining to demonstrate internal elastic laminae, x40).

There were hardly any endothelial cell proliferation and no intimal thickening in the venules of cardiac allografts in uninfected and RCMV-infected rats (not shown).

RCMV Infection in Cardiac Allografts
Samples of cardiac allografts of uninfected and RCMV-infected rats were stained with a mixture of monoclonal antibodies of RCMV early and late antigens with the indirect three-layer immunoperoxidase technique. In addition, RCMV-immunopositive sections were stained separately with antibodies against RCMV early and late antigens to demonstrate infectious (replicating) virus in heart tissue.

No immunoreactivity was observed either in cardiac allografts in uninfected rats or in control sections of cardiac allografts left without primary antibody (Fig 5A). In cardiac allografts in RCMV-infected rats (Table 3), mononuclear inflammatory cell infiltrate in the pericardium was the most frequent and characteristic site for RCMV early and late antigen expression (Fig 5B). In addition, focal expression of RCMV early and late antigens was found in the interstitial mononuclear cell infiltrate and occasionally in cardiomyocytes and the medial cells of proximal aorta (Fig 5C). According to double-immunostaining procedure, RCMV-positive mononuclear cells were ED1⁺ macrophages (Fig 6). No double-positive reaction could be found when antibodies against helper or cytotoxic T cells were applied. Most importantly, focal RCMV early and late antigen expression was often observed in the perivascular mononuclear cell infiltrate around small intramyocardial arterioles and occasionally in the media cells of stenosed small intramyocardial arterioles (Fig 5D). Separately performed immunostaining for RCMV early and late antigens revealed that RCMV late antigen expression was prominent (Fig 7B), whereas RCMV early antigen expression was minor (Fig 7A).

View larger version (0K): [in this window] [in a new window]   Figure 5. Photomicrographs showing immunoperoxidase staining of early and late RCMV antigens with RCMV-specific monoclonal antibodies after heart transplantation. A, No immunoreactivity was observed in cardiac allografts of uninfected rats. B, Mononuclear inflammatory cell infiltrate in the pericardium was the most frequent and characteristic site for RCMV antigen expression in cardiac allografts of RCMV-infected rats (arrows indicate pericardium). C, Focal expression of RCMV antigens in the interstitial mononuclear inflammatory cell infiltrate of RCMV-infected rat. D, Media cells of stenosed small intramyocardial arteries of RCMV-infected rats occasionally showed RCMV antigen expression (Mayer's hematoxylin counterstaining, x10 [A], x100 [B, C, and D]).

View this table: [in this window] [in a new window]   Table 3. Expression of RCMV Early and Late Antigens in Cardiac Allografts of RCMV-Infected Rats

View larger version (0K): [in this window] [in a new window]   Figure 6. Photomicrograph from double-staining (biotin-avidin) procedure demonstrating RCMV early and late antigen expression (brown; chromogen 3,3'-diaminobenzidine) in ED1+ macrophages (red; chromogen 3-amino-9-ethylcarbazole). Mayer's hematoxylin counterstaining, x100.

View larger version (0K): [in this window] [in a new window]   Figure 7. Photomicrographs showing immunoperoxidase staining demonstrating RCMV early (A) and late (B) antigen immunoreactive mononuclear inflammatory cells in the pericardium. Note that the sections are identical and that the immunoreactivity occurs in nucleus (A; arrows indicate immune reaction) and cytoplasm (B). Mayer's hematoxylin counterstaining, x40.

RCMV late antigen expression indicates the presence of replicating virus. Therefore, we performed a modified plaque assay from representative cardiac section. According to immunoperoxidase staining, two of four cardiac allografts contained replicating virus at 3 months; two of four did not. Cardiac sections were homogenized, and specific pathogen–free BN rats irradiated with 5 Gy were injected intraperitoneally with the cardiac allograft cell suspension. After 26 days, salivary glands were taken for plaque assay, and infectious RCMV could be demonstrated only from rats that, according to immunohistochemistry, contained infectious RCMV.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
In this study, we demonstrate that RCMV infection accelerates rat cardiac allograft arteriosclerosis. Although the whole coronary artery tree was affected, the most prominent arteriosclerotic alterations were observed in the small intramyocardial arterioles. RCMV infection was linked with an early perivascular inflammatory response and intramural infiltration of mononuclear inflammatory cells in the cardiac allograft vascular wall. No cardiac allograft arterioles or arteries in uninfected rats showed intramural inflammation. RCMV infection induced endothelial cell proliferation and at least doubled the intimal thickening in small arterioles and arteries of cardiac allografts. In addition, intense endothelial cell proliferation was observed in cardiac allograft capillaries in RCMV-infected rats. As in transplantation-associated allograft arteriosclerosis in which the whole vessel wall is affected, an occlusion of small intramyocardial arterioles may lead to small silent infarcts and gradual deterioration in cardiac allograft function.

The most prominent arteriosclerotic alterations were observed in small cardiac allograft arterioles in RCMV-infected rats. Immunohistochemistry revealed upregulated presence of helper T cells, cytotoxic T cells, NK cells, and macrophages in the perivascular space of arterioles in RCMV-infected rats compared with uninfected rats. This inflammatory infiltrate also showed enhanced immune activation as MHC class II molecule expression but not in interleukin-2 receptor expression. Furthermore, vascular wall SMCs positive for RCMV early and late antigens and perivascular inflammatory cells were observed in the stenosed arterioles in RCMV-infected rats. Inflammatory cells positive for RCMV early and late antigens were frequently seen in the pericardial area and less often in the interstitium in cardiac allografts of RCMV-infected rats. According to double-immunohistochemistry, these cells were ED1-positive macrophages. The uptake of the virus by ED1⁺ macrophages, rather than viral replication in the cell, may account for some of the RCMV expression in these cells. None of the nontransplanted or uninfected cardiac allografts were RCMV early antigen– and late antigen–immunopositive.

RCMV-immunoreactive cardiac allografts were stained separately for RCMV early and late antigens. Staining revealed that late antigen expression was more prominent than early antigen expression, suggesting the presence of replicating RCMV in cardiac transplant up to 3 months after infection. Furthermore, modified plaque assay demonstrated that replicating virus exists in RCMV-immunoreactive allografts. We believe that this is the first experimental model demonstrating productive CMV infection of allograft structures in vivo.

Strong clinical evidence exists that CMV infection accelerates cardiac allograft arteriosclerosis. A Stanford University group⁹ demonstrated that graft atherosclerosis occurred much more frequently and earlier and the rate of graft loss was significantly greater in the CMV group. In addition, two other studies found an association between CMV infection and accelerated allograft arteriosclerosis in coronary angiograms.^{10 11} Recently, prolonged viremia was linked with the development of increased vasculopathic changes in heart allograft recipients.³² However, all clinical studies do not confirm the association between CMV infection and graft arteriosclerosis.^{33 34 35} This lack may, at least in part, be due to the method and criteria used to diagnose CMV infection.

We recently analyzed 762 endomyocardial biopsies of human cardiac allografts.¹³ According to light microscopic criteria, CMV infection was associated with vascular wall inflammation and early endothelial cell proliferation of small intramyocardial vessels. Later, these changes progressed to intimal thickening that encroached on vessel lumens. The histological findings were also correlated with angiographic data: CMV infection–associated acceleration of graft arteriosclerosis was seen both in EMB histology and coronary angiograms. However, the angiographic changes appeared more slowly compared with histological changes in biopsies, where the CMV-linked vascular changes were seen 1 year before in angiography.³⁶

CMV infection is associated with an acceleration of allograft arteriosclerosis in both clinical^{9 10 11 13 14 32 36} and experimental models.^{17 18 19 20} The early initial lesions on both occasions were associated with an accumulation of inflammatory cells in the vascular wall, a phenomenon not observed in CMV-free allografts. The results of this study applying a rat cardiac allograft model further substantiate the role of CMV infection in the generation of chronic rejection in model similar to the clinical situation. An early perivascular and intramural inflammatory cell response associated with RCMV infection was observed, followed by endothelial cell proliferation and gradual intimal thickening, especially at the site of small intramyocardial arterioles. These findings argue that early inflammatory responses in the vascular wall, mediated through mononuclear immune cells, may play a role in the initiation of CMV-associated acceleration of transplant arteriosclerosis. The fact that early and late RCMV antigens were detected in vascular wall SMCs and perivascular inflammatory cells of the stenosed arterioles suggests that these antigens might be the target of the immune responses.

There are several plausible mechanisms by which CMV could promote allograft arteriosclerosis. CMV can infect endothelial cells and SMCs of the vascular wall.^{16 37 38 39} Thus, the virus could cause a lytic infection of the endothelium and lead to inadequate repair and plaque formation. Percivalle and coworkers⁴⁰ recently reported that endothelial cells of capillary or small vessels are primary targets of viral infection in organ localizations of human CMV infection. They further demonstrated that human CMV–infected endothelial cells progressively enlarge until they detach from the vessel wall and enter the blood stream.

In the present study, we were not able to demonstrate that the endothelial cells of small intramyocardial arterioles or capillaries were RCMV-infected, although RCMV-associated vascular wall inflammation, endothelial cell proliferation, and intimal thickening were most prominent in small intramyocardial arterioles and capillaries in RCMV-infected rats. However, our results are compatible with previous work with animal models.^{16 39} These studies demonstrate that the amount of virus in the endothelial cells is rather low or nonexistent in vivo, although activation of endothelial cells can be found in infected animals.^{16 39} Thus, although there is no productive CMV infection, a virus-induced phenomenon of activation of endothelium occurs.

The virus may induce alterations in cellular metabolism and the production of different growth factors.⁴¹ Immediate-early protein of human CMV can be a trans-acting factor and thus possibly could induce alteration in the transcription of host cell genes.⁴² This would lead to proliferation of endothelial cells and SMCs and to increased intimal thickness and is supported by a recent observation of Speir et al⁴³ suggesting that activation of latent CMV infection by coronary angioplasty inactivates the p53 protein in SMCs. This in turn could predispose the cells to increased growth in that the p53 inactivation is believed to contribute to the formation of malignant tumors.⁴³

During CMV infection, enhancement of several molecular inflammatory cascades may occur. The immediate-early gene of CMV can code for a protein that has sequence homology and immunologic cross-reactivity with HLA-DR ß-chain.⁴⁴ Thus, CMV peptide expressed on the surface of the infected endothelial cells could substitute for HLA class II during the cognitive phase of lymphoid activation enabling T-cell activation without allogenic HLA expression. Increased MHC antigen expression mediated by interferon- produced during viral infection has also been suggested to trigger the rejection cascade.⁴⁵ This could also play a pivotal role in the generation of allograft arteriosclerosis, as suggested by Waldman and coworkers.⁴⁶ CMV also encodes a glycoprotein homologous to the heavy chain of MHC class I antigens that has the ability to bind the light chain of MHC class I molecules and may thus be involved in the virus-cell attachment.⁴⁷ These inflammatory and immunologic phenomena in response to injury⁴⁸ induced by viral infection may lead to endothelial cell damage, platelet aggregation, subendothelial inflammation and migration, and proliferation of SMCs, thus promoting cardiac allograft arteriosclerosis.

	Selected Abbreviations and Acronyms

 

CMV = cytomegalovirus HLA = human lymphocyte antigen MEM = modified Eagle's medium MHC = major histocompatibility complex NK = natural killer PFU = plaque-forming units RCMV = rat CMV SMC = smooth muscle cell

	Acknowledgments

 
This work was supported by grants from the Academy of Finland, University of Helsinki, the Aarne Koskelo Foundation, Finnish Medical Society Duodecim, and Finnish Cardiovascular Research Foundation, Helsinki, Finland, and Syntex Development Research, a division of Syntex (USA) Inc, Palo Alto, Calif. We would like to thank E. Wasenius and T. Lahtinen, Transplantation Laboratory, and G. Grauls, Medical Microbiology, for their excellent technical assistance.

Received December 2, 1994; revision received May 11, 1995; accepted May 25, 1995.

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