Ann Thorac Surg 2005;80:1787-1793
© 2005 The Society of Thoracic Surgeons
Original article: Cardiovascular
Effects of Mycophenolate Mofetil on Cardiac Allograft Survival and Cardiac Allograft Vasculopathy in Miniature Swine
Margaret L. Schwarze, MD
a
,
Stuart L. Houser, MD
b
,
Ashok Muniappan, MD
a
,
James S. Allan, MD
a
,
c
,
Matthew T. Menard, MD
a
,
Isabel McMorrow, PhD
a
,
Michaella E. Maloney, BA
a
,
Joren C. Madsen, MD, DPhil
a
,
d
,
*
a Transplantation Biology Research Center, Department of Surgery, Boston, Massachusetts, USA
b Department of Pathology, Boston, Massachusetts, USA
c Division of Thoracic Surgery, Department of Surgery, Boston, Massachusetts, USA
d Division of Cardiac Surgery, Department of Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts
Accepted for publication April 26, 2005.
* Address correspondence to Dr Madsen, Massachusetts General Hospital, 55 Fruit Street, BUL-01-119, Boston, MA 02114 (Email: madsen{at}helix.mgh.harvard.edu).
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Abstract
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BACKGROUND: Chronic rejection, as manifested by cardiac allograft vasculopathy, remains the leading cause of late graft failure in heart transplant recipients. Despite recent clinical trials, the efficacy of mycophenolate mofetil in preventing human cardiac allograft vasculopathy remains controversial. We investigated whether mycophenolate mofetil would prevent cardiac allograft vasculopathy and prolong cardiac allograft survival in our well-established miniature swine model of heart transplantation.
METHODS: Hearts disparate at the major histocompatibility complex class I locus were heterotopically transplanted into miniature swine recipients treated with a 12-day course of mycophenolate mofetil (n = 3) or cyclosporine A (n = 3). Allograft survival, acute rejection, and chronic rejection were monitored in the two groups.
RESULTS: Hearts transplanted with 12 days of cyclosporine were rejected between 46 and 61 days, whereas two of the three hearts transplanted with mycophenolate mofetil remained beating beyond 120 days (p = 0.02). At necropsy, there was a 4.9% mean prevalence of cardiac allograft vasculopathy in the mycophenolate mofetil group as compared with 16.6% in the cyclosporine group (p = 0.03). Cardiac allograft rejection and vasculopathy in the cyclosporine-treated group was associated with prominent myocardial interferon-
gene expression, a finding absent in two thirds of the mycophenolate mofetil-treated swine. Moreover, the mycophenolate mofetil-treated swine failed to develop IgM or IgG alloantibodies.
CONCLUSIONS: A short course of mycophenolate mofetil resulted in a longer allograft survival than a similar course of cyclosporine. Moreover, mycophenolate mofetil reduced the prevalence of cardiac allograft vasculopathy as compared with cyclosporine-treated controls. The salutary effect of mycophenolate mofetil may be related to its ability to decrease interferon- expression in the myocardium and prevent the generation of alloantibodies.
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Introduction
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Despite remarkable advances in immunosuppressive therapy during the past 15 years, cardiac allograft recipients are still threatened by allograft vasculopathy (CAV), which remains the leading killer of heart transplant recipients after the first post-transplant year. To date, there is no drug or therapeutic regimen available for clinical use that is known to prevent this form of chronic allograft rejection.
Mycophenolate mofetil (MMF), the morpholinoethyl ester of mycophenolic acid, is an anti-metabolic agent that prevents acute solid organ allograft rejection and potentially arrests the development of CAV. This drug is a reversible inhibitor of inosine monophosphate dehydrogenase and selectively blocks the de novo synthesis of guanine nucleotides upon which the activated B and T lymphocytes exclusively depend [1]. More recent studies have shown that depletion of intracellular guanosine triphosphate disrupts protein glycosylation, which subsequently inhibits cellular binding to endothelial adhesion molecules [2]. Furthermore, MMF, by this effect, has been shown to impair in vitro adhesion of peripheral blood cells to allograft endothelium [3].
Although investigators have shown that MMF can decrease cardiac allograft arteriopathy in small animal models [4–6], the effect of MMF on CAV in a large-animal model has not been demonstrated. In addition, the effect of MMF on CAV in human cardiac transplant recipients has been difficult to clarify. In a large prospective randomized trial comparing MMF with azathioprine, MMF yielded a slight survival advantage, but the effect on CAV was uncertain or modest at best [7–9]. The use of other immunosuppressive agents in this trial also confounds the interpretation of the data.
In this study, we decided to test whether or not MMF itself prolongs cardiac allograft survival and prevents the development of CAV. To this end, we have used a well-established miniature swine model of heart transplantation [10–12] to examine the effect of substituting MMF for cyclosporine A (CyA) as a single immunosuppressive agent. We believe that our large-animal cardiac transplant model has significant relevance to human heart transplantation, particularly in comparison with small-animal models. Specifically, the lesions of CAV seen in these swine are pathologically similar to those of human recipients; and unlike small animals, miniature swine constitutively expresses class II antigens on their vascular endothelium in a quantity similar to human endothelium [13].
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Material and Methods
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Animals
A selective breeding program for the last 30 years has yielded miniature swine with defined major histocompatibility complex (MHC) loci (termed swine leukocyte antigen [SLA]) [14]. At present, the swine of three homozygous MHC haplotypes (SLAaa, SLAcc, and SLAdd) are maintained. In addition, swine bearing three intra-MHC recombinant haplotypes have been derived by spontaneous recombination events during the breeding of heterozygotes [15]. Genotyping has been controlled by strict pedigree breeding and confirmed by microcytotoxicity testing using allospecific antisera. For the purpose of this experiment, animals 3 to 6 months of age were used. Hearts were transplanted heterotopically across a class I MHC barrier using either SLAgg (class Ic, class IId) donors with SLAdd recipients (class Id, class IId), or SLAjj (class Ia, class IIc) donors with SLAcc (class Ic, class IIc) recipients. Pre-transplant alloreactivity between donor and recipient pairs was confirmed by cell-mediated lympholysis assays as previously described [16]. All animal care and procedures were performed in compliance with both the Principles of Laboratory Animal Care, formulated by the National Society for Medical Research, and the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health, revised 1996.
Cardiac Transplantation and Monitoring
The technique of heterotopic heart transplantation has been previously described [10]. In brief, donors and recipients were pre-medicated with glycopyrrolate (0.04 mg/kg), xylazine (2 mg/kg), and then anesthetized with telazol (1.4 mg/kg), halothane (1%), and N2O (1%). After the placement of two indwelling central venous catheters in each recipient, a transperitoneal approach was used to isolate the infrarenal aorta and inferior vena cava. A right nephrectomy was performed as needed to provide adequate intraabdominal space for the transplanted heart. Both the donor and recipient animals were heparinized with 300 U/kg of heparin. The donor heart was harvested after perfusion with cold cardioplegia (Plegisol [Abbott Laboratories, Abbott Park, IL]) for 5 minutes. The heart was maintained in an ice-saline slurry and prepared for implantation by creating an atrial septal defect and defunctionalizing the mitral valve to minimize left ventricular atrophy and intracavitary thrombus formation. Once prepared, the heart was implanted by anastomosing the donor pulmonary artery to the recipient's inferior vena cava and the ascending donor aorta to the recipient's abdominal aorta. Iridium-tipped ventricular electrodes (Model 6500 pacing lead [Medtronic Inc, Seacaucus, NJ]) were implanted into each ventricle and brought out through the skin for long-term epicardial electrocardiographic monitoring. Heart function was monitored by transabdominal palpation, electrocardiogram (EK/5A [Burdick Corp, Milton, WI]), and echocardiography (Hewlett-Packard Sonos 1500, Andover, MA). Allograft rejection was defined by an R-wave amplitude < 3 mm by electrocardiogram or the lack of ventricular contraction by palpation, confirmed by echocardiography. Serial biopsies were performed on all transplanted hearts through an open transabdominal technique using Tru-cut needles (Baxter, Deerfield, IL), except for the biopsy scheduled on or around postoperative day (POD) 60 in the MMF-treated animals, which was performed by excising a generous portion of the cardiac apex (in order to reduce sampling error).
Immunosuppression
Recipients in the experimental group (Nos. 13259, 13729, and 13889) received intravenous MMF generously provided by Roche Pharmaceuticals (Basel, Switzerland) on PODs 0 through 11. The animals received an initial intravenous dose of 1.5 grams twice daily, which was tapered when all 3 animals developed diarrhea. Trough MMF levels were obtained daily through high performance liquid chromatography. Complete blood counts were measured daily throughout the duration of MMF therapy.
Our goal during MMF therapy (Fig 1A) was to maintain free plasma MMF trough levels between 3 and 5 µg/mL. With the exception of one very high trough level (17.2 µg/mL) on POD 2 in animal number 13259, we were able to achieve this goal (Fig 1B). However on retrospective analysis, the plasma trough levels of mycophenolic acid, the active metabolite of MMF, were less than the limit of detection on PODs 4 through 7 with swine number 13729 (Fig 1B). All 3 experimental animals developed severe, non-bloody diarrhea between 4 to 6 days after starting MMF. The diarrhea resolved after reducing the dose. In 2 animals, numbers 13729 and 13889, we were able to increase the dose after the diarrhea resolved without the recurrence of symptoms. In 2 of our experimental animals (numbers 13889 and 13259), dose adjustments were also made to address some mild leukopenia that had developed during therapy; the leukopenia resolved in both of these animals. In animal number 13729, the white blood cell count did not drop below 13,800 (white blood cells/µL) during the course of immunosuppressive therapy (Fig 1C).
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Fig 1. Mycophenolic acid (MMF) and cyclosporine A (CyA) dosing. (A) The indicated dose of MMF was administered intravenously in two divided doses daily. All animals received 3 grams of MMF on the day of transplantation, and subsequent doses were adjusted based on trough serum levels and onset of diarrhea. (B) Free mycophenolic acid (MMFs primary and active metabolite) trough levels in serum were determined by high performance liquid chromatography. The target range for mycophenolic acid trough levels was 3 to 5 µg/mL (grey box), which was achieved in all animals, except number 13729, who had sub-therapeutic levels from postoperative days 4 through 7. (C) The white blood cell counts (WBC) of cardiac allograft recipients were determined daily while MMF therapy was ongoing. Two of the 3 animals, numbers 13259 and 13889, displayed a modest decline in WBC counts, which recovered after MMF therapy was completed. The remaining animal, number 13729, which also had the lowest trough MMF levels of the 3 animals, did not have any noticeable decline in WBCs. (D) Cyclosporine A trough levels were determined by fluorescence polarization immunoassay performed on whole blood. Once daily intravenous CyA dosing was given to achieve trough levels that were mainly between 400 and 800 ng/mL (grey box). (POD = postoperative day.)
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Recipients in the control group (Nos. 13262, 12049, and 13495) received intravenous cyclosporine, generously provided by Novartis Pharmaceutical Corporation (East Hanover, NJ) on PODs 0 through 11. An intravenous dose ranging between 10 and 13 mg/kg was given once daily and adjusted to maintain whole blood trough CyA levels between 400 and 800 ng/mL (Fig 1D) as determined by fluorescence polarization immunoassay (Abbott Laboratories, Dallas, TX).
Skin Grafts
Skin grafts were performed by previously published techniques [17]. Briefly, split-thickness skin from the donor, recipient, and third-party animals (stored frozen until skin transplant) were placed on a deep split-thickness bed on the recipient's dorsal thorax. Grafts were examined daily for viability by warmth, softness, and color, until rejection occurred. Rejection was determined macroscopically and was defined as when less than 10% of the graft appeared to be viable.
Histopathological Examination
Heart tissue from either biopsy or necropsy specimens was fixed in 10% formalin. The tissues were imbedded in paraffin; cut sections were stained with hematoxylin and eosin, and Verhoeff's elastic stain. A cardiac pathologist blinded to experimental group evaluated histologic specimens to determine the severity of acute and chronic rejection. Scoring of the acute rejection in the cardiac allograft was based on International Society for Heart and Lung Transplantation criteria [18].
Computerized Morphometry
In an effort to quantify the degree of chronic rejection, 5-micron sections of cardiac allograft specimens were cut from paraffin blocks and examined with light microscopy. Elastic-stained epicardial and myocardial arteries and veins with intimal lesions were captured digitally and analyzed using image analysis software (IPLab Spectrum [Signal Analytics Corporation, Vienna, VA]). Manual color segmentation, tracing the endothelial surface (intima), internal elastic lamina, and external elastic lamina was performed on each vessel. From segmented images, intimal and luminal areas were computed, allowing calculation of percent of stenosis of each vessel lumen. Prevalence and severity of CAV were defined as the percent of vessels with intimal lesions and percent stenosis of involved vessels, respectively, and they were expressed as the mean ± standard deviation for each group. The grade of intimal thickening of all epicardial and intramyocardial vessels with CAV was based on a modification of the system described by Lurie and colleagues [19] and expressed as the mean ± standard deviation for each group.
Flow Cytometric Alloantibody Detection
The presence of anti-donor IgM and IgG in the sera of recipient swine was detected by indirect flow cytometry using FACScan II (Becton Dickinson, San Jose, CA). Peripheral blood leukocytes from donor-SLA-matched swine were isolated by gradient centrifugation using Lymphocyte Separation Medium (Organon, Teknika, Durham, NC). The 1 x 106 cells per tube of donor-type peripheral blood leukocytes (SLAgg, class Ic, IId, SLAjj, class Ia, IIc) were suspended in Hank's balanced salt solution containing 0.1% bovine serum albumin and 0.1% NaN3. These peripheral blood leukocytes were then incubated for 30 minutes at 4°C with decomplemented test sera from recipient animals. After two washes, saturating concentrations of fluorescein isothiocyanate-labeled goat anti-swine IgG or IgM (PharMingen, San Diego, CA) were added to each tube, which was then incubated for 30 minutes at 4°C. After a final wash, cells were analyzed by flow cytometry using propidium iodide gating to exclude dead cells. Both naive pig sera and pre-transplant sera from each respective experimental animal were used to assure specific binding.
RNAse Protection Assay
At the time of graft harvest, myocardial tissue was frozen in guanidinium thiocyanate for subsequent extraction of RNA. In order to compare expression of Th1 with Th2 cytokines in the two protocols, a ribonuclease protection assay was used according to the manufacturer's directions (Riboquant Multi-probe Template Set, PharMingen) to quantitate mRNA of interleukin (IL)-4, IL-10, IL-15, IL-2, IL-6, and interferon gamma (IFN-) in the tissue. Housekeeping gene products L32 and glyceraldehyde-3-phosphate dehydrogenase were used as a control for quantity and quality of RNA loaded. Signals were quantified by densitometric analysis (Molecular Analyst, Bio-Rad Laboratories Inc, Hercules, CA) and normalized to account for differences in loading. The expression index (EI) was calculated as follows: EI(a) = [value of sample a] x [highest value of L32/value of L32(a)].
Statistical Analysis
Comparisons of survival of experimental with control groups were made using a log-rank test. Comparisons of CAV grade with prevalence in experimental and control groups were made using an unpaired Student's t test. Comparisons with p values less than 0.05 were considered significant.
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Results
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Survival
Hearts transplanted heterotopically across a class I MHC barrier with 12 days of MMF survived for greater than 120 days, with two of three grafts still beating when the animals were sacrificed (> 124, > 170, and 130 days). In contrast, control hearts transplanted across the same MHC barrier and treated with 12 days of CyA were all rejected in less than 61 days (46, 61, and 52 days; p = 0.02; Table 1).
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Table 1. Cardiac Allograft Survival in Miniature Swine Treated With Either Cyclosporine A or Mycophenolate Mofetil
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Histology
Biopsy and histopathologic examination of the cardiac allografts were performed serially. There was a tendency toward earlier appearance and more prevalent CAV in allografts placed in CyA-treated animals than in MMF-treated animals (data not shown). However, when hearts transplanted in MMF-treated animals were examined at necropsy, all grafts harbored lesions characteristic of CAV, as did allografts in CyA-treated animals (Fig 2).
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Fig 2. Histologic examination of arteries in mycophenolic acid (MMF)-treated cardiac allografts. All specimens are from necropsy. Sections are processed with an elastin stain, and vessels with an appearance consistent with cardiac allograft vasculopathy (CAV) are shown at the indicated magnification. (A) Although lesions consistent with CAV were detected in all of the MMF grafts, the prevalence of such lesions, as determined by morphometric analysis, was significantly less in MMF-treated animals than in cyclosporine A (CyA)-treated animals. (B) For comparison, a representative CAV lesion is shown with a CyA-treated animal. (POD = postoperative day.)
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Morphometric Analysis
We have recently developed a morphometric technique for more accurate depiction of the burden of CAV in cardiac allografts [20]. Morphometric analysis of the cardiac allografts at the time of necropsy revealed CAV in 3% to 7.9% (mean, 4.9 ± 2.7 %) of the arterial vessels examined in the MMF-treated animals. In comparison, the prevalence of CAV in CyA-treated group was 11.4% to 18 % (mean, 16.6 ± 4.6%; p = 0.03, Fig 3). Although CAV prevalence was substantially less in MMF-treated animals, the mean severity of individual lesions in the two groups was similar (p = 0.38; Fig 3). The prevalence of CAV lesions identified in the MMF treatment group is not significantly different from that observed in the hearts of naïve unmanipulated miniature swine (personal communication, Stuart Houser, MD). This result is made more impressive considering that the morphometry was performed at necropsy, which occurred significantly later in MMF-treated animals (mean day of necropsy, POD 141) than with CyA-treated animals (mean day of necropsy, POD 53).
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Fig 3. Morphometric assessment of cardiac allograft vasculopathy (CAV) prevalence and severity in cyclosporine A (CyA)-treated and mycophenolic acid (MMF)-treated recipients of cardiac allografts. Allografts were harvested at necropsy and morphometric techniques were used to measure the incidence and grade of CAV. (A) Allografts from CyA-treated animals exhibited significantly higher prevalence of CAV compared with MMF-treated animals, (B) although the grade of CAV in both groups was similar.
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Ribonuclease Protection Assay Data
Prominent myocardial IFN- transcript expression was noted in all of the allografts in the CyA-treated group ([20], previously reported). In the MMF-treated group, only 1 animal that had transiently sub-therapeutic mycophenolic acid levels (No. 13729) expressed a significant amount of IFN- transcripts within the allograft (Fig 4). Relatively low levels of IL-15 and IL-10 gene expression in both groups were quantified by scanning densitometry (Fig 4). No expression of IL-4, IL-6, or IL-2 was detected (data not shown).
Detection of Alloantibody
Neither IgM nor IgG alloantibodies were found in any MMF-treated animals throughout the entire postoperative course (Figs 5A–5C). Consistent with previously published results, alloantibodies (IgG and IgM) were present at the time of rejection in all of our CyA-treated control animals (Fig 5D).
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Fig 5. Assay for production of alloantibodies in miniature swine recipients of cardiac allografts treated with either mycophenolate mofetil (MMF) or cyclosporine A (CyA). Immunoglobulin M (IgM) and immunoglobulin G (IgG) alloantibodies in the serum of MMF-treated or CyA-treated recipients were assayed by flow cytometry. No IgG or IgM alloantibody was detectable in MMF-treated recipients throughout the study period (A–C), whereas all CyA-treated animals developed IgM and IgG alloantibody before rejection, (D) (a representative animal is shown). Negative and positive control staining with sera from a naïve and donor-type sensitized animal, respectively, are shown (E). (POD = postoperative day.)
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Skin Grafts
Skin grafts were placed on all three MMF-treated animals after POD 110 to determine whether the animals were immunocompetent or tolerant to donor antigen, or both. Animal number 13259 was unexpectedly found ill and was sacrificed 9 days after skin grafting, preventing interpretation. The other two MMF-treated animals rejected both donor-matched and third-party skin with a time course typical of a first-set rejection response, demonstrating immunocompetence and lack of durable tolerance.
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Comment
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In this report, we demonstrate that MMF, as a single-agent, markedly abrogates the development of CAV in a miniature swine model of chronic rejection. As has been previously shown, miniature swine treated with 12 days of CyA all develop significantly elevated CAV prevalence by the time of graft loss [20]. It has been suggested that MMF can directly suppress the process of chronic rejection through its antiproliferative actions on smooth muscle [21, 22], and this may have been one of the mechanisms by which MMF prevented CAV in this miniature swine model of heart transplantation. As an abbreviated course of MMF was used, rather than chronic maintenance therapy, the observed durable effect of MMF treatment suggests that it induced a state of immune responsiveness in these pigs that was less conducive to chronic rejection. In this study comparing MMF with CyA we elected not to use clinically relevant chronic immunosuppression because we did not intend to abrogate allospecific responses completely. Rather, we intended to permit chronic rejection to have an effect on the graft in a reasonable time frame.
Mycophenolic acid also has a salutary effect on allograft survival in this model, as two of three grafts remained beating beyond 120 days and a third survived for 130 days, which was markedly prolonged compared with a mean survival time of 53 days for CyA-treated heart allografts. The beneficial effects of reduced CAV development and prolonged allograft survival attributable to MMF are unlikely to be simply due to increased overall immunosuppression compared with CyA, as we used MMF dosing that was comparable with clinical protocols, whereas CyA dosing was supra-therapeutic. Mycophenolic acid has been shown to inhibit proliferation of both T and B cells [23] which may be the mechanism by which cardiac allograft survival was prolonged in this model.
Although MMF therapy is associated with impaired immune responsiveness to cardiac allografts in miniature swine, it is apparent that 12 days of MMF treatment is not sufficient for tolerance, as all grafts displayed pathologic evidence of acute cellular rejection, and all recipients rejected donor skin transplants in a non-delayed fashion. We have previously shown that durable tolerance to cardiac allografts can be achieved in miniature swine by co-transplantation of donor matched kidneys with 12 days of cyclosporine [12]. It remains to be seen whether MMF would also permit development of tolerance in simultaneous heart and kidney recipients. With respect to tolerance protocols, agents such as MMF may have a valuable role, as it has been suggested that calcineurin inhibitors, such as CyA, can interfere with particular tolerance strategies [24].
This study also provides evidence for an association between intramyocardial IFN- gene expression and the development of CAV. Studies of cardiac transplantation in rodents have provided evidence that IFN- contributes to CAV development [25,26]. Prominent myocardial IFN- gene expression was noted in all of the CyA-treated swine, although absent in two of the three MMF-treated animals. Interestingly, the heart graft of animal number 13729, which was the only MMF-treated animal to have significant IFN- gene expression in the graft, still had a relatively low incidence of CAV compared with CyA-treated animals. It may be that MMFs ability to suppress CAV generation in cardiac allografts is related not only to prevention of intra-graft IFN- expression, but perhaps other mechanisms as well. Admittedly, a larger study is required to confirm this.
There is emerging data to suggest that humoral responses also contribute to the process of chronic rejection in various organ systems [27]. Both acute [27] and chronic humoral rejection [28, 29] of cardiac allografts are recognized processes and prevention of humoral alloimmunity is a clinically recognized objective. In concordance with MMFs known ability to inhibit B cell proliferation and antibody synthesis, we observed that treatment with 12 days of MMF prevented development of alloantibodies in the serum of cardiac allograft recipients. Both IgG and IgM alloantibody are consistently found in recipients treated with 12 days of CyA. Interestingly, MMF treatment prevented development of serum alloantibody in animal number 13889, whose graft had rejected 10 days prior to necropsy. The inhibitory effect of MMF on B cells appears to be relatively long lasting, and the lack of development of alloantibody may have contributed to the protection from interstitial rejection and vasculopathy.
In this study, we have demonstrated that MMF can substantially increase the survival of a heterotopic cardiac allograft. Moreover, MMF therapy seems to delay the onset of CAV, as well as decrease the prevalence of CAV in cardiac allografts in miniature swine. Mycophenolic acid therapy was shown to significantly suppress the expression of IFN- in allografts and the generation of alloantibodies. These data suggest that MMF has a beneficial effect in preventing acute and chronic cardiac allograft rejection, and that there may be an expanded role for this agent in clinical transplantation.
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Acknowledgments
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The authors wish to thank Drs Dax Guenther and Tsuyoshi Shoji for their critical review of the manuscript. Cyclosporine was generously provided by Novartis, and MMF was generously provided by Roche. This study received financial support from Roche. This study was supported in part by grants from the National Heart, Lung, and Blood Institute (RO1 HL54211, PO1 HL18646, and RO1 HL67110) and the National Institute of Allergy and Infectious Diseases (PO1 AI50157) of the National Institutes of Health, and from the Roche Corp.
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E. Raichlin, J.-H. Bae, Z. Khalpey, B. S. Edwards, W. K. Kremers, A. L. Clavell, R. J. Rodeheffer, R. P. Frantz, C. Rihal, A. Lerman, et al.
Conversion to Sirolimus as Primary Immunosuppression Attenuates the Progression of Allograft Vasculopathy After Cardiac Transplantation
Circulation,
December 4, 2007;
116(23):
2726 - 2733.
[Abstract]
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Abstract
Introduction
