Ann Thorac Surg 2001;71:S389-S392
© 2001 The Society of Thoracic Surgeons
Basic research
Transgenic porcine valves show no signs of delayed cardiac xenograft rejection
Raymond H. Chen, MD, PhDa,
David H. Adams, MDa
a Division of Cardiac Surgery, Brigham & Women’s Hospital, Harvard Medical School, Boston, Massachusetts, USA
Address reprint requests to Dr Adams, Division of Cardiac Surgery, Brigham & Women’s Hospital, 75 Francis St, Boston, MA 02115
e-mail: dadams{at}partners.org
Presented at the VIII International Symposium on Cardiac Bioprostheses, Cancun, Mexico, Nov 3–5, 2000.
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Abstract
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Background. Glutaraldehyde fixation stiffens the structural integrity of porcine valves, although the solution also destroys tissue viability and accelerates calcification. Recently, we demonstrated that fresh cardiac valves from domestic pigs do not express the galactose 
1, 3 galactose (-Gal) antigen and may be immunologically unique. The absence of -Gal explained why the valves remained pristine while the rest of the porcine heart was destroyed by primate immunoglobulin M (IgM) and complement membrane attack complex (MAC) within 60 minutes. We sought to clarify whether fresh porcine valves from transgenic pigs bearing human complement regulatory proteins (CD59/DAF) can survive longer in primates and whether porcine cardiac valves remained immunologically privileged after prolonged exposure.
Methods. Tissue sections from wild-type untransplanted (n = 6), wild-type transplanted (n = 3), and transgenic pigs expressing human CD59/DAF proteins transplanted (n = 3) porcine-to-primate cardiac grafts were examined by hematoxylin and eosin, and by immunohistochemistry for the porcine endothelial marker (GalNac), -Gal, primate IgM and MAC.
Results. -Gal antigens were highly expressed on the vascular, but not valvular, endothelium of transgenic pigs. Hearts from CD59/DAF transgenic pigs survived 5, 7, and 11 days, but showed increasing IgM and MAC deposition until failure. Valves remained morphologically intact at explant, and strong GalNac staining suggested an intact endothelial surface. However, the valves showed no signs of IgM- or MAC-mediated damage.
Conclusions. Although hearts from transgenic pigs expressing human complement regulatory proteins can survive for days in the primate recipient, the xenografts eventually fail because of escalating attacks of primate IgM and MAC. The absence of the -Gal antigens protects unfixed porcine valves from rejection.
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Introduction
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Diseased cardiac valves are replaced with either mechanical or biological tissue prostheses. Mechanical valves are more durable, but require long-term anticoagulation therapy with coumadin to prevent surface clotting. Risks associated with anticoagulation therapy, such as bleeding or thromboembolism, are of concern to many patients. Biological tissue valves, on the other hand, do not require anticoagulation therapy, but are limited by durability secondary to structural degeneration or calcification, or both [1].
We recently demonstrated a third possible replacement material: fresh porcine valves [2]. Fresh porcine valves are immunologically privileged, as they do not express -Gal, the carbohydrate antigen that is responsible for the rejection of porcine hearts. Consequently, even when wild-type porcine hearts are destroyed by primate immunoglobulin M (IgM) and membrane attack complex (MAC) deposition within 60 minutes, the valves remain structurally pristine. We are interested in fresh valves because they contain viable endothelial and fibroblast cells, which can continue to repair and modify the leaflets undergoing hemodynamic stress and provide better long-term durability.
To determine whether fresh porcine valves would remain immunologically unique after prolonged survival in primates, we performed xenotransplantation with transgenic pigs expressing human complement regulatory proteins (CD59, DAF, or MCP) as heart donors. These transgenic human proteins down-regulate MAC-mediated attacks and prolong porcine xenograft survival in primates [3]. We examined the valve leaflets in these longer-surviving porcine xenografts to determine if the valves are attacked by antibodies.
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Material and methods
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Experimental animals
Heterotopic pig-to-primate cardiac xenotransplantation was performed to test the immune reaction against fresh, unmanipulated porcine valves. Olive baboons (Papio anubis) served as transplant recipients. Wild-type or transgenic pigs expressing the human CD59/DAF or MCP complement regulatory proteins served as heart donors. The construction of transgenic pigs bearing human complement regulatory proteins has been described previously [4]. Briefly, large genomic clones encompassing the genes encoding human CD59 and DAF were microinjected into fertilized porcine oocytes to generate transgenic pigs using standard technique [5]. Both clones are approximately 90 kb and contain 10 to 20 kb of the 5' and 3' flanking sequences of their respective genes. G1 offspring from one founder that was transgenic for both human CD59 and DAF were used as heart donors. All animals received humane care in accordance with the guidelines of the Harvard University Animal Care Committee, and the "Guide for the Care and Use of Laboratory Animals" (NIH publication 85-23, revised 1985).
Anesthesia
For invasive procedures and anesthesia induction, sedation was achieved using ketamine hydrochloride (10 mg/kg intramuscularly [IM]) in baboons and telazol (5 mg/kg IM) in pigs. Respiratory secretions were controlled with atropine sulfate (0.03 mg/kg). Anesthesia was maintained with inhalational isoflurane (1.3% to 2.0%). Intraoperatively both pigs and baboons were monitored with electrocardiography, noninvasive blood pressure monitoring, and pulse oximetry. All baboons received antibiotic coverage with cefazolin (20 mg/kg IM twice a day) and pain control with butrenorphine (0.005 mg/kg twice a day) postoperatively for 5 days. In the event of diminished fluid or caloric intake, recipients received their calculated daily needs as intravenous (IV) crystalloid solution or Ensure tube feeds.
Heterotopic heart transplantation model
Heart transplantation was performed in the abdominal cavity as described previously [6]. Pigs were anesthetized, and received systemic anticoagulation with heparin (100 IU/kg IV), after which the heart was harvested in a standard fashion after protection by 500 mL of antegrade cold crystalloid cardioplegic solution (dextrose 2.5%, NaCl 0.45%, potassium 30 mEq/L, bicarbonate 5 mEq/L). Transplant organ ischemic time varied between 45 and 55 minutes. The transplant recipients received daily immunosuppression drugs starting 15 days before transplantation. The immunosuppressive regimen consisted of cyclosporine (7 mg/kg IM), methylprednisolone (10 mg/kg IM), cyclophosphamide (10 mg/kg IM), and soluble carbohydrate (50 mg/kg IV).
Evaluation of transplant organ function
An implantable telemetric system (Data Science International, St. Paul, MN) was used to continuously follow graft cardiac rhythm as an indicator of viability in awake recipients [7]. Grafts were explanted when the telemetric signal showed complete cessation of cardiac contractility.
Histopathologic studies of the porcine valves
After graft explantation, aortic and pulmonary valves were carefully dissected and either snap-frozen in tissue freezing medium (Triangle Biomedical Sciences, Durham, NC) and liquid nitrogen or fixed in Carnoy’s solution and embedded in paraffin. Paraffin sections were sectioned at 5 µm thickness and stained with hematoxylin and eosin. Biotinylated-lectin from Griffonia simplicifolia (GSI; Sigma, St. Louis, MO) was used to stain -Gal on porcine endothelium. Cryostat sections were immunostained using standard indirect immunoperoxidase avidin–biotin techniques previously summarized [4], with monoclonal antibodies specific for IgM (Biodesign International, Kennebunk, ME) and MAC (C5-9; Dako, Carpinteria, CA). The signal was developed with the avidin–peroxidase system (ABC kit, Vector Lab, Burlingame, CA).
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Results
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Untransplanted hearts
Untransplanted wild-type or transgenic porcine hearts (n = 6 each) showed strong -Gal expression of the microvascular endothelium (Fig 1A). In comparison, neither aortic (Fig 1B) nor pulmonary valves (Fig 1C) show any detectable -Gal expression.
Transplanted wild-type porcine valves
Three wild-type pig hearts were transplanted into unmodified baboon recipients and were rejected 60 to 80 minutes after implantation. The hearts became edematous and cyanotic, with microscopic thrombi and hemorrhage. There was extensive microvascular IgM and MAC deposition on the microvascular endothelium, although neither was found on aortic and pulmonary valves.
Transgenic porcine valves
Hearts from transgenic pigs expressing human CD59/DAF proteins (n = 3) were transplanted into baboons. Despite administration of high-dose immunosuppression drugs, recipient primates rejected these transgenic porcine hearts in 5, 7, and 11 days. Xenotransplant biopsies showed progressively increased IgM deposition until rejection (Fig 2A), correlating with increased microvascular thrombosis. MAC deposition was highly detectable in the microvascular endothelium of explanted xenografts (Fig 3A).
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Fig 2. Transgenic porcine hearts: immunoglobulin M (IgM) deposition for the xenograft that survived for 11 days. The myocardium was grossly destroyed, although the valves remained intact. Immunohistochemistry showed that IgM attacked microvascular endothelium (A) but spared aortic (B) and pulmonary (C) valves. (Hematoxylin counterstain; x100 before 63.5% reduction.)
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Fig 3. Transgenic porcine hearts: membrane attack complex (MAC) deposition for the xenograft that survived for 11 days, the myocardium was grossly destroyed, although the valves remained intact. Immunohistochemistry showed that MAC attacked microvascular endothelium (A), but spared aortic (B) and pulmonary (C) valves. (Hematoxylin counterstain; x100 before 63.5% reduction.)
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Both aortic and pulmonary valves from rejected xenografts were intact, and the preservation of valvular endothelium was confirmed by GalNac staining. The valve leaflets did not show IgM (Figs 2B and 2C) or MAC (Figs 3B and 3C) deposition.
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Comment
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The use of animal valves as prostheses was first proposed and tested during the 1940s and 1950s. It was understood that transplantation of animal tissues into humans was difficult, but the exact barrier responsible for rejection was not known. It was not until 1968 when the role of histocompatibility antigens in human allotransplantation became understood [8]. Moreover, the mechanism of porcine-to-human xenograft rejection, that is, anti--Gal IgM antibodies, was not elucidated until 1987 [9]. Meanwhile, the field of bioprosthetic valves has been following the dogma that valve fixation is required to prevent rejection.
We previously found that porcine valvular leaflets do not express -Gal on their endothelium [2]. The absence of detectable -Gal on valvular leaflets apparently protected porcine valves from IgM-mediated hyperacute rejection. Consequently, even though wild-type porcine cardiac xenografts were destroyed within 60 to 80 minutes of implantation by the severe IgM and MAC deposition, aortic and pulmonary valves harvested from rejected porcine xenografts showed no sign of damage.
We now investigated the susceptibility of porcine valves to primate antibodies after prolonged exposure. We used CD59/DAF transgenic pigs as heart donors because we have previously shown that pigs expressing these proteins are protected from hyperacute rejection, that is, anti--Gal IgM antibody-triggered, MAC-mediated attacks [4].
After prolonged exposure to porcine hearts, however, baboon recipients synthesized increasing amounts of IgM and MAC, which eventually overwhelmed the suppressive effects of human transgenic CD59/DAF proteins [3]. Consequently, although transgenic xenografts initially did not show MAC deposition, endothelium showed progressively more MAC deposition. An elevating level of microvascular MAC deposition elicited microvascular thrombosis and eventual xenograft loss. Porcine valve leaflets, however, showed no signs of either IgM or MAC deposition. The pristine state of valvular histology, compared with the extensive myocytic damage, confirms the immunologic privilege of valve leaflets.
Recent interest in pig-to-human xenotransplantation has created an industry of commercial vendors providing molecularly screened, pathogen-free pigs. The wide availability of pigs allows the harvesting of fresh porcine cardiac valves at the time of need, so as to eliminate either freezing or fixative treatments, which prolong the shelf life of porcine prosthesis at the expense of tissue viability. In addition, our recent experience with genetic engineering in pigs shows we can selectively insert genes of interest, such as an extra collagen synthetase gene, to strengthen valve tissue integrity. More studies in this area are warranted to realize the potential of fresh porcine valve prosthesis.
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Acknowledgments
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This work was supported by a research grant sponsored by Nextran, Inc. Primate quarantine work was supported by the New England Primate Research Center grant P51RR00168-37. Doctor Chen is an American College of Surgeons Research Scholar 1998–2000 and recipient of NIH Individual National Research Service Award (NRSA) 1F32HL0996601.
We thank Alexander Kadner, MD, and Richard N. Mitchell, MD, PhD, for their insight and discussion, and Jay Tracy for his excellent technical assistance.
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References
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Abstract
Introduction
