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J Thorac Cardiovasc Surg 2000;119:1216-1220
© 2000 The American Association for Thoracic Surgery
SURGERY FOR ACQUIRED CARDIOVASCULAR DISEASE |
From the Departments of Surgerya and Pathology,b Brigham & Women’s Hospital, Harvard Medical School, Boston, Mass.
Dr Chen is an American College of Surgeons Research Scholar 1998-00 and recipient of National Institutes of Health Individual National Research Service Award (NRSA) 1F32HL0996601. This work is supported by a Nextran, Inc, sponsored research grant. Primate quarantine work was supported by the New England Primate Research Center grant P51RR00168-37.
Address for reprints: David H. Adams, MD, Division of Cardiac Surgery, Brigham & Women’s Hospital, 15 Francis St, Boston, MA 02115 (E-mail: dadams{at}partners.org ).
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Abstract |
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-1,3-galactose, with ensuing human complement activation and membrane attack complex deposition. It is unclear, however, whether porcine valve endothelium triggers a similar immune response. We sought to investigate whether fresh porcine valves implanted into primates are rejected.-1,3-galactose, primate immunoglobulin M, and membrane attack complex.-1,3-galactose antigen, galactose -1,3-galactose was not detected on the endothelium of porcine aortic and pulmonary valves. Porcine hearts transplanted into baboon recipients were hyperacutely rejected 60 to 80 minutes after implantation. Despite dramatic tissue damage associated with extensive immunoglobulin M and membrane attack complex binding on the microvascular endothelium, the aortic and pulmonary valves were entirely spared. Valves remained morphologically intact at explant and showed no signs of immunoglobulin M– and membrane attack complex–mediated damage.-1,3-galactose expression may protect unfixed porcine valves from xenograft rejection in primates. Further investigation of viable porcine valves appears warranted. |
Introduction |
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1
It is unclear whether the degeneration of porcine valve prostheses is mediated by immune rejection. Porcine hearts transplanted into human subjects prompt a violent hyperacute rejection similar to that seen in ABO-incompatible transplantation in which the graft becomes cyanotic and edematous minutes after implantation. 2 The mechanism of porcine tissue rejection was elucidated in 1987, when Galili and colleagues 3 showed that human subjects constitutively synthesize immunoglobulin (Ig) M natural antibodies against a porcine endothelial molecule, galactose -1,3-galactose (-Gal). The extensive microvascular thrombosis of hyperacute rejection is the result of IgM-triggered, complement membrane attack complex (MAC)–mediated graft destruction.
Because the mechanism of the human anti-pig immune response is now understood, we sought to determine whether human subjects mount a rejection response against fresh porcine valves. We suspected that valves may be immunologically distinct from heart tissue. Previously, Mitchell and colleagues 4 had shown that cardiac valves, even in rejected human heart transplants, rarely show any cellular infiltrate. Therefore, it may be possible that valves, as nonvascularized tissue, may be a low-visibility target for the immune system. The potential immune privilege of valve tissue suggests that porcine valve tissue may survive in human recipients. We thus sought to investigate the possibility of implanting fresh porcine valves. Fresh porcine valves contain viable endothelial and fibroblast cells, which can continuously repair leaflets undergoing hemodynamic stress and possibly confer better long-term durability. 5
To examine the immune reaction against fresh porcine valves, we performed heterotopic pig-to-baboon cardiac xenotransplantation. Relative expression of -Gal antigens on vascular and valve endothelium was evaluated, and the degree of IgM and MAC deposition on valve leaflets was compared with that on cardiac microvasculature.
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Material and methods |
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TopAbstractIntroductionMaterial and methods Results Discussion References |
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Anesthesia
For invasive procedures and anesthesia induction, sedation was achieved by using ketamine hydrochloride (10 mg/kg administered intramuscularly) in baboons and tiletamine hydrochloride and zolazepam hydrochloride (Telazol; 5 mg/kg administered intramuscularly) in pigs. Respiratory secretions were controlled with atropine sulfate (0.03 mg/kg). Anesthesia was maintained with inhalational isoflurane (1.3%-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 (Ancef; 20 mg/kg administered intramuscularly twice daily) and pain control with butrenorphine (Buprenex; 0.005 mg/kg administered twice daily) postoperatively for 5 days. In the event of diminished fluid or caloric intake, recipients received their calculated daily needs as intravenous crystalloid solution or Ensure tube feeds.
Heterotopic heart transplantation model
Heart transplantation was performed in the abdominal cavity, as previously described. 6 Pigs were anesthetized and received systemic anticoagulation with heparin (100 IU/kg administered intravenously), after which the heart was harvested in a standard fashion after protection by 500 mL of antegrade cold crystalloid cardioplegic solution (dextrose, 2.5%; sodium chloride, 0.45%; potassium, 30 mEq/L; and bicarbonate, 5 mEq/L). Transplant organ ischemic time varied between 45 and 55 minutes. The animals received no immunosuppression.
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 solution and embedded in paraffin. Paraffin sections were sectioned at 5-µm thickness and stained with hematoxylin and eosin. Biotinylated lectin from Griffonia simplicifolia (Sigma, St Louis, Mo) was used to stain -Gal on porcine endothelium. Cryostat sections were immunostained by use of standard indirect immunoperoxidase avidin-biotin techniques, as previously summarized, 7 with monoclonal antibodies specific for IgM (Biodesign International, Kennebunk, Me) and MAC (C5-9; Dako, Carpinteria, Calif). The signal was developed with the avidin-peroxidase system (ABC kit; Vector Lab, Burlingame, Calif).
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Results |
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-Gal expression on the endothelium of valve leaflets and cardiac microvasculature. Cardiac microvascular endothelium showed strong -Gal expression (Fig 1, A ). In comparison, neither aortic valves (Fig 1, B ) nor pulmonary valves (Fig 1, C ) showed any detectable -Gal expression.
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, B ) deposition on the microvascular endothelium. Interestingly, both aortic and pulmonary valves were intact, and immunohistochemistry of pulmonary and aortic valves showed no signs of either IgM (Fig 2, C ) or MAC (Fig 2, D ).
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Discussion |
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8 mentioned that "the (porcine) graft was sterilized by immersion in liquid ethylene oxide." The other pioneers in the field, Binet and colleagues, 9 described "non-sterile pig and calf aortic valves obtained immediately after death at the slaughter house was at once placed in a mercurial solution" for 3 days. Only after that were the valves transferred into "sterile Gross’s solution." The first reported use of formaldehyde on valves was to denature and stiffen elastic fibers in human homografts. 10 The first formaldehyde preservation of porcine valves was performed by O’Brien, 11 who used the solution in 1967 for "both preservation and sterilization." Both Hamilton and Gerbode 12 and Paneth and O’Brien 10 hypothesized that because formaldehyde "de-natures the proteins of the graft, [it] may minimize rejection phenomenon." However, there was no experiment or data to support this hypothesis.
A legacy of these early pioneers is glutaraldehyde-fixed porcine valves, which have successfully served as valve prostheses for the past 40 years. However, these valves degenerate because of hemodynamic stress and calcification. Fresh porcine valves may help to prevent both problems. Fresh valves contain viable endothelial and fibroblast cells, which may continue to repair and modify the leaflets undergoing hemodynamic stress and possibly confer better long-term durability. 5 Historically, fresh valves have not been seriously contemplated as possible valve prostheses, mainly because of the assumption that all fresh porcine tissue, whether myocardium or cardiac valves, would undergo rejection.
In this report, however, we found that porcine valvular leaflets do not express -Gal on their endothelium. The absence of detectable -Gal on valvular leaflets may have 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 severe IgM and MAC deposition, aortic and pulmonary valves harvested from rejected xenografts showed no sign of damage.
The difference of -Gal expression among different endothelial layers was not totally unexpected because we previously found that the endothelial cells of porcine cardiac microvasculature express -Gal, whereas those of larger vessels do not. 7 Decreased -Gal expression on valve endothelium could be due to either decreased glycosylation or decreased surface glycoproteins on leaflet endothelium. Protein expression is closely regulated in endothelial cells, and flow rate can clearly modulate endothelial gene expression. The expression of proteins, such as major histocompatibility complex antigen 13 and vascular cell adhesion molecule 1, 14 are all influenced by the amount of shear force exerted on the endothelium. Consequently, it is possible that shear force may also modulate other genes, such as the gal -1,3-galactosyltransferase gene. The gal -1,3-galactosyltransferase protein glycosylates the -Gal terminal sugar to surface proteins, 15 and its expression is tightly controlled in the porcine endothelium. For example, inflammatory cytokines, such as tumor necrosis factor and lipopolysaccharide, decrease the level of gal -1,3-galactosyltransferase protein. Consequently, it is possible that differential shear forces exerted on cardiac valvular endothelium may influence gal -1,3-galactosyltransferase gene expression and reduce Gal production along the vascular system.
Alternatively, the differential Gal staining may be reflective of the level of expression of glycoproteins on porcine endothelium. Holzknecht and Platt 16 showed that the majority of porcine Gal epitopes bound by human xenoreactive antibodies are found on the von Willebrand factor and the integrin family 1, v, 3/5, ß1, and ß3. Ando and Kamiya 17 also showed that ß1 integrin is the signal-transducing mechanism for shear stress within endothelial cells. Consequently, the population of integrins may vary among endothelial cells because of different stress, and the degree of Gal staining may vary accordingly.
Regardless of the exact mechanism for differential -Gal expression, the absence of -Gal on porcine valvular endothelium protects the leaflets from xenoantibody-triggered rejection. To the best of our knowledge, there has been no published report describing the immunopathology of fresh porcine valves in human subjects or primates. More studies in this area are warranted to realize the potential of fresh porcine valve prostheses.
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References |
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(l,3) galactose expression by porcine cardiac vascular endothelium. Xenotransplantation 1999;6:169-72. [Medline]This article has been cited by other articles:
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  R. S. Farivar, F. Filsoufi, and D. H. Adams Mechanisms of Gal{alpha}1-3Gal{beta}1-4GlcNAc-R ({alpha}Gal) expression on porcine valve endothelial cells J. Thorac. Cardiovasc. Surg., February 1, 2003; 125(2): 306 - 314. [Abstract] [Full Text] [PDF]
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 R. H. Chen and D. H. Adams Transgenic porcine valves show no signs of delayed cardiac xenograft rejection Ann. Thorac. Surg., May 1, 2001; 71 (2007): S389 - S392. [Abstract] [Full Text] [PDF]
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R. H. Chen and D. H. Adams Decreased porcine valve antigenicity with in vitro culture Ann. Thorac. Surg., May 1, 2001; 71 (2007): S393 - S395. [Abstract] [Full Text] [PDF]
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J. T. Beranek Why complement attacks cardiomyocytes in hyperacute heart rejection J. Thorac. Cardiovasc. Surg., February 1, 2001; 121(2): 400 - 401. [Full Text] [PDF]
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R. H. Chen, R. S. Farivar, and D. H. Adams Reply J. Thorac. Cardiovasc. Surg., February 1, 2001; 121(2): 401 - 401. [Full Text] [PDF]
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