Ann Thorac Surg 2001;71:S393-S395
© 2001 The Society of Thoracic Surgeons
Basic research
Decreased porcine valve antigenicity with in vitro culture
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. Porcine valvular prostheses may stimulate inflammation after implantation, with resultant accelerated structural degeneration. We investigated the expression of porcine major histocompatibility complex (MHC) class II molecules on valve leaflets and the possibility of decreasing valve antigenicity with in vitro culture.
Methods. Aortic and pulmonary valves were harvested from domestic pigs under sterile conditions and cultured in vitro with either porcine or baboon serum for 4 days. Valves were harvested daily and fixed in Carnoy’s or formalin solution. Microtome sections of valves were examined by hematoxylin and eosin, and by immunohistochemistry for porcine MHC class II proteins and an endothelial marker, 
-N-acetylgalactosaminyl glycoprotein (-GalNac).
Results. Porcine aortic and pulmonary valves constitutively express -GalNac proteins and porcine MHC class II antigens. Porcine valves continue to express both -GalNac and MHC class II after 48 hours of culture in porcine serum. After 48-hour culture in baboon serum, however, MHC class II antigens became undetectable on valvular leaflets, although -GalNac molecules were still detected.
Conclusions. Porcine valvular endothelial cells remain viable after 2 days of in vitro culture. Porcine valves cultured with primate serum show decreased MHC class II antigenic expression. In vitro culture before glutaraldehyde fixation may decrease inflammation associated with implantation.
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Introduction
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It remains unclear if immunologic processes play a role in the deterioration of bioprosthetic porcine valves after implantation. Historically it was assumed that glutaraldehyde-treated porcine valve prostheses may be immunologically inert. However, recent evidence suggests that porcine bioprosthetic valves do trigger an inflammatory reaction in the recipient [1].
It is likely that the porcine major histocompatibility complex (MHC) class II molecules may be responsible for the inflammatory response [2, 3], based on three findings: First, porcine valves constitutively express MHC class II molecules [4]. Second, porcine MHC class II molecules directly stimulate human T cells and cause human T cells to release IL-2 [5]. Lastly, glutaraldehyde does not remove antigenicity, because MHC class II antigens on glutaraldehyde-fixed T cells can still activate B cells [6].
We sought to determine if the expression of MHC class II molecules on porcine heart valves can be modified, as antigenic reduction before glutaraldehyde fixation may decrease the inflammatory nature of porcine bioprosthetic valves. We hypothesized that continued porcine MHC class II expression may be possible only with continued cytokine stimulation, as the expression of human MHC class II molecule diminishes when deprived of cytokines in vivo. We thus cultured viable porcine valves in vitro with serum of porcine or incom-patible xenogenic primate sera in order to examine the expression of MHC class II molecules.
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Material and methods
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Experimental animals
Domestic wild-type pigs bred under allergen- and pathogen-free conditions were used for the experiments. 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).
Valve harvest
Pigs were sedated with telazol (5 mg/kg intramuscularly) before oral-tracheal intubation, and anesthesia was maintained with inhalational isoflurane (1.3% to 2.0%). The heart was harvested through a median sternotomy under sterile conditions, and heart valves were immediately dissected from the heart for tissue processing.
Valve culture
Isolated porcine valves were kept in normal saline at 4°C and immediately transferred to RPMI 1640 culture medium (Fisher, Pittsburgh, PA) containing 10% of either pooled porcine or baboon serum, 100 mg/mL penicillin and streptomycin, 2 mmol/L glutamine, 1 mmol/L sodium pyruvate, and Hepes buffer in 24-well flat bottom plates (Corning, www.corninglabware.com) and kept in Ultima tissue culture incubator (Revco, Asheville, NJ) at 37°C with 50% CO2. Valve samples were collected at time zero and every 24 hours until 4 days of culture.
Histopathologic studies of the porcine valves
Cultured porcine valves were collected each day and either snap-frozen in tissue freezing medium (Triangle Biomedical Sciences, Durham, NC) and liquid nitrogen, or fixed in either Carnoy’s solution or 40% formalin and embedded in paraffin. Paraffin sections were sectioned at 5 µm thickness and stained with hematoxylin and eosin. Biotinylated lectin from Dolichos biflorus (DBA; Sigma, St. Louis, MO) was used to stain -GalNac on porcine endothelium. MHC class antibody was obtained from VMRD (Pullman, WA). The signal was developed with the avidin–peroxidase system (ABC kit, Vector Lab, Burlingame, CA).
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Results
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Porcine valves cultured in porcine serum
We examined porcine heart valves (n = 6) cultured in vitro with porcine serum for the expression of the endothelial marker -GalNac and the MHC class II molecules. Endothelium on the porcine valves was preserved and continued to express -GalNac on day 2 of culture (Figs 1A and 1B). The preserved endothelium continued to express porcine MHC class II molecules (Figs 1C and 1D).
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Fig 1. Porcine valves cultured in vitro with porcine serum. Freshly harvested aortic valves were cultured with porcine serum and showed persistent valvular expression of -GalNac on day 0 (A) and day 2 (B). Similarly, the valves continued to express porcine major histocompatibility complex class II antigens on day 0 (C) and day 2 (D) of culture. (Hematoxylin counterstain; x100 before 46% reduction.)
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Porcine valves cultured in baboon serum
We examined porcine heart valves (n = 6) cultured in vitro with baboon serum for the expression of the endothelial marker -GalNac and the MHC class II molecules. Porcine valvular endothelium was preserved after 2 days of in vitro culture (Figs 2A and 2B). In contrast, MHC class II molecules were not detectable after 2 days of culture in baboon serum (Figs 2C and 2D).
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Fig 2. Porcine valves cultured in vitro with baboon serum. Freshly harvested aortic valves were cultured with baboon serum and showed persistent valvular expression of -GalNac on day 0 (A) and day 2 (B). The valves expressed porcine major histocompatibility complex class II antigens on day 0 (C), but not by day 2 (D) of culture. (Hematoxylin counterstain; x100 before 46% reduction.)
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Comment
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MHC class II proteins are a powerful stimulant of the immune system, and their porcine analogues, SLA II (Swine leukocyte antigen II), have been fully characterized in recent years [2, 3]. It is now clear that the expression profile of MHC class II molecules in pigs is different from that in humans. Whereas human MHC class II antigens are only constitutively expressed in specialized antigen-presenting cells, porcine MHC class II molecules have a much wider tissue distribution. They are constitutively expressed on resting T cells, coronary artery endothelium, and cardiac valvular endothelium [4, 7, 8].
The presence of porcine MHC class II on porcine heart valves may have a strong implication on the longevity of porcine valve bioprosthesis. Because it is now clear that porcine MHC class II molecules can directly stimulate human T cells [5] and that such reactivity is not neutralized by glutaraldehyde fixation [6], MHC class II molecules may be responsible for the inflammatory reaction associated with porcine bioprosthetic valves after implantation [1].
Although MHC class II molecules can be removed by protease digestion after glutaraldehyde, such a step would both destroy the cross-linking effects of glutaraldehyde and weaken the collagen structural integrity of the valve. Instead, we chose to reduce MHC class II expression by in vitro culture. The expression of MHC class II molecules is closely regulated by 
-interferon, which is often species restricted [9]. It has been demonstrated that human interferon cannot upregulate porcine MHC class II molecules [10]. Our in vitro culture with primate serum most likely deprived the porcine endothelial cells of the needed cytokines for continued MHC expression. This is not achieved by endothelial loss, since the expression of -GalNac was intact after 2 days of culture. Instead, the loss of MHC class II after 2 days of in vitro culture most likely represents a downregulation of the MHC class molecules. The preservation of endothelial cells may be important, as it may be protective of the valvular collagen structure by preventing the attachments of human antibodies [11].
It is unclear if MHC class II antigens affect valve bioprostheses. Further studies are needed to investigate the potential utility of MHC-free valve bioprosthesis.
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Acknowledgments
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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 Jay Tracy for his excellent technical assistance.
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Abstract
Introduction
