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Ann Thorac Surg 2001;71:S385-S388
© 2001 The Society of Thoracic Surgeons
a Department of Cardiothoracic Surgery, Cape Heart Centre, University of Cape Town Medical School, Cape Town, South Africa
Address reprint requests to Mr Human, Cardiovascular Research Unit, Cape Heart Centre, Faculty of Health Sciences, University of Cape Town, Anzio Rd, 7925 Observatory, Cape Town, South Africa
e-mail: ctshuman{at}samiot.uct.ac.za
Presented at the VIII International Symposium on Cardiac Bioprostheses, Cancun, Mexico, Nov 3–5, 2000.
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Abstract |
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Methods. Aortic wall buttons (6 mm) were punched from porcine aortic wall tissue fixed with 0.2% glutaraldehyde and detoxified with urazole and then subsequently perforated under sterile conditions. The perforated buttons were then incubated with either immune serum prepared by immunization of New Zealand White rabbits (n = 5) with Freund’s incomplete adjuvant emulsions of tissue homogenates of similarly treated aortic wall tissue, or incubated with the corresponding control preimmune sera obtained before immunization of the same animals. The tissue was then implanted subdermally on the back of unrelated New Zealand White rabbits (n = 8) for a period of 3 weeks. After the buttons were explanted, tissue calcium levels were determined by atomic absorption spectroscopy.
Results. Tissue calcium was increased in all five immune serum-treated replicates (range, 61.8% to 431.2%; mean, 225.9% ± 73.2%) when compared with control samples treated with preimmune sera. Overall, the mean calcium level was significantly increased (p < 0.0001) when tissue was treated with immune sera (66.0 ± 10.0 µg/mg versus 22.6 ± 4.8 µg/mg in control tissue). Graft specificity of immune sera was confirmed by Western blot analysis.
Conclusions. These results strongly suggest a role of circulating graft-specific antibody in the disease of bioprosthetic graft calcification.
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Introduction |
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As early as 1987, Nimni and associates [2] showed that these low concentrations of GA resulted in a distinctly higher level of circulating antibodies than enhanced fixation using diamine bridges. Nevertheless, without providing a direct link between these circulating antibodies and augmented degeneration of bioprosthetic tissue valves, the presence of specific antibodies could be easily disregarded as inconsequential. In the past few years, such a link between circulating, graft-specific antibodies and valve degeneration was eventually found by a number of investigators. Apart from allograft studies, which increasingly supported the notion of immune degeneration [3], a growing number of researchers also provided evidence for immune-mediated xenograft degeneration. Dahm and colleagues [4] showed in 1995 that specific antibodies were present in 100% of patients with failed tissue valves but in only 25% of patients with intact valves. In 1996, Eishi and coworkers [5] demonstrated in Takayasu patients requiring aortic valve replacement that the administration of steroids significantly suppressed bioprosthetic calcification. Equally supportive data came from Chauvaud and colleagues [6], who recently reported a lack of calcification in a relatively large clinical series of pericardial implants that were fixed with GA but autologous rather than xenogeneic. The most convincing study, however, came from Vincentelli and associates in 1998 [7]. This group demonstrated in the same animal model that after fixation in 0.65% GA, xenogeneic tissue calcified 35 times more than autologous tissue.
As much as these data lend an increasing weight to the suspicion that immune mechanisms are strong facilitators of bioprosthetic degeneration, a direct connection between a specific antibody response and tissue calcification was still outstanding. In the present study we prove this direct connection by demonstrating significantly increased bioprosthetic mineralization after preincubation in autologous serum that contained high levels of specific antibodies against the xenogeneic heart valve tissue.
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Material and methods |
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Tissue preparation
After an initial fixation period of 2 days in 0.2% GA (4°C, phosphate-buffered saline [PBS]), 6-mm circular disks were punched out from porcine aortas and transferred back into new 0.2% GA solution. At the end of 7 days of fixation, samples were rinsed in high volumes (30 mL/g) of PBS and thoroughly detoxified in urazole as described in detail elsewhere [8], washed in high volumes of PBS, and finally stored in low volumes of PBS for less than 1 month.
Rabbit immunization
Before immunization, 20 mL of blood was collected from 5 New Zealand White rabbits, and the serum was frozen at -20°C. For the purpose of immunization, fixed and detoxified tissue was taken from the PBS and homogenized by pulverization under liquid nitrogen using a mortar and pestle until a fine-powder consistency was achieved. Sterile technique was used throughout. Tissue was resuspended in sterile PBS and emulsified with Freund’s incomplete adjuvant. Subsequently, the animals were immunized subcutaneously on the back with a total dose of 10 mg of tissue wet weight per animal distributed between two 0.5-mL injections. Animals were boosted on day 14 with an identical antigen dose, also emulsified in Freund’s incomplete adjuvant. Blood samples were taken before boosting and again after a further 3 weeks.
Detection of specific antibodies
The graft specificity of the immune sera was confirmed by Western blot analysis. Fresh homogenized aortic wall tissue was extracted overnight at 4°C in a sodium dodecyl sulfate extraction buffer (Tris, 50 mmol/L, pH7.4; SDS, 0.1%; NaN3, 0.02%). The protein concentration of the extract was determined spectrophotometrically. Sodium dodecyl sulfate polyacrylamide gel electrophoresis was performed on 10% Tris-HCl minigels. Separated protein was blotted onto polyvinylidene difluoride membranes, which were then blocked in Tween Tris-buffered saline. Sera were applied as 1:100 dilutions in Tween Tris-buffered saline, and specific immunoglobulin G antibody was detected through the use of a goat anti-rabbit immunoglobulin G horseradish peroxidase conjugate (BioRad, Hercules, CA).
Bioprosthetic tissue exposure to specific antibodies
All aortic wall buttons were thoroughly detoxified to prevent antibody cross-linking through leaching GA. After perforation of the buttons under sterile conditions to increase the tissue surface for antibody exposure, eight replicate buttons were incubated in each of the five immune sera. Another eight replicate buttons were incubated in each of the five corresponding preimmune sera. Eight buttons were incubated in normal saline as controls. A total of 88 buttons were therefore prepared.
Rabbit implants
All anesthetic and surgical procedures were approved by the animal research and ethics committee of the University of Cape Town and complied with the "Principles of Laboratory Care" and the Guidelines for the Care and Use of Laboratory Animals (NIH publication 86-23). Samples were implanted subdermally on the back of 8 New Zealand White rabbits (1.59 ± 0.04 kg) that were unrelated to those used for immunization. Each animal was the recipient of 11 aortic wall buttons, 5 having been incubated with the different preimmunization sera, 5 with the corresponding postimmunization sera, and 1 with normal saline. After 3 weeks of implantation the animals were euthanized, and the implants were retrieved for tissue calcium analysis.
Tissue calcification measurement
After storage at -20°C, the tissue was dried at 104°C for 24 hours, weighed, ashed in a muffle furnace at 560°C for 12 hours, and dissolved in a 20% hydrochloric acid solution (10 mg dried tissue in 1 mL HCl). Final dilution was achieved in a 0.5% lanthanum chloride solution, and absorption was measured at 422.7 nm on an atomic absorption spectrophotometer (Varian AA1275, Springvale, Australia). Calcium levels were expressed as micrograms per milligram of dry mass of tissue.
Statistical analysis
All data are expressed as means ± standard error. Comparison of calcium levels between preimmune sera–incubated and immune sera–incubated tissue was performed using one-way analysis of variance. Comparison of preimmune sera–incubated tissue with saline-incubated tissue was performed using the Dunnett test. A significance level of 0.05 or less was accepted as being statistically significant.
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Comment |
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TopAbstractIntroductionMaterial and methodsResultsCommentReferences |
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Aortic wall tissue—which was chosen because of its detrimental calcification in both homografts and modern stentless xenografts—calcified three times more after incubation with serum containing specific antibodies against the porcine tissue. As control samples were incubated in preimmune sera of the same animals, the calcific-promoting effect of immune sera can conclusively be linked to the immunization process itself. To exclude nonspecific inflammatory effects as a reason behind this phenomenon, the presence of specific antibodies against porcine tissue was demonstrated in the immune sera.
The underlying theory of all attempts to link bioprosthetic degeneration with immune mechanisms is one of suboptimally masked antigenicity. This phenomenon was demonstrated by various groups [4, 9] and is the result of a compromise on which commercial fixation is still based today. This compromise, which was to choose very low concentrations of GA and thus insufficient cross-linking over the perceived adverse effects of the dialdehyde [1], stands in stark contrast to common knowledge in immunology. Even basic laboratory experience, such as immunohistochemistry, teaches that masking of antigens through better cross-linking prevents antigen recognition. It should therefore not come as a surprise that evidence has accumulated over the past 20 years confirming that the low-grade fixation of bioprosthetic heart valves does indeed elicit an immune response. As early as 1987, Nimni and coworkers [2] alerted the scientific community that standard commercial GA fixation only achieves a 59% reduction in antigenicity as opposed to a 92% reduction obtained through enhanced fixation. Other groups have demonstrated the generation of specific antibodies against pericardium fixed in 0.2% GA [9, 10]. The main criticism against all these studies was the fact that their subcutaneous administration of immunogens hardly reflects the low-grade exposure to the immune system of intracardially placed bioprosthetic tissue. However, circulating antibodies were regularly detected in human recipients of bioprosthetic heart valves. In patients with porcine valves, specific antibodies were detected in as many as 58% [11]. In homograft patients, 82% of recipients presented with a specific antibody response, 92% of which produced immunoglobulin G against class I human leukocyte antigens [3].
The most crucial step in proving the involvement of an immune response in tissue valve failure is the demonstration of a link between an immune response and calcific degeneration. In the past few years, both Vincentelli and colleagues [7] and our group [12–14] have provided indirect evidence for such a link. Vincentelli and associates [7] demonstrated that xenogeneic tissue fixed in 0.65% GA calcified 35 times more in the same animal model compared with fixed autologous tissue. Our group found a direct correlation between cross-link density, inflammation, and calcification in both the rat [12, 13] and the sheep model [14]. By increasing the cross-link density in GA-treated porcine root prostheses, bioprosthetic calcification of 0.2% GA-fixed aortic wall tissue could be reduced by 90.0% (p = 0.004) and 53.7% (p < 0.0001) after 6 and 24 weeks of sheep implantation, respectively. However, as much as these data strongly supported the notion that calcification correspondingly decreases with increasing masking of antigenicity, the multifactorial nature of tissue mineralization made it likely that higher cross-linking itself rather than the mitigation of an immune reaction alone may have contributed to the lower calcification. Therefore, a more direct connection between an immune response and tissue calcification was required. By demonstrating threefold higher calcification in aortic wall tissue that was preincubated in serum containing high levels of graft-specific antibodies, we were able to provide this direct immune-calcification link. Such a link certainly does not disregard the complexity of bioprosthetic heart valve degeneration. Undoubtedly, a variety of other mechanisms, such as tissue fixation itself and accessibility of nucleation sites, plays a crucial role. Furthermore, the level of insight into the actual molecular mechanisms behind an immune link to calcification is unsatisfying and can only be seen as a beginning. At the same time, it was the categorical exclusion of antigenicity as a contributing factor to bioprosthetic tissue degeneration in the 1980s and early 1990s that was the reason that observations in this direction started late and the mechanisms involved are still unidentified. The main challenge ahead will be to both identify tissue antigens eliciting a specific immune response and elucidate molecular mechanisms through which antibody binding facilitates calcification.
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