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J Thorac Cardiovasc Surg 1995;109:218-223
© 1995 Mosby, Inc.
CARDIAC AND PULMONARY REPLACEMENT |
Carnaxide and Lisbon, Portugal
Supported in part by the Programa CIENCIA/JNICT, Fundação Calouste Gulbenkian, The Portuguese Health Ministry, and CMDT/UNL.
Address for reprints: João Q. Melo, MD, Instituto do Coração, Av. Prof. Reynaldo dos Santos, 27 Carnaxide P-2795, Portugal.
Abstract
Assessment of the cellular origin of allograft valves is essential in comprehending their biologic behavior and is improving preparation methods. In this study we retrospectively analyzed 10 allografts obtained form patients who underwent valve replacement or heart transplantation. Histologic evaluation and deoxyribonucleic acid amplification by polymerase chain reaction technology with fluorescence labeled primers was performed on different parts of the valve leaflets. Automated analyses of the obtained amplifiers showed in the heart transplantation group the presence of receptor cells intersperses with native donor cells in three cases. Preliminary results for the valve replacement group are inconclusive as yet.(J THORACCARDIOVASCSURG1995;109:218-23)
Allograft valve replacement is considered in two different clinical situations: valve replacement and heart transplantation. In the valve replacement group, the valves are usually cryopreserved and the recipients are not immunosuppressed. The late results of these substitutes are good, with acceptable valve gradients and a low incidence of thromboembolism and endocarditis. However, the durability of cryopreserved allografts is limited and the failure rate increases with time. 
1,2 In the heart transplantation group the valves are fresh and viable, and their recipients are immunosuppressed. During a period of up to 6 years these valves functioned normally and there were no cases of endocarditis or thromboembolism. Histologic studies were made to determine the structure of the explanted leaflets. Pathologic specimens were assessed genetically to evaluate the origin of the cells from fragments of the valve leaflets. Polymerase chain reaction (PCR) technology 3 was used with fluorochrome-labeled primers to amplify (CA)n hypervariable deoxyribonucleic acid (DNA) target regions 4,5 from very small amounts of DNA extracted from various portions of explanted aortic valve leaflets.
MATERIALS AND METHODS
Ten valves were retrospectively analyzed from five patients who had undergone allograft valve replacement and from five who had had heart transplantation. All valves from the valve replacement group were removed during reoperations and valves from the heart transplantation group were collected during postmortem examination. These valves were obtained with approval from the national institutional review board. In the valve replacement group the donor valves were retrieved from brain dead donors (three cases) and from cadavers (two cases). In the heart transplantation group all the hearts were obtained from brain dead donors. After heart transplantation all patients were given cyclosporine, azathioprine, and prednisone for immunosuppression. Four patients incurred one to three treated rejection episodes. All patients in the heart transplantation group died with no evidence of rejection, except for patient T1, who died 1 month after the operation with signs of mild rejection. The mean age, sex distribution, blood type, and valve information for the valve replacement and heart transplantation groups are outlined in Table I.
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6 DNA was amplified by PCR technology by means of primers in which one primer is fluorescently labeled. Oligonucleotide primers flanking the hypervariable (CA)n repeat regions 7-10 were synthesized by a 381 DNA synthesizer (Applied Biosystems, Inc., Foster City, Calif.). A fluorescent dye (5-carboxyfluorescein [FAM] or 2',7'-dimethoxy-4',5'-dichloro-6-carboxyfluorescein [JOE]) was attached to the 5' end of the pair of PCR primers using Aminolink 2 (Applied Biosystems, Inc.) and were purified by high-performance liquid chromatography, as described by Gibbs and associates. 11 The size of the (CA)n -containing amplimers is determined by referencing them to the internal lane size standards by means of the GENESCAN 672 software (Applied Biosystems, Inc.). 12,13 RESULTS
Histologic studies indicated that the valve leaflets from the valve replacement group lost their normal structure. There was evidence of hyalinization and predominant ground substance, sometimes with focal calcification. Small quantities of fibroblasts were found only in cases A1 and A2. Endothelial cells were not found in any of the cases and in three cases there was evidence of focal polymorphonuclear infiltration. In the heart transplantation group, histologic evaluation was almost normal with a slight decrease in the number of cells at the free edge of the leaflets. The endothelium was focally maintained and none of the leaflets showed inflammatory infiltrations.
The valve replacement group was genetically assessed with marker D5S82, which yielded a heterozygous profile in two samples (A1 and A4) and homozygous profiles for the remaining three samples An electrophoretogram with more than two peaks was not encountered for any of the valve replacement cases, so that the results were inconclusive. Sample A4 exhibited two different sized amplimers in the interior region of the leaflet and one amplimer with a very small area in the exterior region of the leaflet. DNA controls from the receiver were obtained in cases A1 and A2, in which the same genotypes as those present in the valves were found. For the heart transplantation group, three different sized amplimers in three cases (T1, T3, and T5) were obtained. In two cases (T1 and T5) we found a very high difference in the area of one amplimer as compared with the other, demonstrating the presence of one population with a homozygous profile and another with a heterozygous profile (Fig. 1). We were unable to find any of these types of electrophoretograms in case T2. Analysis of T4 indicates a homozygous profile in the valve and a heterozygous profile for the control DNA (recipient). Results for case T5, showing three different sized amplimers using marker D5S299 and two different sized amplimers with areas differing by a factor of 2 using marker D5S346, are presented in Table II.
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The aim of this study is to determine allograft leaflet cellular origin with and without the influence of immunosuppression by means of a new method of fluorescent primers in PCR and electrophoresis. This type of automated DNA analysis using primers in a PCR reaction in which one of them is fluorescently labeled enables us to accurately genotype and quantify the obtained amplimers. Cell origin (donor or recipient) was not always visually apparent, especially when the control and valve DNA samples were amplified to the same size in the PCR reaction. This verifies that quantified areas evaluated by the GENESCAN 672 software are crucial for assessing quantification. With respect to scoring accuracy, the electrophoretograms obtained show that homozygous genotypes can be readily distinguished from heterozygotes, which differ by only two nucleotides in the size of their alleles.
The most striking factor for the valves in the valve replacement group was the marked decrease in cellularity. The presence of infection in a few of these valves made the DNA assessment of the native cells controversial because the obtained DNA amplification may be due to infection-related leukocytes. So that this problem could be avoided, five slices of 5 µm each from the same region as that used for genetic assessment were histologically examined and found to contain no white blood cells. Results in the valve replacement group indicated a heterozygous profile in A1 and a homozygous profile in A2. The respective DNA controls had similar profiles indicating coincidental genotypes for recipient and donor in both cases or total inhabitation of the cells of the valves by host cells. For the heart transplantation group a pattern of three DNA profiles was found in three cases, and within these a pattern of two DNA profiles was found by the use of different markers in which the area of one allele is notably different from the other. This enables us to deduce the genotype of the donor as homozygous for the more concentrated band. These findings were confirmed by the use of three different markers, proving a double origin of these cells. In one case we encountered recipient cells within all fragments of the leaflet and a patchy distribution of endothelial cells. Morphologic studies showed that the number of endothelial cells was negligible as compared with the number of fibroblasts. Therefore, we conclude that the cells yielding DNA for amplification are the most abundant cells, the fibroblasts. For case T5 with the marker D5S299, the amount of DNA-amplified product showed that the cell population at the free edge of the cusps was predominately from the recipient and the cells at the base were from mixed origins. These differences in location of cell population along the leaflet require further research before any conclusion can be reached.
The overall method used in this study represents a promising alternative to conventional genetic screening The combination of PCR with the sensitivity of the fluorescent PCR alternative and the use of specific software to screen and to quantify the obtained amplimers results in a time-saving method with enhanced sensitivity and accuracy of detection. Further use of new genetic assessment methods will definitely improve our knowledge of the cellular content of the allograft valve in heart transplants and valve replacement.
Appendix: DISCUSSION
Dr. A. C. Yankah (Berlin, Germany).
Dr. Melo, I agree with you that allografts are antigenic. The viable allografts and even the allovital grafts are subjected to immunologic attack and trauma. Consequently, the endothelial cells as well as the fibroblasts and the collagen structures would be destroyed after implantation after an immune response by the host. The immunologic complications are our major concern, especially in children. Unless the immune response is delayed or arrested by means of a nontoxic dose of immunosuppression such as cyclosporine to inhibit T-lymphocyte activation and prevent structural deterioration, the durability of the viable allograft valve would be limited. It has been demonstrated in our animal experimental laboratory that in rats a short course of cyclosporine therapy with a dose of 10 mg/kg for 14 days beginning from the day of operation can achieve unresponsiveness of the host to the allograft. Subsequently, the endothelial cells and the fibroblasts could be preserved. The cells could survive to replicate, the structures and valve integrity could be maintained, and therefore durability of the allograft could be prolonged. In certain rat strains (weakly allogeneic) with major histocompatibility-compatible, non-major histocompatibility-incompatible encoded endothelial cells, prolonged if not indefinite valve durability could be achieved after a short-course nontoxic dose of cyclosporine.
If these findings are applicable in the clinical situation, then this implies that patients who are at risk for forming antibodies against allograft valves could benefit from a tissue typing especially for HLA-DR compatibility and an additional short-course nontoxic dose of immunosuppressive therapy for about 3 months. Observations made in heart transplant patients in whom aortic valve function was always normal during acute or chronic rejection support and justify the use of some type of immunosuppression in high-risk patients who are electively scheduled to receive an allovital graft.
The two slides confirm the results of the animal experiments we performed at the Institute of Immunology at the University of Kiel, Germany. Allografts in rats not treated with cyclosporine A lost their endothelial cells after a humoral rejection, leaving bare collagen structures in the blood circulation. Subsequently the fibroblasts died away and the valve became acellular. The lost endothelium, however, was replaced by a thin layer of fibrous neointima. Very interestingly, no thrombotic formations were observed on the valve surfaces.
In the cyclosporine-treated rats the endothelial cells were maintained until postoperative day 250, when the experiment was terminated. The endothelial cells could be identified by factor VII and Ulex europaeus lectin identification test, and endothelial cell origin was shown by means of donor and recipient specific monoclonal antibodies as well as monoclonal antibodies (OX6) directed at major histocompatibility class II rat endothelial antigens.
In the control syngeneic grafts or the isografts (herein referred to as autografts), no immune response was detected by direct immunofluorescence study. The isografts were viable and durable at the completion of the experiment on day 250.
I would like to address one question to Dr Melo. Can you apply your method to identify the donor or recipient endothelial cells?
Dr. Melo.
This method is very useful. Of course, the key issue regarding this study concerns which cells we are looking at. Are they endothelial cells or fibroblasts?
Evaluating the allograft group is easy because we could not show, in any of these specimens that were kept in pathology, any endothelial cells on the surface of these allografts . We were able to get PCR amplification, but because we had just one band, it could be either from the donor or from the recipient. However, this DNA definitely did not come from the endothelial cells of the donor.
The second issue concerns the transplantation group. The endothelial cells assessed by cytology are scarce, the main reason being that all these examinations have at least a 24-hour delay after death. At histologic examination all the controls showed the endothelial cells to be present, but in a patchy distribution. In two of the three samples both donor and recipient cells were present in all the pieces studied, which would be impossible if the DNA were from the endothelial cells. This indicates that we are looking at fibroblasts. Besides, this method is quantitative, which gives you an idea of the number of cells present. This is a second reason to believe that most DNA is from the fibroblasts.
Mr. Magdi H. Yacoub (Harefield, England).
Dr. Melo, I have two questions for you. What were the genetic markers used for the PCR? What was the exact sequence and what genes were you targeting and why?
Second, what is the interval between insertion and examination in both types?
Dr. Melo.
Regarding your first question, I will have my genetics colleagues provide you all the information. I think they chose their markers because of the specificity of the Portuguese population. One of the characteristics of a small country like ours is that it is much more homozygous than a population in which there is a greater crossing of races.
Regarding the second question, in patients having aortic valve replacement there is a very large delay between implantation and removal, which varied from 2 months to 6 years. In the transplantation group the average time was 7 months.
Dr. Yacoub.
Did you evaluate the effect of time?
Dr. Melo.
Yes, and we could show exactly on these three allografts where we have seen donor and recipient cells; one allograft had 1 month, another 6 months, and the other 4 years after implantation. They were in the shortest, medium, and longest time after transplantation.
Acknowledgments
We thank Dr. Eric Mayrand and Dr. Margaret Galvin form Applied Biosystems, Inc., Foster City, California, for their assistance in automated DNA analysis.
Footnotes
Read at the Seventy-fourth Annual Meeting of The American Association for Thoracic Surgery, New York, N.Y., April 24-27, 1994. 
*Recipient of a BIC scholarship from Programa CIENCIA/JNICT.
References
This article has been cited by other articles:
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  A. Simon, M. Wilhelmi, G. Steinhoff, W. Harringer, P. Brucke, and A. Haverich CARDIAC VALVE ENDOTHELIAL CELLS: RELEVANCE IN THE LONG-TERM FUNCTION OF BIOLOGIC VALVE PROSTHESES J. Thorac. Cardiovasc. Surg., October 1, 1998; 116(4): 609 - 616. [Abstract] [Full Text] [PDF]
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J. P. Neves, S. Gulbenkian, T. Ramos, A. P. Martins, M. C. Caldas, R. Mascarenhas, M. Guerreiro, A. Matoso-Ferreira, R. Santos, C. Monteiro, et al. MECHANISMS UNDERLYING DEGENERATION OF CRYOPRESERVED VASCULAR HOMOGRAFTS J. Thorac. Cardiovasc. Surg., June 1, 1997; 113(6): 1014 - 1021. [Abstract] [Full Text]
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