Ann Thorac Surg 2001;71:S406-S407
© 2001 The Society of Thoracic Surgeons
Basic research
Protein adsorption of calcified and noncalcified valvular bioprostheses after human implantation
Ming Shen, MD, PhDa,
Sophie M. Carpentier, PhDa,
Alain J. Berrebi, MDa,
Lin Chen, MDa,
Bernard Martinet, BSa,
Alain Carpentier, MD, PhDa
a Laboratory for the Study of Cardiac Grafts and Prostheses, UPRES 264 Université Paris VI, Hôpital Européen Georges Pompidou, Paris, France
Address reprint requests to Dr Shen, Laboratoire d’Etude des Greffes et Prothèses Cardiaques, Hôpital Broussais, 96 rue Didot, 75014 Paris, France
e-mail: labo.legpc{at}brs.ap-hop-paris.fr
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. The incidence of calcification of porcine valve bioprostheses shows important, and as yet unexplained, variations. Previous studies by others showed that osteopontin and osteocalcin are expressed in calcified porcine valve bioprostheses. However, no study has yet explored other proteins that could also be involved.
Methods. Twelve porcine valve bioprostheses were retrieved from 12 patients and were separated into two groups. Group 1 (n = 6) had early calcification after 4 to 9 years (mean, 6 ± 2.3 years). The mean age of the patients at the time of implantation was 46 ± 9 years. Group 2 (n = 6) had no calcification after 8 to 14 years (mean, 12 ± 2.8 years). The mean age was 47 ± 13.4 years. These valves were analyzed by electrophore-sis, and the bands were quantified by densitometry.
Results. A 14-kd protein showed a 50% increase in the calcified group. A 31-kd protein found in the calcified group was not detected in the noncalcified group. Three other proteins (45, 39, and 28 kd) showed reduced adsorption in the calcified group.
Conclusions. Important differences were found in the proteins adsorbed in calcified and noncalcified bioprostheses after implantation in patients. Besides osteopontin and osteocalcin, several other proteins may play a role in the process of calcification of valvular bioprostheses.
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Introduction
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Since the first implantation of a glutaraldehyde-treated valvular bioprostheses in humans [1], the large variation in the duration of this type of valve substitute remains a vexing question. Even within the same age groups, some valves calcify after 6 to 8 years whereas others continue to function well beyond 15 years. This study was undertaken to determine whether the proteins adsorbed in the valve after implantation may explain this variation.
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Material and methods
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Patients
The 12 bioprostheses used for this investigation were retrieved from 12 patients aged from 35 to 66 years (mean, 47 ± 11.7 years). These patients were selected because 6 showed early calcification whereas 6 others showed no calcification after long-term implantation. Patients who had been pregnant or who suffered from renal disease were excluded. All valves had been placed in the mitral position and failed because of structural failures or periprosthetic leak. Among these 12 valves, 6 showed extensive calcification after 4 to 9 years (mean, 6 ± 2.3 years), and 6 showed no calcification after 8 to 14 years (mean, 12 ± 2.8 years). In group 1 (early calcification), there were 4 women and 2 men, and the age ranged from 47 to 62 years (mean, 46 ± 10.3 years). In group 2 (no calcification), there were 3 women and 3 men, and the age ranged from 35 to 66 years (mean, 47 ± 13.9 years).
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
The valves retrieved were immediately frozen until analysis. The extracted proteins (50 µg) were processed for sodium dodecyl sulfate polyacrylamide gel electrophoresis. Electrophoresis was performed on 8% to 12% and 12% to 16% separating gels with a 4% stacking gel, according to the method of Laemmli [2]. Samples were dissolved in sodium dodecyl sulfate buffer (2-mercapto-ethanol, 10 mmol/L; Tris buffer, 100 mmol/L, pH 6.8; 30% glycerol; 2% sodium dodecyl sulfate), heated at 100°C for 3 minutes, and electrophoresed overnight at a constant voltage (100 V at 4°C). Proteins separated on the gel were visualized using Amido Schwartz (2%) staining.
Quantitative analysis of each band was performed using densitometry (Bio-Profil, Vilber Lourmat, Marne-La-Vallée, France). Results were expressed as percent of the total protein content. The significance level was set at p less than 0.05.
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Results
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The results of electrophoresis are shown in Figures 1 and 2. The noncalcified group (6 patients) showed a higher concentration of the 45-, 39-, and 28-kd proteins. The calcified group (6 patients) showed a higher concentration of the 14-kd protein than the noncalcified group. In addition, a 31-kd protein was found in the calcified group, which did not exist in the noncalcified group.
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Fig 1. Electrophoresis of proteins adsorbed on 12 bioprostheses implanted in humans. The calcified (6 patients) and noncalcified groups (6 patients) show differences in amounts of 45-, 39-, 31-, 28-, and 14-kd proteins. Quantification was made by densitometry. (MW = molecular weight.)
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Fig 2. Comparative adsorption of 45-, 39-, 31-, 28-, and 14-kd proteins adsorbed on bioprostheses implanted in 12 patients; 6 were calcified, 6 others noncalcified after long-term implantation. The arrow shows that there was no 31 kDa protein in noncalcified bioprostheses.
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Comment
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In 1967, glutaraldehyde treatment was found to enhance tissue stability and to reduce antigenicity of valvular xenografts [1]. This led to the first human implantation of a glutaraldehyde-treated porcine valve [1]. In numerous clinical or scientific papers on this topic, reference is rarely made to this early work. Glutaraldehyde-treated bioprosthetic valves are no longer grafts in the strict sense, because duration is not based on tissue survival or tissue regeneration. They are chemically stabilized tissues, which require unfailing stability of the treated tissue. The term "bioprosthesis" was created to reflect the biologic origin and prosthetic characteristics [3]. The biologic origin is responsible for the major complication associated with valvular bioprosthesis, ie,: tissue calcification. Tissue calcification is mainly caused by the nonviable nature of the tissue, which has lost its ability to regenerate tissular matrix and to secrete natural inhibitors of mineralization.
The aim of this study was to contribute to understanding why some bioprosthetic valves calcify early after operation (mean, 6 years), whereas others do not calcify after a long-lasting implantation (mean, 12 years). Indeed, these patients were selected to be in the same age range (46 years), and patients with risk factors for calcification (pregnancy, renal disorders, infectious diseases) were excluded.
Because some proteins are known to facilitate or inhibit calcification, it was appropriate to study proteins adsorbed in these valves after implantation. Bone apatite crystal formation is regulated by noncollagenous matrix proteins such as osteopontin, osteonectin, osteocalcin, and matrix carboxyglutamic acid–containing proteins [4]. Transgenic mice lacking matrix carboxyglutamic acid–containing proteins showed extensive arterial calcification [5], and osteocalcin was present in calcified bioprostheses [6]. On the other hand, osteopontin was found in calcified areas of bioprostheses implanted in humans [7]. These findings are confirmed by this study, which shows that the 31-kd protein corresponding to osteopontin was found in calcified bioprostheses and not in noncalcified bioprostheses. For the first time, three other proteins at 45, 39, and 28 kd were found at higher concentrations in noncalcified bioprostheses. The role of these proteins is unknown. Identification of these proteins, which is currently under investigation, may elucidate the process of calcification of bioprostheses in vivo.
Important differences were found in proteins adsorbed in valvular bioprostheses that showed early calcification and those without calcification. Besides osteopontin and osteocalcin, which are known to be involved in the process of calcification, three other proteins identified by electrophoresis in this study may play a role in preventing calcification.
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Acknowledgments
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We thank Martine Rancic for her technical help.
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References
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Carpentier A., Lemaigre G., Robert L., Carpentier S., Dubost C. Biological factors affecting long-term results of valvular heterografts. J Thorac Cardiovasc Surg 1969;58:467-483.[Medline]
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Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 1970;277:680-685.
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Carpentier A., Dubost C. From xenograft to bioprosthesis: evolution of concepts and techniques of valvular xenografts. In: Ionescu M.I., Ross D.N., Wooler G.H., eds. Biological tissue in heart valve replacement. London: Butterworth, 1972:515-541.
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Luo G., Ducy P., Mckee M.D., et al. Spontaneous calcification of arteries and cartilage in mice lacking matrix GLA protein. Nature 1997;386:78-81.[Medline]
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Shen M., Marie P., Farge D., et al. Osteopontin is associated with bioprosthetic heart valve calcification in humans. C R Acad Sci III 1997;320:49-57.[Medline]
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Abstract
Introduction
