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Decellularization followed by PNGase F treatment efficiently removes immunogenic αGal epitopes and other N ‐acetylglucosamine structures on the glycocalyx of porcine pulmonary heart valve matrices
Author(s) -
Findeisen Katja,
Ramm Robert,
Hilfiker Andres
Publication year - 2014
Publication title -
xenotransplantation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.052
H-Index - 61
eISSN - 1399-3089
pISSN - 0908-665X
DOI - 10.1111/xen.12083_19
Subject(s) - decellularization , pngase f , wheat germ agglutinin , epitope , chemistry , staining , lectin , glycocalyx , microbiology and biotechnology , biochemistry , agglutinin , glycoprotein , biology , antibody , glycan , pathology , extracellular matrix , medicine , immunology
Background Xenotransplantation leads to hyperacute or acute graft reaction due to interaction of preformed human antibodies, mainly αGal‐antibodies, with carbohydrate structures present on non‐human tissue. By removing these carbohydrate structures from porcine decellularized pulmonary heart valves (PHV), grafts might be generated that allow heart valve replacement therapy as with allogeneic PHV matrices. Methods Thus, after detergents based decellularization (0.5%SDS/0.5%Triton‐X100) cell free PHV matrices were enzymatically treated with α1‐3,6‐galactosidase or PNGase F. The potential impact on the glycocalyx was investigated by histochemical stains utilizing isolectin B4 (IL‐B4), wheat germ agglutinin (WGA), Datura stramonium lectin (DSL), and Ricinus communis agglutinin (RCA I) on decellularized only, and decellularized and enzymatically treated specimens. Native PHV tissue served as controls. Results All used lectins stained native heart valve tissue. Decellularization resulted in reduced IL‐B4 and WGA staining, whereas cell removal had no effect on DSL and RCA I staining. Enzymatic PNGase F treatment resulted in a further decrease of IL‐B4 and WGA staining whereas initially not affected DSL stain was reduced as well. Enzymatic treatment with α1‐3,6‐galactosidase led to a reduction of IL‐B4 stain only. Conclusion Decellularization per se is able to reduce αGal epitopes as demonstrated by IL‐B4 staining. This reduction of αGal epitopes can be enhanced by α1‐3,6‐galactosidase digestion as expected. Enzymatic treatment with PNGase F that recognizes GlcNacβ(1‐N)Asn sites results in removal of carbohydrate structures as αGal and N ‐acetyl‐glucosamines to a high degree as demonstrated by IL‐B4, WGA, and DSL stains. In summary, detergent based decellularization followed by PNGase F treatment resembles an efficient way to remove immunogenic epitopes from porcine pulmonary heart valve matrices, thus potentially enabling the generation of xenogeneic PHV matrices for clinical application.