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

Abstract

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.

Decellularized Vascular Grafts

“…Nevertheless, the preserved histones have been reported to possess low immunogenicity and antimicrobial properties (Rose et al 1998;Kim et al 2000). Triton X-100 has also been frequently used to remove SDS residues, especially during perfusion decellularization of whole organs or limbs (Ott et al 2008;Uygun et al 2010;Andrée et al 2014;Findeisen et al 2015).…”

“…Previous report had suggested that α-Gal is present on the ECM proteins (Galili 2015) and it was suggested that the use of an enzyme like α-galactosidase should be considered for the complete removal of α-Gal (Granados et al 2017). Findeisen and colleagues (2015) treated porcine heart valves with decellularization solution containing 0.5% SDS and 0.5% Triton-X100 and with α-galactosidase or PNGase F. It was concluded that α-galactosidase could only remove the α-Gal epitope, where PNGase F was able to carbohydrate structures including α-Gal and Neu5Gc epitopes (Findeisen et al 2015).…”