Elimination of alpha-gal xenoreactive epitope: alpha-galactosidase treatment of porcine heart valves.
OBJECTIVE
Porcine heart valves are among the most widely used tissue valves in clinical heart valve implantation. However, immunologic responses have been implicated as potential causes of the limited durability of xenograft heart valves. The study aim was to determine the effectiveness of alpha-galactosidase treatment used to degrade the major xenoreactive antigens found in xenograft heart valves.


METHODS
Fresh porcine heart valves and pericardium treated with alpha-galactosidase were studied to evaluate the xenoreactive galactose (alpha1,3) galactose (alpha-gal) antigen. Removal of the alpha-gal epitope from the porcine heart valve was monitored via 3,3'-diaminobenzidine staining intensity, while the removal of alpha-gal from N-glycans on porcine heart valves treated with recombinant alpha-galactosidase was determined either qualitatively or quantitatively by mass fingerprinting using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The porcine pericardium was used for monitoring the change in mechanical properties after alpha-galactosidase treatment. In addition, the biomechanical modification property of collagen fiber rearrangement on tissue was assessed using transmission electron microscopy (TEM).


RESULTS
Following a 24-h incubation at pH 7.2, 4 degrees C, employing 0.1 U/ml of Bacteroides thetaiotaomicron-derived recombinant alpha-galactosidase, the enzyme effectively removed the alpha-gal epitopes expressed on porcine heart valves. The identification type of alpha-gal N-glycan on fresh aortic valve, aortic wall, pulmonary valve, and pulmonary wall was 7.1%, 10.3%, 6% and 8%, respectively. In the presence of alpha-galactosidase treatment, alpha-gal-containing N-glycans were converted into alpha-gal-negative N-glycans. Likewise, alpha-gal-containing N-glycans were not detected when MALDI-TOF MS quantitative analysis was used. Furthermore, no significant difference was observed in the mechanical properties and findings from TEM in alpha-galactosidase-treated porcine pericardial tissue when compared to fresh porcine pericardium.


CONCLUSIONS
Alpha-galactosidase can effectively remove the alpha-gal epitope from porcine heart valves and pericardium. This may possibly alleviate harmful xenoreactive immunologic responses by alpha-gal, without adversely affecting the biomechanical properties of the alpha-galactosidase-processed tissue.
Xenotransplantation Research Center, Seoul National University Hospital, Seoul, Republic of Korea.
Sun-Young
SY
Choi
Hee-Jin
HJ
Jeong
Hong-Gook
HG
Lim
Seong-Sik
SS
Park
Soo-Hwan
SH
Kim
Yong Jin
YJ
Kim
D015100:3,3'-Diaminobenzidine / Q000494:pharmacology; D000818:Animals; D001021:Aortic Valve / Q000276:immunology; D001696:Biomechanical Phenomena / Q000187:drug effects; D001705:Bioprosthesis / Q000009:adverse effects*; D000939:Epitopes / Q000187:drug effects / Q000276:immunology; D006084:Graft Rejection / Q000209:etiology / Q000276:immunology / Q000517:prevention & control*; D006350:Heart Valve Prosthesis / Q000009:adverse effects*; D006801:Humans; D013058:Mass Spectrometry; D023421:Models, Animal; D010449:Peptide Mapping / Q000379:methods; D010496:Pericardium / Q000276:immunology; D011475:Prosthesis Failure / Q000187:drug effects; D011664:Pulmonary Valve / Q000276:immunology; D034421:Sus scrofa; D013552:Swine; D014181:Transplantation Immunology / Q000187:drug effects; D000519:alpha-Galactosidase / Q000494:pharmacology*
D000939:Epitopes; D015100:3,3'-Diaminobenzidine; D000519:alpha-Galactosidase