Objective: We have previously reported that porcine valve endothelium does not express immunodetectable levels of the carbohydrate Gal(alpha)1-3Galbeta1-4GlcNAc-R (known as alphaGal), suggesting that fresh porcine valve may be immunoprivileged. In this study, we further investigated the mechanisms of alphaGal expression on porcine valve endothelial cells.
Methods: Primary cultures of porcine valvular endothelial cells were established and compared with porcine aortic endothelial cells and human vein endothelial cells. Immunoblotting, reverse transcriptase-polymerase chain reaction, and flow cytometry were used to compare the expression of alphaGal at both the protein and messenger RNA levels.
Results: Porcine valvular endothelial cells grew rapidly on a gelatin substrate. Similar to our previous in vivo results, valve endothelial cells expressed alphaGal much less intensely than did aortic endothelial cells. Porcine aortic endothelial cells expressed an isolectin B4 (isolectin B4 lectin Bandeiraea simplicifolia) immunodetectable band at 135 kd that was not visible on porcine valve endothelial cells or on human vein endothelial cells. Reverse transcriptase-polymerase chain reaction documented three transcripts of the alphaGal gene that were identically expressed on porcine valve and aortic endothelial cells. Furthermore, flow cytometry showed an almost identical surface profile between porcine aortic and valve endothelial cells, in contrast with human vein endothelial cells.
Conclusions: Cultures of primary valve endothelial cells were established and exhibited similar phenotypic patterns in vitro to those we have previously documented in vivo. RNA and flow cytometric analyses documented no difference between the RNA expression and surface protein profile for alphaGal, although whole-cell extracts demonstrated an immunodetectable band on Western blotting that was present on aortic endothelial cells but not on valve endothelial cells. These findings clarify the mechanism of expression of alpha1,3galactosyltransferase gene expression in valve endothelial cells, suggesting that delayed rejection of fresh porcine cardiac valves may occur.