Mechanisms of Galα1-3Galβ1-4GlcNAc-R (αGal) expression on porcine valve endothelial cells

Abstract

Objective: We have previously reported that porcine valve endothelium does not express immunodetectable levels of the carbohydrate Galα1-3Galβ1-4GlcNAc-R (known as αGal), suggesting that fresh porcine valve may be immunoprivileged. In this study, we further investigated the mechanisms of αGal 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 αGal 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 αGal 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 αGal 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 αGal, 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 α1,3galactosyltransferase gene expression in valve endothelial cells, suggesting that delayed rejection of fresh porcine cardiac valves may occur.J Thorac Cardiovasc Surg 2003;125:306-14