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J Thorac Cardiovasc Surg 2003;125:306-314
© 2003 The American Association for Thoracic Surgery

Surgery for Acquired Cardiovascular Disease (ACD)

Mechanisms of Gal1-3Galß1-4GlcNAc-R (Gal) expression on porcine valve endothelial cells

From the Department of Surgery, Brigham and Women's Hospital, Boston, Mass,^a and the Department of Cardiothoracic Surgery, Mount Sinai Medical Center, New York, NY.^b

Received for publication Dec 26, 2001. Revisions requested March 11, 2002; revisions received April 19, 2002. Accepted for publication May 1, 2002. Address for reprints: David H. Adams, MD, Department of Cardiovascular and Thoracic Surgery, Mount Sinai Medical Center, 1190 Fifth Ave, New York, NY 10029 (E-mail: david.adams{at}mountsinai.org).

Objective: We have previously reported that porcine valve endothelium does not express immunodetectable levels of the carbohydrate Gal1-3Galß1-4GlcNAc-R (known as Gal), suggesting that fresh porcine valve may be immunoprivileged. In this study, we further investigated the mechanisms of Gal 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 Gal 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 Gal 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 Gal 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 Gal, 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 1,3galactosyltransferase gene expression in valve endothelial cells, suggesting that delayed rejection of fresh porcine cardiac valves may occur.

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