A strategy for inducing immune tolerance to valve endothelial cells through gene transfer.
OBJECTIVE
We and others have demonstrated an immune response to homograft valve endothelial cells both in vivo and in vitro. Clinically, this is particularly manifest in children. In an attempt to address this problem we have explored a strategy of inducing specific immune tolerance by genetic manipulation of valve endothelial cells. FasL is an inducer of apoptosis; it binds Fas and results in programmed cell death (apoptosis) of Fas-bearing cells such as T lymphocytes. FasL has been shown to be important in the protection of tissue grafts (testis, cornea, kidney and pancreatic islet) from rejection. The ultimate aim of this work is to determine whether the transfection of FasL into human heart valve endothelial cells can hinder immune rejection by induction of apoptosis in T cells.


METHODS
The full-length human FasL cDNA was cloned into a mammalian expression vector containing the neomycin resistance marker. The endothelial cell line HMEC-1 was transfected with the plasmid and selected with antibiotic G418. Cultures from positive clones were analyzed by semi-quantitative polymerase chain reaction (PCR) to determine approximate copy numbers of FasL. Reverse transcription (RT)-PCR was carried out to examine the production of mRNA from the construct. Western blot analysis was performed to detect the protein expression. Cytotoxic assays were subsequently performed to detect the FasL function in those transfected cells.


RESULTS
High copy number transfected cell lines were produced, and mRNA and protein expression were confirmed. Preliminary results from cytotoxic assays show that transfected cells have enhanced cytotoxicity in comparison with their parent cell line.


CONCLUSIONS
FasL can be overexpressed in endothelial cells and appears to modify the cells' immunological behavior. These findings could have important implications for enhancing homograft valve durability.
Li
Y
Y
Cardiothoracic Surgery, Heart Science Centre, NHLI, Imperial College of Science, Technology and Medicine, London, United Kingdom.
McCormack
A M
AM
Brand
N J
NJ
Yacoub
M H
MH
D017209:Apoptosis; D015153:Blotting, Western; D002460:Cell Line; D004727:Endothelium / Q000166:cytology / Q000276:immunology; D053222:Fas Ligand Protein; D006084:Graft Rejection / Q000517:prevention & control; D006351:Heart Valves / Q000276:immunology / Q000637:transplantation*; D006801:Humans; D066298:In Vitro Techniques; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000276:immunology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013601:T-Lymphocytes / Q000276:immunology; D014162:Transfection; D023001:Transplantation Tolerance / Q000276:immunology*; D019014:fas Receptor / Q000276:immunology*
C505661:FASLG protein, human; D053222:Fas Ligand Protein; D008562:Membrane Glycoproteins; D019014:fas Receptor