A strategy for inducing immune tolerance to valve endothelial cells through gene transfer

Background and aim of the study: We and others have demonstrated an immune response to homograft valve endothelial cells both in vivo and in vitro. Cl inically, this is particularly manifest in children. In an attempt to addre ss this problem we have explored a strategy of inducing specific immune tol erance by genetic manipulation of valve endothelial cells. Fast is an induc er of apoptosis; it binds Fas and results in programmed cell death (apoptos is) of Fas-bearing cells such as T lymphocytes. Fast has been shown to be i mportant in the protection of tissue grafts (testis, cornea, kidney and pan creatic islet) from rejection. The ultimate aim of this work is to determin e whether the transfection of Fast into human heart valve endothelial cells can hinder immune rejection by induction of apoptosis in T cells. Methods: The full-length human Fast cDNA was cloned into a mammalian expres sion 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 poly merase chain reaction (PCR) to determine approximate copy numbers of Fast. 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 Fast function in those transfected cells. Results: High copy number transfected cell lines were produced, and mRNA an d protein expression were confirmed. Preliminary results from cytotoxic ass ays show that transfected cells have enhanced cytotoxicity in comparison wi th their parent cell line. Conclusion: Fast can be overexpressed in endothelial cells and appears to m odify the cells' immunological behavior. These findings could have importan t implications for enhancing homograft valve durability.