The principal objective is to determine whether endothelial cell response to transplantation mediated immune injury can be altered in vivo using an NF-kappaB consensus decoy. This will be accomplished using endothelial cell lined vascular grafts stably transfected and then implanted in vivo. To make cells adherent, in vitro shear stress pretreatment, a tool Ballermann s lab has developed, will be used. Endothelial cell inflammatory cascade is a primary obstacle in the field of transplantation and a primary mediator of vascular graft arteriosclerosis. The nuclear transcription factor NF- kappaB promotes nuclear gene expression of multiple pro- inflammatory responses in endothelial cells which elicit subsequent damage to blood vessels. We propose that NF-kappaB promoter activity can be inhibited by creating a concatamer with multiple NF-kappaB oligonucleotide consensus sequences designed to competitively bind active NF- kappaB. The effectiveness of the decoy will first be quantified in vitro by measuring specific cytokine stimulated responses as compared to scrambled decoy controls. Once quantified in vitro, the stably transfected endothelial cell lined grafts will be transplanted into syngeneic and allogeneic rat hosts and examined for alterations in inflammatory responses and graft neointima development. The proposed studies represent a step toward gene therapy to produce more durable grafted vessels, prolong transplant organ survival, and create a better understanding of the modulation of the vascular endothelial cell inflammatory cascade.
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