Christopher Wrighton

Role of endothelial cells in transplantation

Endothelial cell activation with accompanying vascular inflammatory changes is considered central to the experimental manifestations of both hyperacute and delayed xenograft rejection responses. Natural xenoreactive antibodies directed at alpha-galactosyl residues of xenogeneic glycoproteins and glycolipids, with associated complement activation via the classical pathway, are considered major immediate mediators of graft endothelial cell injury in the clinically relevant discordant swine to primate combinations. In delayed xenograft rejection processes, where recipients are treated prophylactically to ameliorate these initial events, activation of infiltrating mononuclear phagocytes and natural killer cells are associated with ongoing endothelial cell activation processes, procoagulant generation and vascular thrombosis. Allograft hyperacute rejection is observed when vascularised organs are transplanted to sensitized individuals with high levels of cytotoxic antibodies. Less dramatic forms of humoral allograft rejection (termed accelerated or vascular rejection) and the more common cell-mediated endothelialitis are associated with significant graft damage. Endothelial cell activation is also linked with graft preservation injury, forms of chronic rejection and delayed graft loss. Experimental work is currently being directed at the control of hyperacute rejection, the close understanding of endothelial cell thromboregulation in both transplanted xeno- and allografts and the development of novel therapeutic agents including gene therapy and the possible use of organs from transgenic animals.

Adenoviral-mediated overexpression of I(kappa)B(alpha) in endothelial cells inhibits natural killer cell-mediated endothelial cell activation.

Activated natural killer (NK) cells have been found in rejecting discordant xenografts and may contribute to endothelial cell (EC) activation and damage. The transcription of genes associated with EC activation, such as E-selectin and interleukin (IL)-8, is regulated by the transcription factor NF-kappaB. In resting EC, NF-kappaB is complexed within the cytoplasm to I(kappa)B(alpha), and EC activation leads to dissociation of the I(kappa)B(alpha)-NF-kappaB complex and nuclear translocation of NF-kappaB. We investigated whether overexpression of I(kappa)B(alpha) in EC, using adenoviral gene transfer, could block NF-kappaB translocation, thereby inhibiting NK cell-mediated EC activation. Co-culture of human NK cells with porcine EC resulted in a threefold increase in E-selectin expression after 4 hr and secretion of greater than 650 pg/ml porcine IL-8 over 24 hr. Overexpression of I(kappa)B(alpha) inhibited the NK cell-mediated induction of E-selectin expression and IL-8 secretion, whereas overexpression of P-galactosidase did not. The inhibition of EC activation was not due to variation in NK-EC adhesion, as the level of adhesion was similar between adenovirally infected and noninfected EC over 4 hr. The level of NK cell-mediated EC cytotoxicity was not significantly different after 4 hr of co-culture, but after 24 hr, cytotoxicity was increased in virally infected cells. Cytotoxicity was more marked in cells overexpressing I(kappa)B(alpha) than cells overexpressing beta-galactosidase. SLA class I and the induction of SLA class II antigen in response to interferon-gamma treatment were reduced in cells infected with adeno-I(kappa)B(alpha) and empty adenovirus, demonstrating that viral infection alone can influence EC biology. Overexpression of I(kappa)B(alpha) using adenovirus provides a novel approach to inhibiting NK cell-mediated EC activation, but additional strategies will be required to inhibit NK cell-mediated cytotoxicity.

Genetic engineering of endotheiial cells

Abstract: Endotheiial cell activation is a major obstacle to successful xenotransplantation. The activated phenotype is largely based on the transcriptional induction of a number of genes and their products. Due to the large number of new gene products in activated endotheiial cells, it is not feasible to target each of them individually for therapeutic intervention. A common factor important for the induction of many, if not all, genes induced upon endotheiial cell activation if NF‐KB. It is thus a reasonable target for inhibition if one is to attempt to inhibit endotheiial cell activation genetically. Donor animals for xenotransplantation are amenable to genetic manipulation and we believe that transgenic animals carrying several transgenes will be the standard of future experimental and clinical xenotransplantation.