994. Effect of Vectors on Human Endothelial Cell Signal Transduction: Implications for Cardiovascular Gene Therapy
Authors: Peng H. Tan, George J.T. George
Journal: Molecular Therapy (2006)

Objective: Endothelium is an important target for gene therapy. We have investigated the effect of viral and non-viral vectors on the phenotype and function of endothelial cells (ECs), and developed methods to block any activation caused by these vectors. Methods and results: Transduction of ECs with viral vectors, including adenovirus, lentiviruses and Moloney murine leukemia virus can induce a pro-inflammatory phenotype. We showed that viral vectors resulted in considerable upregulation of adhesion molecules and MHC II molecules. We saw an upregulation of Th1 cytokines, as well as enhanced secretion of proinflammatory cytokines in EC following viral transductions. Inhibition of NFκB did not completely abolish virally induced adhesion molecule expression and pro-inflammatory cytokine production. However, inhibition of both PI3-K and PKR resulted in total inhibition of EC activation. This suggests that activation of PI3-K and PKR pathways not only activate NFκB pathway, but also other downstream effector pathways. Use of other inhibitors suggested that blockade of the MAPK p38, JNK, STAT-1 and STAT3 pathways had little effect on the activation of ECs by viral transduction. Both the PKR and the PI3-K pathways are associated with cytokine production (IFN-α/β for PKR and TNF-α/IL-1α/β for PI3-K) that serves as a positive feedback loop for the activation of those pathways. These might serve to help 'lock' the EC into an activated phenotype. In order to test the functional consequences of viral transduction of EC, we incubated EC with viral vectors then used them as in adhesion and transmigration assays with granulocytes, lymphocytes and monocytes. Viral vector-induced adhesive effect was comparable to that seen following cytokine activation of the ECs. Viral transduction effect on EC can be varied. We also demonstrated that virally activated ECs failed to respond to subsequent activation by proinflammatory cytokines. We showed that EC that were chronically stimulated with pro-inflammatory cytokines failed to upregulate pro-inflammatory phenotypes upon subsequent activation, in a similar manner to virally transduced ECs. If neutralising antibodies to these cytokines were added from the time of viral transduction, the ECs were able to respond to activation stimuli in a similar manner to untransduced ECs. Conclusions: We have shown that transduction with viral vectors has dramatic consequences for ECs both in terms of their phenotype, the activation pathways induced and their function. This can modulate the effect of gene therapy by inducing local inflammation, which may both modulate the underlying disease process. Non-viral vectors, on the other hand, are relatively innocuous and fail to activate significantly ECs. However, they are less efficient at mediating gene expression! An understanding of both the consequences of EC transduction, and the pathways by which it is mediated, is important in allowing the design of improved strategies involving the viral vector modification of ECs. In this regard we have shown that blockade of the signaling pathways involved in viral activation may be used to prevent such activation