Inhibition of endothelial cell activation by adenovirus-mediated expression of I kappa B alpha, an inhibitor of the transcription factor NF-kappa B.

Abstract

SummaryDuring the inflammatory response, endothelial cells (EC) transiently upregulate a set of genes encoding, among others, cell adhesion molecules and chemotactic cytokines that together mediate the interaction of the endothelium with cells of the immune system. Gene upregulation is mediated predominantly at the transcriptional level and in many cases involves the transcription factor nuclear factor (NF) KB. We have tested the concept of inhibiting the inflammatory response by overexpression of a specific inhibitor of NF-KB, IKBot. A recombinant adenovirus expressing Ir, B~x was constructed (rAd.IKBe 0 and used to infect EC of human and porcine origin. Ectopic expression of IgJ3a resulted in marked, and in some cases complete, reduction of the expression of several markers of EC activation, including vascular cell adhesion molecule 1, interleukins 1, 6, 8, and tissue factor. Overexpressed IKBot inhibited NF-K_B specifically since (a) in electrophoretic mobility shift assay, NF-r,B but not AP-1 binding activity was inhibited, and (b) von Willebrand factor and prostacyclin secretion that occur independently of NF-r,B, remained unaffected. Functional studies of leukocyte adhesion demonstrated strong inhibition of ilL-60 adhesion to IKBot-expressing EC. These findings suggest that NF-KB could be an attractive target for therapeutic intervention in a variety of inflammatory diseases, including xenograft rejection.

Heterogeneous requirement of IκB kinase 2 for inflammatory cytokine and matrix metalloproteinase production in rheumatoid arthritis: Implications for therapy

“…For passaged synovial fibroblasts, an MOI of 500 was used because these cells are more resistant to adenovirus infection. These MOIs have been previously shown by us and others to result in Ͼ90% of these cells expressing the transgene of interest (17,35,41,42,45).…”

“…AdIKK2dn and I B␣ were produced by us (39). All viruses were E1/E3-deleted, belonged to the Ad5 serotype, and had been previously used in other studies (39)(40)(41)(42)(43). Viruses were propagated in HEK 293 cells (American Type Culture Collection, Manasas, VA), purified by ultracentifugation through 2 CsCl gradients, and viral titers were determined by plaque assay as previously described (44).…”

Anti-inflammatory gene therapy directed at the airway epithelium

“…Thus, I B␣ overexpression, or transfection with NF B decoy consensus binding sites decreased NF B activity in endothelial and stromal cells and monocytes in vitro. [19][20][21] This has been extended to animal models of inflammation such as ischemiareperfusion injury and septic shock. 22,23 …”

Adenovirus-mediated expression of green fluorescent protein

“…17 C3 excitation and emission maxima are at 488 and 510 nm, respectively, allowing detection by fluorcells. 11,12 This species difference leads to difficulties in evaluating AdV gene therapy in experimental animals.escence microscopy and FACS analysis using standard filter sets. After cotransfection of the transfer vector 18 conTherefore, a careful optimization of the conditions used is of great importance to achieve optimal transduction taining C3 with the plasmid pJM17 19 into 293 cells, fluorescence originating from the expression of the nonrecomefficiencies.…”

“…17 C3 excitation and emission maxima are at 488 and 510 nm, respectively, allowing detection by fluorcells. 11,12 This species difference leads to difficulties in evaluating AdV gene therapy in experimental animals.…”