Functional inhibition of NF-kappaB signal transduction in alphavbeta3 integrin expressing endothelial cells by using RGD-PEG-modified adenovirus with a mutant IkappaB gene.
In order to selectively block nuclear factor kappaB (NF-kappaB)-dependent signal transduction in angiogenic endothelial cells, we constructed an alphavbeta3 integrin specific adenovirus encoding dominant negative IkappaB (dnIkappaB) as a therapeutic gene. By virtue of RGD modification of the PEGylated virus, the specificity of the cell entry pathway of adenovirus shifted from coxsacki-adenovirus receptor dependent to alphavbeta3 integrin dependent entry. The therapeutic outcome of delivery of the transgene into endothelial cells was determined by analysis of cellular responsiveness to tumor necrosis factor (TNF)-alpha. Using real time reverse transcription PCR, mRNA levels of the cell adhesion molecules E-selectin, vascular cell adhesion molecule (VCAM)-1 and intercellular adhesion molecule (ICAM)-1, the cytokines/growth factors IL-6, IL-8 and vascular endothelial growth factor (VEGF)-A, and the receptor tyrosine kinase Tie-2 were assessed. Furthermore, levels of ICAM-1 protein were determined by flow cytometric analysis. RGD-targeted adenovirus delivered the dnIkappaB via alphavbeta3 to become functionally expressed, leading to complete abolishment of TNF-alpha-induced up-regulation of E-selectin, ICAM-1, VCAM-1, IL-6, IL-8, VEGF-A and Tie-2. The approach of targeted delivery of dnIkappaB into endothelial cells presented here can be employed for diseases such as rheumatoid arthritis and inflammatory bowel disease where activation of NF-kappaB activity should be locally restored to basal levels in the endothelium.
Ogawara
Ken-ichi
K
Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, Okayama University, Okayama, Japan. ogawara@pharm.okayama-u.ac.jp
Kułdo
Joanna M
JM
Oosterhuis
Koen
K
Kroesen
Bart-Jan
BJ
Rots
Marianne G
MG
Trautwein
Christian
C
Kimura
Toshikiro
T
Haisma
Hidde J
HJ
Molema
Grietje
G
D000256:Adenoviridae / Q000235:genetics*; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000378:metabolism*; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005799:Genes, Dominant; D006801:Humans; D020672:I-kappa B Proteins / Q000235:genetics*; D039302:Integrin alphaVbeta3 / Q000378:metabolism*; D009154:Mutation; D016328:NF-kappa B / Q000037:antagonists & inhibitors* / Q000378:metabolism; D009842:Oligopeptides / Q000494:pharmacology; D011092:Polyethylene Glycols / Q000494:pharmacology; D015398:Signal Transduction; D019076:Transgenes
D020672:I-kappa B Proteins; D039302:Integrin alphaVbeta3; D016328:NF-kappa B; D009842:Oligopeptides; D011092:Polyethylene Glycols; C047981:arginyl-glycyl-aspartic acid