Crucial role of nuclear factor-κB in neointimal hyperplasia of the mouse carotid artery after interruption of blood flow

We used a molecular genetics approach to investigate the role of nuclear factor-κB (NF-κB) in neointimal hyperplasia induced by flow interruption of carotid artery in mice. Wild type mice (WT mice) and mice rendered deficient in p105, the precursor of p50, one of the components of the multimeric transcription factor NF-κB (NF-κB knockout mice; KO mice), were subjected to a complete ligation of the left common carotid artery. Morphometric analysis of the structural alteration caused by the disruption of the arterial blood flow was performed 14 days after surgery. Furthermore the expression of intercellular adhesion molecule-1 (ICAM-1) in injured arteries was evaluated 4 days after artery ligation by the means of reverse transcriptase polymerase chain reaction (RT-PCR) and quantification of the ICAM-1 protein levels. In a separate experiment normal mice were randomly assigned to receive a recombinant adeno-associated virus (rAAV) encoding the gene for the NF-κB inhibitory protein IκBα (rAAV-IκBα), or the β-galactosidase gene (rAAV-LacZ), both at a dose of 1011 copies and 2 weeks later were subjected to the complete ligation of the left carotid artery. NF-κB activity (studied by means of electrophoretic mobility shift assay—EMSA), IκBα expression (evaluated by Western blot analysis) ICAM-1 evaluation (RT-PCR and quantification of the protein levels) and a morphometric analysis were evaluated in the injured arteries. Disruption of the arterial blood flow caused a marked neointimal hyperplasia. The mean intimal area was 0.023±0.002 mm2 in wild type mice compared with 0.002±0.001 mm2 in NF-κB knockout mice. ICAM-1 expression was 1.7±0.8 relative amount of ICAM-1 mRNA in wild type mice compared with 0.4±0.06 relative amount of ICAM-1 mRNA in NF-κB knockout mice. ICAM-1 protein levels were also significantly reduced in NF-κB knockout mice. Injured arteries treated with rAAV-IκBα had a greater expression of IκBα and lower NF-κB activity, when compared with vessels treated with rAAV-LacZ. Furthermore, ICAM-1 expression was markedly attenuated by the treatment with rAAV-IκBα (rAAV-LacZ=1.6±0.8 relative amount of ICAM-1 mRNA; rAAV-IκBα=0.55±0.04 relative amount of ICAM-1 mRNA). ICAM-1 protein levels were also significantly decreased in rAAV-IκBα treated mice. Finally the mean intimal area was 0.028±0.003 mm2 in left carotid arteries treated with rAAV-LacZ whereas it was 0.003±0.004 mm2 in vessels treated with rAAV-IκBα. Our data indicate that NF-κB plays a crucial role in neointimal hyperplasia induced by flow cessation in the mouse carotid artery, and in addition suggest that rAAV-mediated gene transfer of IκBα might represent a novel therapeutic approach to the treatment of restenosis.