We used a molecular genetics approach to investigate the role of
nuclear factor-kappaB (
NF-kappaB) 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-kappaB (
NF-kappaB 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-kappaB inhibitory
protein IkappaBalpha (rAAV-
IkappaBalpha), or the
beta-galactosidase gene (rAAV-LacZ), both at a dose of 10(11) copies and 2 weeks later were subjected to the complete
ligation of the left carotid artery.
NF-kappaB activity (studied by means of electrophoretic mobility shift assay-EMSA),
IkappaBalpha 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 mm(2) in wild type mice compared with 0.002+/-0.001 mm(2) in
NF-kappaB 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-kappaB knockout mice.
ICAM-1 protein levels were also significantly reduced in
NF-kappaB knockout mice. Injured arteries treated with rAAV-
IkappaBalpha had a greater expression of
IkappaBalpha and lower
NF-kappaB activity, when compared with vessels treated with rAAV-LacZ. Furthermore,
ICAM-1 expression was markedly attenuated by the treatment with rAAV-
IkappaBalpha (rAAV-LacZ=1.6+/-0.8 relative amount of ICAM-1
mRNA; rAAV-
IkappaBalpha=0.55+/-0.04 relative amount of ICAM-1
mRNA).
ICAM-1 protein levels were also significantly decreased in rAAV-
IkappaBalpha treated mice. Finally the mean intimal area was 0.028+/-0.003 mm(2) in left carotid arteries treated with rAAV-LacZ whereas it was 0.003+/-0.004 mm(2) in vessels treated with rAAV-
IkappaBalpha. Our data indicate that
NF-kappaB 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
IkappaBalpha might represent a novel therapeutic approach to the treatment of restenosis.