ABSTRACT
Title
Neointimal hyperplasia is inhibited by the IκB-Kinase Inhibitor, NEMO-binding Domain Peptide in apoE-/- mice
Authors
M.V. Di Lauro1, G. Grassia1, M. Maddaluno1, C. Musilli2, D. De Stefano1, R. Carnuccio1, A. Ianaro1, P. Maffia3,1, A. Parenti2 and A. Ialenti1
1Department of Experimental Pharmacology, University of Naples Federico II, Via Domenico Montesano, 49, 80131 Naples, Italy
2Department of Preclinical and Clinical Pharmacology, University of Florence, Viale G. Pieraccini, 6, 50139 Florence, Italy
3Institute of Infection, Immunity and Inflammation, University of Glasgow, Glasgow G12 8TA, UK
1Department of Experimental Pharmacology, University of Naples Federico II, Via Domenico Montesano, 49, 80131 Naples, Italy
2Department of Preclinical and Clinical Pharmacology, University of Florence, Viale G. Pieraccini, 6, 50139 Florence, Italy
3Institute of Infection, Immunity and Inflammation, University of Glasgow, Glasgow G12 8TA, UK
Abstract
The activation of the nuclear factor (NF)-κB is a crucial step in the arterial wall’s response to injury. The identification and characterization of the NF-κB essential modulator (NEMO)-binding domain (NBD) peptide, that can block the activation of the IκB kinase (IKK) complex, have provided an opportunity to selectively abrogate the inflammation-induced activation of NF-κB.We have previously reported thatlocal treatment with the NBD peptide (300 μg/site) in the rat carotid artery balloon angioplastymodel, significantly reduced the number of proliferating cells at day 7 and reduced injury-induced neointimal formation by 50% at day 14. These effects were associated with a significant reduction of NF-κB activation and monocyte chemotactic protein-1 expression in the carotid arteries of rats treated with the peptide.
The aim of the present study was to evaluate the effect of the NBD peptide on smooth muscle cell proliferation, migration and invasion in vitro, processes contributing to the neointimal hyperplasia. In addition, the effect of the NBD peptide was also examinedon injury-induced neointimal formation in apolipoprotein E-deficient (apoE-/-) mice, an animal model of neointimal hyperplasia characterized by increased vascular inflammation.
In vitrodata showed that the NBD peptide (0.01-1 µM) significantly inhibited TNF-α-induced SMC proliferation by 15% (P<0.05, n=4), 20% (P<0.001, n=4), and 30% (P<0.001, n=4) respectively. This effect of the NBD peptide was not due to induction of cell apoptosis as demonstrated by flow cytometry analysis of Annexin V–labeled cells. We also evaluated the effects of the NBD peptide on TNF-α-induced SMC chemotaxis. The NBD peptide significantly
inhibited chemotactic migration by 15% (P<0.05, n=3) at 0.01 µM, and about 20% (P<0.001, n=3) at both 0.1 and 1 µM. Moreover, the NBD peptide (1 µM) significantly reduced SMC TNF-α-induced invasion (by 70%, P<0.01, n=3) through the Matrigel barrier, which mimics the extracellular matrix. Interestingly, we observed that the NBD peptide was able to inhibit only the latent form of MMP2 (P<0.05) without significantly affecting the activated forms of both MMP2 and 9, which are known to be required for VSMC proliferation and migration into the intimal area of vascular wall. These effects were associated with a significant reduction of NF-κB activation.
In the apoE-/- mice local treatment with the NBD peptide (150 μg/site) significantly reduced injury-induced neointimal formation by 46% (P<0.01) 28 days after injury, compared with mut-NBD-treated mice.
In conclusion, we demonstrate that NBD peptide reduces neointimal formation in hyperlipidemic mice and rat SMC proliferation, migration and invasion in vitro, effects associated with the inhibition of NF-κB activation supporting the use of specific IKK inhibitors to reduce neointimal hyperplasia.
The aim of the present study was to evaluate the effect of the NBD peptide on smooth muscle cell proliferation, migration and invasion in vitro, processes contributing to the neointimal hyperplasia. In addition, the effect of the NBD peptide was also examinedon injury-induced neointimal formation in apolipoprotein E-deficient (apoE-/-) mice, an animal model of neointimal hyperplasia characterized by increased vascular inflammation.
In vitrodata showed that the NBD peptide (0.01-1 µM) significantly inhibited TNF-α-induced SMC proliferation by 15% (P<0.05, n=4), 20% (P<0.001, n=4), and 30% (P<0.001, n=4) respectively. This effect of the NBD peptide was not due to induction of cell apoptosis as demonstrated by flow cytometry analysis of Annexin V–labeled cells. We also evaluated the effects of the NBD peptide on TNF-α-induced SMC chemotaxis. The NBD peptide significantly
inhibited chemotactic migration by 15% (P<0.05, n=3) at 0.01 µM, and about 20% (P<0.001, n=3) at both 0.1 and 1 µM. Moreover, the NBD peptide (1 µM) significantly reduced SMC TNF-α-induced invasion (by 70%, P<0.01, n=3) through the Matrigel barrier, which mimics the extracellular matrix. Interestingly, we observed that the NBD peptide was able to inhibit only the latent form of MMP2 (P<0.05) without significantly affecting the activated forms of both MMP2 and 9, which are known to be required for VSMC proliferation and migration into the intimal area of vascular wall. These effects were associated with a significant reduction of NF-κB activation.
In the apoE-/- mice local treatment with the NBD peptide (150 μg/site) significantly reduced injury-induced neointimal formation by 46% (P<0.01) 28 days after injury, compared with mut-NBD-treated mice.
In conclusion, we demonstrate that NBD peptide reduces neointimal formation in hyperlipidemic mice and rat SMC proliferation, migration and invasion in vitro, effects associated with the inhibition of NF-κB activation supporting the use of specific IKK inhibitors to reduce neointimal hyperplasia.