The IκB Kinase Inhibitor Nuclear Factor-κB Essential Modulator–Binding Domain Peptide for Inhibition of Injury-Induced Neointimal Formation
Objective—The activation of nuclear factor-κB (NF-κB) is a crucial step in the arterial wall’s response to injury. The identification and characterization of the NF-κB essential modulator–binding domain (NBD) peptide, which can block the activation of the IκB kinase complex, have provided an opportunity to selectively abrogate the inflammation-induced activation of NF-κB. The aim of the present study was to evaluate the effect of the NBD peptide on neointimal formation.
Methods and Results—In the rat carotid artery balloon angioplasty model, local treatment with the NBD peptide (300 μg/site) significantly reduced the number of proliferating cells at day 7 (by 40%; P<0.01) and reduced injury-induced neointimal formation (by 50%; P<0.01) 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. In addition, the NBD peptide (0.01 to 1 μmol/L) reduced rat smooth muscle cell proliferation, migration, and invasion in vitro. Similar results were observed in apolipoprotein E−/− mice in which the NBD peptide (150 μg/site) reduced wire-induced neointimal formation at day 28 (by 47%; P<0.01).
Conclusion—The NBD peptide reduces neointimal formation and smooth muscle cell proliferation/migration, both effects associated with the inhibition of NF-κB activation.
The transcriptional factor nuclear factor κB (NF-κB) plays a critical role in the pathophysiological processes leading to neointimal formation.1 Activated NF-κB is detected in human restenotic lesions, vascular smooth muscle cells (SMCs), monocytes, and endothelial cells.2 Activated NF-κB has also been found in balloon-injured rat carotid arteries and has been associated with neointimal formation and expression of NF-κB-regulated genes, such as vascular cell adhesion molecule-1, monocyte chemotactic protein-1 (MCP-1), and tumor necrosis factor-α (TNF-α).3–6 Consistent with its role in vascular injury, blocking NF-κB activation via transfection of adenoviral IκB7 or NF-κB decoy oligodeoxynucleotides (ODN) attenuated neointimal formation after balloon injury in animal models.8 Recently, the first clinical use of an NF-κB decoy at the site of coronary stenting for the prevention of restenosis has been described.9
NF-κB activation influences SMC viability and migration/invasion by inducing genes with survival functions5,10,11 and genes involved in matrix degradation.12 On the other hand, several inflammatory mediators involved in neointimal hyperplasia (eg, TNF-α) are able to activate NF-κB in SMCs in vitro.5 These findings link the activation of NF-κB to neointimal formation and to the inflammatory response associated with injury-induced SMC proliferation/migration, thus validating NF-κB as a potential target for the control of neointimal hyperplasia. However, the indispensable role played by NF-κB in many biological processes has raised the concern that a complete shutdown of this pathway would have significant detrimental effects on normal cellular function. Instead, drugs that selectively target only the inflammation-induced NF-κB activity would be of greater therapeutic value.
A key step in NF-κB activation is the phosphorylation of IκB proteins by the IκB kinase (IKK) complex (IKKα, IKKβ, and NF-κB essential modulator [NEMO]).13 NEMO regulates the IKK complex activity through its binding to the carboxyl-terminal region of the IKKα and IKKβ, termed the NEMO-binding domain (NBD). In this regard, a cell-permeable NBD peptide has been shown to block the association of NEMO with the IKK complex, inhibiting NF-κB activation and ameliorating inflammatory responses.14 The potential of this peptide as an antiinflammatory agent has been demonstrated in vivo in various animal models, including phorbol ester–induced ear edema and zymosan-induced peritonitis,14 lipopolysaccharide-induced septic shock,15 mouse carrageenan-induced paw edema,16 and a mouse model of experimental arthritis.17 Importantly, the NBD peptide does not completely inhibit NF-κB activity, suggesting that selective disruption of the interaction of NEMO and IKKβ will most likely leave residual NF-κB activity that might be sufficient to maintain normal cellular processes.14
Nevertheless, the effects of a highly selective pharmacological inhibition of the proinflammatory IKK activity have not yet been investigated in vascular injury.
Therefore, the aim of the present study was to investigate the effect of the NBD peptide on neointimal formation in vivo using 2 well-known animal models of arterial injury: rat carotid artery balloon angioplasty and wire-induced carotid injury in apolipoprotein E–deficient (apoE−/−) mice. In addition, the effects of the NBD peptide on SMC proliferation and migration in vitro were also examined. Our results support the selective targeting of IKK as a powerful approach in the control of neointimal formation.
Primary aortic SMCs were isolated from the thoracic aorta of male Wistar rats as previously described18 and grown in Dulbecco’s modified Eagle’s medium (Cambrex Bio Sciences) supplemented with l-glutamine, 10% fetal bovine serum (Cambrex Bio Sciences), 100 U/mL penicillin, and 100 μg/mL streptomycin in a humidified incubator at 37°C in 5% CO2. Before initiation of the assays, the SMCs were starved into Dulbecco’s modified Eagle’s medium supplemented with 0.1% fetal bovine serum for 48 hours. Studies were performed with cells at passages 3 to 6.
Cell Proliferation Study
SMC proliferation was quantified by the total cell number as previously reported.18 Briefly, 5×103 cells were seeded onto 48-well plates and allowed to adhere overnight. Starved cells were stimulated with TNF-α (5 ng/mL, R&D Systems), platelet-derived growth factor-BB (PDGF-BB) (10 ng/mL, R&D Systems) or fibroblast growth factor-2 (FGF-2) (10 ng/mL, R&D Systems) in the presence or absence of the NBD peptide (0.01 to 1 μmol/L; Genosphere Biotech). After 72 hours, cells were fixed with methanol and stained with Diff-Quik. Proliferation was evaluated as cell duplication by counting the number of cells in 10 random fields of each well at a magnification of ×200 (TNF-α experiment) or ×400 (PDGF-BB and FGF-2 experiments) with the aid of a 21-mm2 ocular grid.
Chemotactic Migration and Invasion
The modified Boyden chamber (48-well plates, Neuroprobe) was used for chemotaxis studies.18,19 Polyvinyl-pyrrolidone-free polycarbonate filters (8-μm pore size) were coated with 100 μg/mL collagen type I and 10 μg/mL fibronectin. Biocoat Matrigel invasion chambers (24-well plates with an 8.0-μm pore size filter; Becton Dickinson) were used according to the manufacturer’s instructions for invasion studies. TNF-α (5 ng/mL), PDGF-BB (10 ng/mL), or FGF-2 (10 ng/mL) was added to the lower wells, and starved cells (12×103 for migration assay and 3×104 for invasion assay) were seeded into the upper wells of the chamber and incubated at 37°C. The NBD peptide (0.01 to 1 μmol/L) was added to the cell suspension 60 minutes before seeding. After 4 hours for the migration assay or 48 hours for the invasion assay, the migrated cells were fixed and stained with hematoxylin. Cell migration was measured by microscopic evaluation of the number of cells moved across the filter, in 10 random fields for the migration assay and in the entire filter for the invasion assay.
Apoptosis was quantified by flow cytometry using a commercially available Annexin V–Alexa Fluor 488 apoptosis detection kit following the manufacturer’s guidelines (Molecular Probes). Starved SMCs were stimulated for 24 hours with TNF-α (5 ng/mL) and then washed twice in PBS, trypsinized, and collected. To evaluate the effect of the NBD peptide, SMCs were pretreated for 1 hour with the peptide (1 μmol/L) before the TNF-α stimulation. Cells were centrifuged, the supernatant was discarded, and the cell pellet was resuspended in the kit’s binding buffer. The cells were centrifuged again, the supernatant was discarded, and the pellet was resuspended in the kit’s buffer containing Alexa Fluor 488 Annexin V solution and MitoTracker red dye. Samples were incubated in the dark for 10 minutes and analyzed using an Epics XL flow cytometer (Beckman Coulter) equipped with a 488-nm argon laser. Apoptotic cells showed green fluorescence with decreased red fluorescence, and live cells showed very little green fluorescence and bright red fluorescence. Isotype-matched antibodies were used as a negative control.
Cells were cultured in 96-well culture plates in 10% fetal bovine serum medium until 90% confluence was achieved. Starved cells were stimulated with TNF-α (5 ng/mL) in the presence or absence of the NBD peptide (1 μmol/L). After 24 hours, the media were collected, clarified by centrifugation, and subjected to electrophoresis in 8% SDS-PAGE containing 1 mg/mL gelatin under nondenaturing conditions. After electrophoresis, the gels were washed with 2.5% Triton X-100 to remove SDS and incubated for 24 hours at 37°C in 50 mmol/L Tris buffer containing 200 mmol/L NaCl and 20 mmol/L CaCl2, pH 7.4. The gels were stained with 0.5% Coomassie Brilliant Blue R-250 in 10% acetic acid and 45% methanol and destained with 10% acetic acid and 45% methanol. Bands of gelatinase activity appeared as transparent areas against a blue background. Gelatinase activity was then evaluated by quantitative densitometry.
Cells were used after the induction of quiescence in 24-well plastic culture plates at a density of 2×104 cells/well. The cells were stimulated with TNF-α (5 ng/mL) in the presence or absence of the NBD peptide (0.01 to 1 μmol/L). After 24 hours, media were collected and centrifuged at 2000g for 15 minutes at 4°C, and supernatants were used for enzyme-linked immunosorbent assay (ELISA) to detect MCP-1 (OptEIA, BD, Biosciences, Sparks, Md).
Cytosolic and Nuclear Extracts
Cells (1×105) suspended in 10% fetal bovine serum medium were seeded in 6-well plates and allowed to adhere overnight. Cells were kept in starving conditions for 48 hours. The medium was then removed and replaced with fresh medium containing TNF-α (5 ng/mL) or PDGF-BB (10 ng/mL) in the presence or absence of the NBD peptide (0.01 to 1 μmol/L) or the mutated NBD (mut-NBD) peptide (1 μmol/L). The NBD peptides used in this study were described previously.16
The cell pellet was resuspended in 100 μL of ice-cold hypotonic lysis buffer (10 mmol/L Hepes, 10 mmol/L KCl, 0.5 mmol/L phenylmethylsulfonyl fluoride, 1.5 μg/mL soybean trypsin inhibitor, 7 μg/mL pepstatin A, 5 μg/mL leupeptin, 0.1 mmol/L benzamidine, 0.5 mmol/L dithiothreitol) and incubated on ice for 15 minutes. The cells were lysed by rapid passage through a syringe needle 5 times and centrifuged for 10 minutes at 13 000g. The supernatant containing the cytosolic fraction was removed and stored at −80°C. The nuclear pellet was resuspended in 30 μL of high-salt extraction buffer (20 mmol/L Hepes pH 7.9, 10 mmol/L NaCl, 0.2 mmol/L EDTA, 25% v/v glycerol, 0.5 mmol/L phenylmethylsulfonyl fluoride, 1.5 μg/mL soybean trypsin inhibitor, 7 μg/mL pepstatin A, 5 μg/mL leupeptin, 0.1 mmol/L benzamidine, 0.5 mmol/L dithiothreitol) and incubated at 4°C for 30 minutes with constant agitation. The nuclear extract was then centrifuged for 10 minutes at 6000g, and the supernatant was aliquoted and stored at −80°C. Protein concentration was determined using the Bio-Rad protein assay kit.
Western Blot Analysis
Immunoblotting analysis of phospho-IκBα (Ser32/36) was performed on cytosolic extracts. The samples were mixed with gel loading buffer (50 mmol/L Tris, 10% SDS, 10% glycerol, 10% 2-mercaptoethanol, 2 mg/mL bromophenol) at a ratio of 1:1, boiled for 3 minutes, and centrifuged at 1000g for 5 minutes. An equivalent protein amount (30 μg) of each sample was electrophoresed in a 10% discontinuous polyacrylamide gel. The proteins were transferred onto nitrocellulose membranes according to the manufacturer’s instructions (Bio-Rad). The membranes were saturated by incubation for 2 hours with 10% milk buffer and then incubated with the primary antibody (mouse anti-phospho-IκBα, 1:1000, Cell Signaling) at 4°C overnight. The membranes were washed 3 times with 0.01% Tween20 in PBS and then incubated with anti-rabbit or anti-mouse immunoglobulins coupled to peroxidase (1:1000, Dako). The immunocomplexes were visualized using the ECL chemiluminescence method.
Electrophoretic Mobility Shift Assay
Double-stranded NF-κB consensus oligonucleotide probe (5′-AGC TTC AGA GGG GAC TTT CCG AGA GG-3′) was end-labeled with [32P]γ-ATP. Nuclear extracts (10 μg of protein from each sample) were incubated for 20 minutes with radiolabeled oligonucleotides (2.5×104 to 5.0×104 cpm) in 20 μL of reaction buffer containing 2 μg of poly(dI-dC), 10 mmol/L Tris-HCl (pH 7.5), 100 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L dithiothreitol, 1 μg/μL bovine serum albumin, 10% (v/v) glycerol. Nuclear protein-oligonucleotide complexes were resolved by electrophoresis on a 5% nondenaturing polyacrylamide gel in 0.5× Tris-borate/EDTA at 150 V for 2 hours at 4°C. The gels were dried and autoradiographed with intensifying screen at −80°C for 24 hours.
Male Wistar rats (Harlan Laboratories) weighing 250 g and 8-week-old female apoE−/− mice (Charles River) were used. Animals were housed at the Department of Experimental Pharmacology, University of Naples Federico II. All procedures were performed according to Italian ministerial authorization (DL 116/92) and European regulations on the protection of animals used for experimental and other scientific purposes.
Rat Carotid Balloon Angioplasty
Rats were anesthetized with an intraperitoneal injection of ketamine (100 mg/kg) (Gellini International) and xylazine (5 mg/kg) (Sigma). Endothelial denudation of the left carotid artery was performed with a balloon embolectomy catheter (2F, Fogarty, Edwards Lifesciences) according to the procedure validated in our laboratories.19,20 Immediately after endothelial denudation, 300 μg of the NBD peptide or 300 μg of the mut-NBD peptide in 100 μL of pluronic gel (pH 7.2) was applied to the adventitia.21,22 The control group received pluronic gel only. Some animals were subjected to anesthesia and surgical procedure without balloon injury (sham-operated group). Rats were euthanized 7 and 14 days after angioplasty. Carotid arteries were collected and processed as described below.
Atherogenic Murine Model of Vascular Injury
ApoE−/− mice were fed an atherogenic diet (21% fat, 0.15% cholesterol, 19.5% casein, wt/wt, TD88137, Mucedola) from 1 week before until 4 weeks after carotid injury performed as described previously,19 with minor modification. Briefly, mice were anesthetized as described above, and endothelial injury of the left common carotid artery was performed with a 0.35-mm diameter flexible nylon wire introduced through the left external carotid artery and advanced to the aortic arch. The endothelium was damaged by passing the wire through the lumen of the artery 3 times. Immediately after endothelial denudation, 150 μg of the NBD peptide or 150 μg of the mut-NBD peptide in 50 μL of pluronic gel was applied to the adventitia. Carotid arteries were collected 28 days after wire injury and processed as described below.
Evaluation of Neointimal Formation
Carotid arteries were fixed by perfusion with phosphate-buffered saline (PBS; pH 7.2) followed by PBS containing 4% formaldehyde through a cannula placed in the left ventricle. Paraffin-embedded sections were cut (6 μm thick) from the approximate middle portion of the artery and stained with hematoxylin and eosin to demarcate cell types. Ten sections from each carotid artery were reviewed and scored under blind conditions. The cross-sectional areas of tunica media and neointima were determined by a computerized analysis system (LAS, Leica).
Proliferating Cell Nuclear Antigen Analysis
Proliferating cell nuclear antigen (PCNA) analysis was used to quantify the proliferative activity of cells at the balloon injury sites and was performed using monoclonal mouse anti-PCNA antibody (1:250, PC10, Sigma) and biotinylated anti-mouse secondary antibody (1:400, DakoCytomation). Slides were treated with streptavidin–horseradish peroxidase (DakoCytomation) and exposed to diaminobenzidine chromogen (DakoCytomation) with hematoxylin counterstain. Six sections from each carotid artery and 10 fields per section were reviewed and scored under blind conditions. Data are represented as the percentage of cells positive for PCNA 7 days after angioplasty.
Immunohistochemical Localization of the NBD Peptide
Localization of the biotinylated (bio)-NBD peptide in rat carotid arteries was performed by immunofluorescence to determine the temporal and spatial distribution of the peptide delivered to the adventitia. Briefly, 300 μg of the bio-NBD peptide (Genosphere Biotech, Paris, France) in 100 μL of pluronic gel was applied on the carotid artery immediately after the injury. Immunohistochemical analysis was performed on 5-μm frozen sections of rat carotid artery 3, 7 and 14 days after injury. The biotinylated peptide was detected by Texas red–conjugated streptavidin (1:100, DakoCytomation). For the identification of the SMC, a monoclonal anti-α-smooth muscle actin fluorescein isothiocyanate (1:250, clone 1A4, Sigma) was used. 4′,6-Diamidino-2-phenylindole was used to identify nuclei.
Preparation of Total Extracts of Rat Carotid Arteries
All the extraction procedures were performed on ice with ice-cold reagents as described above. Briefly, liquid nitrogen frozen pooled carotid arteries (n=2) were crushed into powder and resuspended in an adequate volume of hypotonic lysis buffer and then centrifuged for 15 minutes at 6000g, and the supernatant was aliquoted and stored at −80°C. Protein concentration was determined using the Bio-Rad protein assay kit. Total extracts were used to evaluate MCP-1 production and NF-κB activity by ELISA and electrophoretic mobility shift assay (EMSA), respectively.
Results are expressed as mean±SEM of n animals for in vivo experiments and mean±SEM of multiple experiments for in vitro assays. The Student t test was used to compare 2 groups or ANOVA (2-tailed probability value) was used with the Dunnett post hoc test for multiple groups using GraphPad Instat 3 software (San Diego, CA). The nonparametric Mann-Whitney U test was used for evaluation of neointimal formation. The level of statistical significance was 0.05 per test.
Effect of the NBD Peptide on NF-κB Activation in SMCs
Thirty minutes of stimulation with TNF-α (5 ng/mL) caused a significant IκBα phosphorylation at Ser32 (Figure 1A). Consistent with its mechanism of action, treatment with the NBD peptide reduced the phosphorylation of IκBα in a concentration-dependent manner. Treatment with the mut-NBD peptide (1 μmol/L) did not affect IκBα phosphorylation (Figure 1A). To further confirm the inhibitory effect of the NBD peptide on NF-κB activation, we examined the NF-κB/DNA binding 4 hours after TNF-α stimulation. As shown in Figure 1B, the NBD peptide (0.01 to 1 μmol/L) inhibited TNF-α-induced NF-κB activation. The relative densitometric analysis showed a concentration-dependent inhibition, significant at all concentrations studied (Figure 1B). The NBD peptide alone (1 μmol/L) did not affect NF-κB basal activity (Figure 1B). The mut-NBD peptide (1 μmol/L) showed no effect on TNF-α-induced NF-κB activation (data not shown). The NBD peptide (1 μmol/L) inhibited PDGF-BB (10 ng/mL)-induced NF-κB activation (data not shown).
Effect of the NBD Peptide on SMC Proliferation and Apoptosis
Initiation and maintenance of SMC proliferation is a critical event in the pathogenesis of neointimal formation. As shown in Figure 2A, the NBD peptide (0.01 to 1 μmol/L) 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. The NBD peptide (1 μmol/L) did not stimulate cell apoptosis either alone or in the presence of TNF-α (5 ng/mL) (Figure 2B). Similarly, the NBD peptide (1 μmol/L) significantly inhibited PDGF-BB (10 ng/mL)-induced SMC proliferation by 27% (P<0.05, n=3) but was without effect when the stimulant was FGF-2 (10 ng/mL) (Supplemental Figure IA, available online at http://atvb.ahajournals.org).
Effect of the NBD Peptide on SMC Migration
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 μmol/L, and ≈20% (P<0.001, n=3) at both 0.1 and 1 μmol/L (Figure 3A). The NBD peptide reduced PDGF-BB-induced but not FGF-2-induced SMC migration (Supplemental Figure IB). Moreover, the NBD peptide (1 μmol/L) significantly reduced SMC TNF-α-induced invasion (by 70%, P<0.01, n=3) through the Matrigel barrier, which mimics the extracellular matrix (Figure 3B).
Effect of the NBD Peptide on Matrix Metalloproteinase 2 and Matrix Metalloproteinase 9 Activity
Subconfluent cultures of SMCs were exposed to TNF-α (5 ng/mL) for 24 hours in the presence or absence of the NBD peptide (1 μmol/L) to assess gelatinase production. Gelatin zymography of control supernatants showed the constitutive release of the latent forms of matrix metalloproteinase 2 (MMP2), visualized as a band at 72 and 68 kDa. TNF-α stimulated the release of MMP2 and induced its activation, as revealed by the appearance of the 62 kDa form (Figure 4A). The NBD peptide significantly (P<0.05) inhibited the latent form of MMP2 without affecting the activated form and slightly, although not significantly, decreased the TNF-α-induced MMP9 gelatinase active form production (92 kDa) (Figure 4A and 4B).
Effect of the NBD Peptide on MCP-1 Production
MCP-1 production by cultured rat SMCs was determined in cell supernatants by ELISA. As shown in Figure 4C, stimulation of SMCs with TNF-α (5 ng/mL) caused an increased release of MCP-1 compared with that observed in unstimulated cells. In the presence of the NBD peptide (0.01 to 1 μmol/L), a concentration-related inhibition of MCP-1 production was observed. Interestingly, the NBD peptide at higher concentrations totally abolished TNF-α-induced MCP-1 production. The NBD peptide alone (1 μmol/L) did not affect basal MCP-1 production (Figure 4C).
Effect of the NBD Peptide on Neointimal Formation in Rat Injured Carotid Arteries
Rats were treated with either the NBD peptide, the mut-NBD peptide (300 μg/site), or an equal volume of pluronic gel (100 μL, control group) immediately after balloon injury. A reduction of proliferating cells was demonstrated in the carotid arteries of the NBD peptide-treated rats 7 days after injury (P<0.01, n=5) (Figure 5A). Moreover, the NBD peptide treatment caused a significant inhibition of neointimal formation by 54% (P<0.01, n=10) at day 14 compared with the control group (Figure 5B). The local application of the mut-NBD peptide (300 μg/site) did not affect neointimal formation (n=5; Figure 5B). In addition, the NBD peptide significantly (P<0.01) increased the lumen area and decreased neointima/media ratio (Supplemental Table I).
Effect of the NBD Peptide on NF-κB Activation in Rat Injured Carotid Arteries
To support the hypothesis that the reduction of neointimal thickness correlated with NF-κB inhibition, the NF-κB/DNA binding activity was evaluated on extracts from carotid arteries by EMSA. The NBD peptide, but not the mut-NBD peptide, significantly (n=3, P<0.001) reduced balloon-induced NF-κB activation in injured arteries 3 and 14 days after injury. A low level of NF-κB/DNA binding activity was detected in total protein extracts from carotid arteries of sham-operated rats (Figure 5C and 5D).
Effect of the NBD Peptide on MCP-1 Production in Rat Carotid Arteries
The NBD peptide was able to significantly inhibit MCP-1 protein production 7 and 14 days after injury, evaluated by ELISA as described above. In the contralateral carotid artery (data not shown), no significant changes were observed at any of the time points, compared with measurements in sham-operated animals (Figure 5E).
Effect of the NBD Peptide on Neointimal Formation in apoE−/− Mice
Carotid endothelial denudation was performed in apoE−/− mice fed an atherogenic diet. Twenty-eight days after injury, the neointimal area was reduced by 46% (P<0.01) in apoE−/− mice treated with the NBD peptide compared with mut-NBD-treated mice (Figure 5F). The NBD peptide significantly increased the lumen area (P<0.01) and decreased the neointima/media ratio (P<0.05) (Supplemental Table II).
In Vivo Localization of the Bio-NBD Peptide
No positive staining was found in noninjured arteries or pluronic gel-treated carotids 14 days after angioplasty. In contrast, the bio-NBD peptide was detectable in the adventitia and media of injured vessels 3 days following injury. The bio-NBD peptide was also detectable in the media and the neointima at days 7 and 14 (Figure 6).
The results obtained in this study show that the local administration of the NBD peptide, a selective inhibitor of IKK activation, reduces neointimal formation in rodent models of vascular injury mainly by inhibiting SMC activation.
Increased SMC proliferation and acquisition of a proinflammatory phenotype are central features associated with the development of neointimal lesions.23 The NBD peptide showed both in vivo and in vitro antiproliferative activity. Treatment with the NBD peptide diminished the number of PCNA-positive proliferating cells in the rat vessel wall 7 days after balloon injury, concomitant with the beginning of neointimal formation. Furthermore, the NBD peptide inhibited in vitro TNF-α- and PDGF-BB-induced rat SMC proliferation and migration, effects associated with the inhibition of NF-κB activation. On the contrary, the NBD peptide showed no effect on FGF-2-induced SMC proliferation/migration. This discrepancy could be explained by the facts that FGF-2 induces IκB degradation and NF-κB activation through a pathway distinct from TNF-α5 and that p38 and p42/p44 MAPKs are also involved in FGF-2-induced SMC activation.24 Interestingly, although the NBD peptide significantly reduced rat SMC invasion through the Matrigel barrier, it was able to inhibit only the latent form of MMP2 without significantly affecting the activated forms of both MMP2 and MMP9, which are known to be required for SMC proliferation and migration into the intimal area of vascular wall.25,26 These results suggest that other proteases may cooperate with gelatinases in the TNF-α-induced cell invasion process.
Activated NF-κB mediates the expression of several proinflammatory genes in SMCs, among which MCP-1 has been demonstrated to play a pivotal role in SMC proliferation/migration18,27 and neointimal formation in several animal models.19,27 Interestingly, treatment with the NBD peptide significantly inhibited MCP-1 production, both in vitro and in vivo. Moreover, the total inhibition of MCP-1 production and SMC proliferation observed at the highest concentration (1 μmol/L) suggests that NF-κB triggers the autocrine/paracrine loop mechanism involved in the amplification of the inflammatory vascular response.
Recent findings support the concept of NF-κB as a regional regulator of SMC survival rather than a direct promoter of proliferation of these cells.10 In our experiments, the highest concentration of the NBD peptide showed no effect on SMC apoptosis, either when used alone or when used with TNF-α. These results are in contrast with previous data obtained by Obara et al,28 showing increased apoptosis rate in TNF-α-stimulated SMCs overexpressing a truncated IκBα. Our results could be justified, at least in part, by the use of a selective inhibitor of the IKK complex formation. It is known that under physiological conditions, NF-κB is partially activated and involved in cell survival.10 The NBD peptide inhibits only the inflammatory-induced NF-κB activation without modifying the amount constitutively activated.14,29 To confirm this point, we observed that the use of the NBD peptide, at higher concentrations, did not reduce the basal level of MCP-1 production compared with resting cells, most likely reflecting the fact that the NBD peptide does not affect basal NF-κB activity.
In the last 10 years, NF-κB has been investigated as a novel therapeutic target to prevent restenosis. However, the potential for developing effective therapeutic strategies based on NF-κB blockade remains to be determined. Several studies have targeted NF-κB activation in the control of vascular injury,8 and a phase I/IIa open-label multicenter study to assess the inhibitory effects of an NF-κB ODN decoy on in-stent coronary restenosis (INDOR Study) has reported the clinical safety of such approach in humans.30
In contrast to other therapeutic principles, the inhibition of the NF-κB system represents a broad-spectrum, multipurpose weapon that can interfere with several fundamental pathophysiological mechanisms in the development of neointimal formation, targeting both proliferation and inflammation. In our study, the use of a peptide that selectively inhibits the IKK complex represents a novel and interesting approach. Compared with other NF-κB inhibitors tested to inhibit neointimal formation, the NBD peptide has the advantage of inhibiting the induction of NF-κB activation without inhibiting basal NF-κB activity that may be involved in fundamental cellular processes.14,29 Notably, the continuous administration of the peptide for >45 days did not lead to overt toxicity in mice.17
Importantly, the Pluronic F-127 gel has been shown to be absorbed in vivo at 3 days. Such a short-term local administration of traditional NF-κB inhibitors (eg, pyrrolidine dithiocarbamate) has been shown to inhibit NF-κB activation in the injured vessels at day 3, without affecting intimal formation at day 14.6 Intriguingly, using the same delivery approach, we have clearly shown the presence of the biotinylated peptide in the rat media and neointima up to 14 days after injury.
Importantly, the NBD peptide efficacy was also confirmed in the hyperlipidemic mouse model, demonstrating the efficacy of the peptide under circumstances of increased vascular inflammation.31
Neither rodent model is a reliable experimental model of human angioplasty, and as such, the present study has some limitations. It would be desirable to explore the efficacy and therapeutic potential of the NBD peptide in larger preclinical animal models. However, our results demonstrate the involvement of NF-κB as a regulator in the formation of neointima in mouse and rat vascular injury models and support the use of specific IKK inhibitors to reduce neointimal hyperplasia.
Sources of Funding
This work was supported by the Italian Government Grant PRIN2007 (2007LTAJMA_001) and a Regione Campania grant (L.R. n. 5/2002-Ann. 2005). Mr Parratt’s Master of Research studentship was supported by a Capacity Building Award in Integrative Mammalian Biology funded by the Biotechnology and Biological Sciences Research Council (BBSRC), British Pharmacological Society (BPS), Medical Research Council (MRC), Knowledge Transfer Network (KTN) and Scottish Funding Council (SFC).
Received on: December 12, 2009; final version accepted on: September 25, 2010.
de Winther MP, Kanters E, Kraal G, Hofker MH. Nuclear factor κB signaling in atherogenesis. Arterioscler Thromb Vasc Biol. 2005; 25: 904–914.
Hoshi S, Goto M, Koyama N, Nomoto K, Tanaka H. Regulation of vascular smooth muscle cell proliferation by nuclear factor-κB and its inhibitor, I-κB. J Biol Chem. 2000; 275: 883–889.
Bu DX, Erl W, de Martin R, Hansson GK, Yan ZQ. IKKβ-dependent NF-κB pathway controls vascular inflammation and intimal hyperplasia. FASEB J. 2005; 19: 1293–1295.
Breuss JM, Cejna M, Bergmeister H, Kadl A, Baumgartl G, Steurer S, Xu Z, Koshelnick Y, Lipp J, De Martin R, Losert U, Lammer J, Binder BR. Activation of nuclear factor-κB significantly contributes to lumen loss in a rabbit iliac artery balloon angioplasty model. Circulation. 2002; 105: 633–638.
Ohtani K, Egashira K, Nakano K, Zhao G, Funakoshi K, Ihara Y, Kimura S, Tominaga R, Morishita R, Sunagawa K. Stent-based local delivery of nuclear factor-κB decoy attenuates in-stent restenosis in hypercholesterolemic rabbits. Circulation. 2006; 114: 2773–2779.
Mehrhof FB, Schmidt-Ullrich R, Dietz R, Scheidereit C. Regulation of vascular smooth muscle cell proliferation: role of NF-κB revisited. Circ Res. 2005; 96: 958–964.
Chen Y, Budd RC, Kelm RJ Jr, Sobel BE, Schneider DJ. Augmentation of proliferation of vascular smooth muscle cells by plasminogen activator inhibitor type 1. Arterioscler Thromb Vasc Biol. 2006; 26: 1777–1783.
Bond M, Chase AJ, Baker AH, and Newby AC. Inhibition of transcription factor NF-κB reduces matrix metalloproteinase-1, -3 and -9 production by vascular smooth muscle cells. Cardiovasc Res. 2001; 50: 556–565.
May MJ, D'Acquisto F, Madge LA, Glöckner J, Pober JS, Ghosh S. Selective inhibition of NF-κB activation by a peptide that blocks the interaction of NEMO with the IκB kinase complex. Science. 2000; 289: 1550–1554.
Parenti A, Bellik L, Brogelli L, Filippi S, Ledda F. Endogenous VEGF-A is responsible for mitogenic effects of MCP-1 on vascular smooth muscle cells. Am J Physiol Heart Circ Physiol. 2004; 286: H1978–H1984.
Grassia G, Maddaluno M, Guglielmotti A, Mangano G, Biondi G, Maffia P, Ialenti A. The anti-inflammatory agent bindarit inhibits neointima formation in both rats and hyperlipidaemic mice. Cardiovasc Res. 2009; 84: 485–493.
Maffia P, Grassia G, Di Meglio P, Carnuccio R, Berrino L, Garside P, Ianaro A, Ialenti A. Neutralization of interleukin-18 inhibits neointimal formation in a rat model of vascular injury. Circulation. 2006; 114: 430–437.
Torsney E, Mayr U, Zou Y, Thompson WD, Hu Y, Xu Q. Thrombosis and neointima formation in vein grafts are inhibited by locally applied aspirin through endothelial protection. Circ Res. 2004; 94: 1466–1473.
Skaletz-Rorowski A, Eschert H, Leng J, Stallmeyer B, Sindermann JR, Pulawski E, Breithardt G. PKC δ-induced activation of MAPK pathway is required for bFGF-stimulated proliferation of coronary smooth muscle cells. Cardiovasc Res. 2005; 67: 142–150.
Bendeck MP, Zempo N, Clowes AW, Galardy RE, Reidy MA. Smooth muscle cell migration and matrix metalloproteinase expression after arterial injury in the rat. Circ Res. 1994; 75: 539–545.
Obara H, Takayanagi A, Hirahashi J, Tanaka K, Wakabayashi G, Matsumoto K, Shimazu M, Shimizu N, Kitajima M. Overexpression of truncated IκBα induces TNF-α-dependent apoptosis in human vascular smooth muscle cells. Arterioscler Thromb Vasc Biol. 2000; 20: 2198–2204.
Baima ET, Guzova JA, Mathialagan S, Nagiec EE, Hardy MM, Song LR, Bonar SL, Weinberg RA, Selness SR, Woodard SS, Chrencik J, Hood WF, Schindler JF, Kishore N, Mbalaviele G. Novel insights into the cellular mechanisms of the anti-inflammatory effects of NF-κB essential modulator binding domain peptides. J Biol Chem. 2010; 285: 13498–13506.
Maffia P, Zinselmeyer BH, Ialenti A, Kennedy S, Baker AH, McInnes IB, Brewer JM, Garside P. Images in cardiovascular medicine. Multiphoton microscopy for 3-dimensional imaging of lymphocyte recruitment into apolipoprotein-E-deficient mouse carotid artery. Circulation. 2007; 115: e326–e328.