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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:2198.)
© 2000 American Heart Association, Inc.

Vascular Biology

Overexpression of Truncated IB Induces TNF-–Dependent Apoptosis in Human Vascular Smooth Muscle Cells

Hideaki Obara; Atsushi Takayanagi; Junichi Hirahashi; Katsunori Tanaka; Go Wakabayashi; Kenji Matsumoto; Motohide Shimazu; Nobuyoshi Shimizu; Masaki Kitajima

From the Departments of Surgery (H.O., K.T., G.W., K.M., M.S., M.K.), Molecular Biology (A.T., N.S.), and Internal Medicine (J.H.), Keio University School of Medicine, Tokyo, Japan.

	Abstract

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Abstract—Dysregulation of apoptosis is one of the likely underlying mechanisms of neointimal thickening, a disorder in which proinflammatory cytokines may influence the function of vascular smooth muscle cells (VSMCs) and contribute to atherogenesis. One of these cytokines, tumor necrosis factor- (TNF-), induces 2 possibly conflicting pathways, 1 leading to the activation of nuclear factor-B (NF-B) and the other leading to caspase-mediated apoptosis. We investigated whether specific inhibition of NF-B affects TNF-–dependent apoptosis in human VSMCs. To inhibit NF-B activation specifically, we constructed a recombinant adenovirus vector expressing a truncated form of the inhibitor protein IB (AdexIBN) that lacks the phosphorylation sites essential for activation of NF-B. The IBN was overexpressed by adenoviral infection and was resistant to stimulus-dependent degradation. Electromobility gel shift and luciferase assays demonstrated that overexpression of IBN inhibited NF-B activation induced by TNF- or interleukin-1ß (IL-1ß). In cells overexpressing IBN, TNF- dramatically induced apoptosis, whereas IL-1ß had no effect. The induction was suppressed by treatment with a selective inhibitor of the caspase-3 family, Z-DEVD-fmk, and the overexpression of IBN induced TNF-–mediated caspase-3 and caspase-2 activity. These results indicate that overexpression of IBN induces TNF-–dependent apoptosis by efficient and specific suppression of NF-B and upregulation of caspase-3 and caspase-2 activity in human VSMCs. Our findings suggest that adenovirus-mediated IBN gene transfer may be useful in the treatment of disorders associated with inflammatory conditions, such as the response to vascular injury and atherosclerosis.

Key Words: apoptosis • nuclear factor-B • inhibitory-B • tumor necrosis factor- • vascular smooth muscle cells

	Introduction

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Apoptosis has been reported to be involved in human atherosclerosis and experimental models of vascular injury but virtually absent in normal vessels, suggesting that it plays a part in the pathophysiological mechanisms of vessel injury.^{1 2 3 4 5} Apoptosis also occurs frequently in proliferative lesions, particularly restenosis lesions,^{3 5} and its presence is strongly correlated with intimal hyperplasia.³ The temporal sequence of proliferation and apoptosis in experimental models is consistent with a role for apoptosis in the control of neointimal cellularity¹ ; therefore, apoptosis in vascular smooth muscle cells (VSMCs) has been proposed to be importantly involved in the control of neointimal thickening.⁵ However, little is known about the mechanisms that control VSMC apoptosis.

The development of intimal hyperplasia is marked by a considerable inflammatory infiltrate, with cells of monocyte lineage being the most abundant.^{6 7} In response to vascular injury, proinflammatory cytokines, such as tumor necrosis factor- (TNF-) and interleukin-1ß (IL-1ß), are produced by activated macrophages as well as by VSMCs themselves, and these cytokines can regulate gene expression, differentiation, and growth of VSMCs in vitro and in vivo.^{8 9 10} TNF- is a pleiotropic cytokine that is expressed abundantly in atherosclerotic lesions.¹¹ Although ligand binding of TNF- receptors usually triggers cellular apoptosis, there is substantial evidence that TNF- itself has little effect on the apoptosis or growth of VSMCs.^{12 13} Thus, the relation between TNF- and VSMC growth and apoptosis remains obscure.

Examinations of the signaling pathways distal to TNF- receptor activation have indicated that, in addition to proapoptotic cascades, TNF- also engages pathways that activate the transcription factor nuclear factor-B (NF-B). NF-B has been implicated in atherosclerosis because activated NF-B is present in human atherosclerotic lesions¹⁴ but not in normal vessels.¹⁵ In a rat model of arterial injury, NF-B activity was induced and the protein expression of NF-B family members was upregulated at the time of rapid proliferation of SMCs and neointima formation after a balloon catheter–induced injury.¹⁶

Activation of cytoplasmic NF-B requires the degradation of an inhibitor protein, IB, which traps NF-B in the cytoplasm. In response to signals, IB is phosphorylated on 2 serine residues in its NH₂-terminal regulatory domain, serines 32 and 36, and degraded.^{17 18 19} To inhibit NF-B activation, we constructed a recombinant adenovirus vector expressing the nondegraded form of the NF-B inhibitor IB (AdexIBN), in which the 54 NH₂-terminal amino acids containing the phosphorylation sites essential for the activation of NF-B were deleted.¹⁸ To clarify the role of NF-B in TNF-–dependent apoptosis in VSMCs, we overexpressed this IBN in human VSMCs and examined them for TNF-–dependent apoptosis.

To provide additional insight into the regulatory mechanism of apoptosis, it is important to study the effects of NF-B on the caspase cascade. In investigations with the HT1080 fibrosarcoma cell line, NF-B activation was found to suppress TNF-–induced apoptosis by blocking activation of caspase-8.²⁰ Because caspase-3 is an effector of the caspase cascade and is located downstream of caspase-8, we examined the effect of IBN overexpression on TNF-–induced caspase-3 and caspase-2, members of the caspase-3 family, activity in VSMCs.

	Methods

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Materials
Dulbecco’s Modified Eagle Medium (DMEM) and fetal-calf serum (FCS) were obtained from Gibco. Insulin-transferrin-selenite supplement was obtained from Sigma Chemical Co. A selective inhibitor of the cysteine protease protein-32/caspase-3 subfamily, Z-Asp-(OMe)-Glu-(OMe)-Val-Asp-(OMe)-fmk (Z-DEVD-fmk), was obtained from Calbiochem. Recombinant human TNF-, an ELISA that detects cell death, and a protease-inhibitor mixture were obtained from Boehringer Mannheim. Recombinant human IL-1ß was purchased from Genzyme. A kit for assaying caspase-3 cellular activity was obtained from Biomol, and a caspase-2/IL-1ß–converting enzyme and ced-3 homolog-1 (ICH-1) colorimetric protease assay kit was purchased from Medical and Biological Laboratories.

Cell Culture
Human aortic SMCs were obtained from Cell Systems and cultured in DMEM with antibiotics and 10% FCS. Cells were cultured at 37°C in humidified air with 5% CO₂ and changes of medium every 2 days. These cells showed typical hill-and-valley morphological features on phase-contrast microscopy. Cells between passages 5 and 10 were used for all experiments.

Recombinant Adenovirus Vectors
We constructed a recombinant adenovirus vector (Adex) expressing the nondegraded form of the NF-B inhibitor IB (Adex1CAKT IBN; abbreviated AdexIBN) as previously described.^{21 22} This IBN lacks the 54 NH₂-terminal amino acids present in wild-type human IB (MAD3). It has been reported to be neither phosphorylated nor proteolyzed in response to signal induction but to fully inhibit NF-B.¹⁸ A modified method (cosmid-terminal-protein complex method), provided by Dr I. Saito (Laboratory of Molecular Genetics, Institute of Medical Science, University of Tokyo, Tokyo, Japan),²³ was used to construct the adenovirus vector. Purified virus stocks were prepared by CsCl step-gradient centrifugation, as previously described.²⁴ Recombinant lacZ adenovirus (AdexlacZ), which contains the CAG promotor, lacZ gene, and poly A signal sequences, was used as a control vector; it was supplied by Dr I. Saito.²⁵

Transfection of lacZ or IBN Gene With Adex-Polyethylenimine Complexes
Mediated gene transfer with Adex-polyethylenimine (PEI; molecular weight, 25 000; Aldrich) was performed with a modification of a previously described technique.²⁶ For formation of the Adex-PEI complexes, AdexlacZ or AdexIBN was incubated with PEI diluted in 20 mmol/L HEPES (pH 7.4) at a concentration of 0.5 mmol/L for 30 minutes before it was added to the VSMCs. Selected amounts of this mixture were then diluted in DMEM without FCS and added to the VSMCs (cultured to 60% confluence) at a multiplicity of infection (MOI) of 10. After 1 hour at 37°C, the mixture was removed, and fresh medium containing 1.5% FCS and insulin-transferrin-selenite supplement was added. Twenty-four hours after infection, cells were treated with TNF- (1 to 100 ng/mL), IL-1ß (10 ng/mL), or a combination of TNF- (10 ng/mL) and IL-1ß (10 ng/mL).

Detection of lacZ Expression
Forty-eight hours after AdexlacZ infection, expression of the ß-galactosidase transgene was determined by staining the cells with 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-gal) solution for 1 hour as described previously.²⁶ Blue staining of the cells was viewed with use of phase-contrast microscopy.

Western Blot Analysis of IB
VSMCs infected with AdexlacZ or AdexIBN were stimulated with TNF- (10 ng/mL) for various periods of time, and the cytoplasmic extract of the VSMCs was prepared as previously described.²² Cytoplasmic extracts (10 µg of protein) were separated on 10% polyacrylamide-SDS gels and transferred to polyvinylidene fluoride membranes. Membranes were incubated at room temperature for 1 hour in blocking buffer (5% low-fat milk powder in Tris-buffered saline) and then overnight in PBS containing the primary antibody rabbit anti-IB (C-21; Santa Cruz Biotechnology, Inc) at 1:50 dilutions. This antibody recognizes the C-terminal domain of IB. After being washed in Tris-buffered saline containing 0.08% Tween-20, the membranes were incubated for 1 hour at 25°C in diluting buffer containing a 1:1500 dilution of alkaline phosphatase–conjugated secondary antibody (Tago, Inc). After being washed, the bands corresponding to IB and IBN were visualized with the use of an alkaline phosphatase substrate kit (Vector Laboratories, Inc).

Preparation of Nuclear Extracts
VSMCs were infected with AdexIBN or AdexlacZ for 24 hours before treatment with TNF- (10 to 100 ng/mL), IL-1ß (10 ng/mL), or a combination of TNF- (10 ng/mL) and IL-1ß (10 ng/mL). Nuclear proteins were isolated by using the method of Selzman et al.²⁷ In brief, confluent VSMCs (10⁶ cells) were treated with the experimental agents for 30 minutes, after which the medium was aspirated and the cells were washed gently on ice with 2 mL of cold PBS. The cells were then scraped into 0.5 mL of cold, hypotonic buffer containing 50 mmol/L Tris, 100 mmol/L NaCl, 2 mmol/L EDTA, 2 mmol/L EGTA, 1 mmol/L DTT, and tablets of protease-inhibitor mixture plus 0.6% NP-40; allowed to swell on ice for 30 minutes; and spun vigorously to obtain lysis. After centrifugation for 15 minutes, the pellet was washed with an additional 0.5 mL of cold buffer and respun. The resulting nuclear pellet was resuspended in 100 µL of ice-cold buffer containing 20 mmol/L HEPES (pH 7.9), 1 mmol/L EGTA, 1 mmol/L DTT, 0.4 mol/L NaCl, and protease-inhibitor tablets and shaken occasionally for 30 minutes at 4°C. The nuclear extract was centrifuged for 5 minutes at 12 000g at 4°C, and the supernatant was collected and stored at -70°C.²⁷ Protein concentrations were determined by using the Lowry assay with the Bio-Rad DC protein-assay dye reagent (Bio-Rad Laboratories).

Fluorescent Electrophoretic Mobility Shift Assay
The cells were washed twice with ice-cold PBS, and DNA-binding NF-B activity in nuclear extracts was determined by electrophoretic mobility shift assay (EMSA). Cells were stimulated for 30 minutes, and nuclear extracts were prepared as described above. The sequence of the consensus double-stranded oligonucleotide (Promega) used to detect the DNA-binding activity of NF-B was 5'-AGTTGAGGGGACTTTCCCAGGC-3'. The sequence of the mutant oligonucleotide used for the competition assay was 5'-AGTTGAGCCGACTTTTACAGGC-3'. A fluorescent oligonucleotide labeling kit (Vistra 5'-Oligolabeling kit, Amersham Life Science) was used to label the 5' end of consensus oligonucleotides with fluorescein. Labeled oligonucleotide (1.4 pmol) was incubated with 10 µg of nuclear extract for 30 minutes on ice; before loading, 25% glycerol was added. To demonstrate the specificity of DNA-protein binding, the binding reactions were performed in the presence of a 5-fold and 30-fold excess of unlabeled consensus oligonucleotide competitor or a 30-fold excess of unlabeled mutant oligonucleotide competitor. The samples were resolved on 6% polyacrylamide gels. Gels were viewed directly with a FluorImager (Molecular Dynamics).

Transfection and Luciferase Assays
To assess NF-B activity, luciferase reporter constructs containing the minimal promoter with 3 tandem NF-B–binding sites (pNF-B-Luc, Clontech) or nonresponsive vectors (pTAL-Luc, Clontech) were used. VSMCs cultured in 12-well plates were transfected with 0.5 µg of pNF-B-Luc or 0.5 µg of pTAL-Luc (control vector) with the lipofection method by using 3 µL of FuGENE (Boehringer Mannheim). Cotransfection with 0.5 µg of pRL-TK vector (Promega) was used in all experiments to normalize transfection efficiency. Twelve hours after transfection, the cells were washed in PBS and incubated in DMEM with 1.5% FCS for 12 hours. Subsequently, the cells were infected with AdexlacZ or AdexIBN. Twenty-four hours after infection, cells were exposed to TNF- (10 to 100 ng/mL), IL-1ß (10 ng/mL), or a combination of TNF- (10 ng/mL) and IL-1ß (10 ng/mL) for 1 hour. Cells were then lysed, and luciferase activities were measured by using the dual-luciferase reporter assay system (Promega).

Nuclear Morphological Features and Quantification of Apoptosis
AdexlacZ-infected and AdexIBN-infected VSMCs were incubated with or without TNF- (10 ng/mL) for 24 hours. Adherent cells were stained with Hoechst-33258, and the nuclei were viewed under a fluorescence microscope as previously described.²² Quantitative analysis of apoptosis based on morphological changes was then performed. The ratio of apoptotic cells was calculated as the proportion of nuclei in each well that had undergone apoptosis.

Detection of DNA Fragmentation
AdexlacZ-infected and AdexIBN-infected VSMCs were incubated with TNF- (1 to 100 ng/mL), IL-1ß (10 ng/mL), or a combination of TNF- (10 ng/mL) and IL-1ß (10 ng/mL) for 24 hours. Cytosolic oligonucleosome-bound DNA was quantified by using an ELISA kit with a primary anti-histone antibody and a secondary anti-DNA antibody coupled to peroxidase.

Caspase-3 and Caspase-2 Activity
For detection of caspase-3 activity, 10⁶ cells were lysed in buffer (50 mmol/L HEPES, [pH 7.4], 100 mmol/L NaCl, 0.1% CHAPS, 0.1% NP-40, 1 mmol/L DTT, and 0.1 mmol/L EDTA) for 5 minutes at 4°C and then centrifuged at 10 000g for 10 minutes. The supernatant was stored at -70°C. Protein content was analyzed by using the Bio-Rad DC protein-assay dye reagent (Bio-Rad Laboratories). The activity of caspase-3 was assayed according to the instructions provided by the manufacturer of the assay (Biomol). For detection of caspase-2 activity, 2x10⁶ cells were lysed in cell-lysis buffer and centrifuged at 10 000g for 1 minute. The activity of caspase-2 was detected with an assay kit (Medical and Biological Laboratories) by following the manufacturer’s instructions.

Statistical Analysis
Multiple comparisons were evaluated by ANOVA followed by Scheffé’s test. Results are expressed as mean±SEM; P<0.05 was considered to represent statistical significance.

	Results

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Determination of Transfection Efficiency
Studies have shown that polycations increase the efficiency of adenovirus-mediated gene transfer to several cell lines.^{26 28 29} To evaluate the efficiency of adenovirus-mediated gene transfer, VSMCs were incubated with AdexlacZ for 1 hour at 37°C at an MOI of 10 in the presence or absence of PEI. After 48 hours, ß-galactosidase expression was determined by X-gal staining. As shown in Figure 1, AdexlacZ infection alone resulted in lacZ expression in <10% of the cells, whereas adsorption in the presence of PEI increased the percentage of cells expressing the transgene to 100% of VSMCs. We therefore used an MOI of 10 with PEI in all subsequent experiments.

View larger version (69K): [in this window] [in a new window]   Figure 1. Gene-transfer efficiency mediated by Adex-PEI complexes. VSMCs were grown to 90% confluence, infected with AdexlacZ at an MOI of 10 in the absence (A) or presence (B) of PEI, and stained with X-gal. Original magnification, x100.

Overexpression of IBN by Adenovirus Vectors
The expression of IB proteins in cytoplasmic extracts of VSMCs was detected by Western blotting. As shown in Figure 2A, IBN protein expression appeared within 6 hours and was observed for >48 hours in AdexIBN-infected cells. It was detected by the anti-IB (C-21) antibody, which recognizes the C-terminal domain of IB. As shown in Figure 2B, after treatment with TNF-, wild-type IB degraded rapidly, within 15 minutes. In contrast, IBN protein was not degraded by TNF-.

View larger version (33K): [in this window] [in a new window]   Figure 2. Detection of overexpression of IBN protein by Western blot assessment. A, Time course of IBN expression after adenovirus infection. VSMCs were infected with AdexIBN, and after the times indicated, cytoplasmic proteins were extracted. The Western blots contained equal amounts of cytoplasmic extracts in each lane (10 µg). The results were obtained with anti-IB (C-21) antibody, which recognizes the C-terminal domain of IB. Representative examples of results from 3 independent experiments are shown. B, Signal-induced proteolysis of wild-type IB and overexpression of adenovirus-mediated IBN. Twenty-four hours after infection with AdexlacZ or AdexIBN, cells were incubated with TNF- (10 ng/mL). At the times indicated, cytoplasmic proteins were extracted and analyzed by Western blotting with anti-IB (C-21) antibody. Representative examples of results from 3 independent experiments are shown.

Decreased Activation of NF-B by Overexpression of IBN
DNA-binding activity of NF-B was strongly activated by TNF-, IL-1ß, or a combination of TNF- and IL-1ß in the nuclear extracts of AdexlacZ-infected cells, but the NF-B activity induced by these cytokines was reduced by overexpression of IBN (Figure 3A). To determine the specificity of binding of the NF-B oligonucleotide, the binding reactions were performed in the presence of a 5- and 30-fold excess of unlabeled consensus oligonucleotide competitor or a 30-fold excess of unlabeled mutant oligonucleotide competitor. The NF-B DNA band was reduced by the 30-fold excess of unlabeled consensus oligonucleotide, but it was not competed out by the unlabeled mutant oligonucleotide (data not shown).

View larger version (35K): [in this window] [in a new window]   Figure 3. Fluorescent EMSA of NF-B (A) and assay of NF-B–induced luciferase activity (B). A, Binding activity to an oligonucleotide containing a consensus NF-B motif was assayed in nuclear extracts of VSMCs infected with AdexlacZ or AdexIBN. Twenty-four hours after adenovirus infection, the cells were incubated with TNF- (10 to 100 ng/mL), IL-1ß (10 ng/mL), or a combination of TNF- (10 ng/mL) and IL-1ß (10 ng/mL) for 30 minutes, and nuclear extracts were prepared and analyzed by fluorescent EMSA. Representative examples of results from 3 independent experiments are shown. B, VSMCs were transfected with pNF-B-Luc by using the lipofection method. Twelve hours after transfection, cells were infected with AdexlacZ or AdexIBN and then exposed to TNF- (10 to 100 ng/mL), IL-1ß (10 ng/mL), or a combination of TNF- (10 ng/mL) and IL-1ß (10 ng/mL) for 1 hour. Cotransfection with pRL-TK vector was used in all experiments to normalize transfection efficiency. Luciferase activity was measured with the dual-luciferase reporter assay system and reported as relative luciferase activity compared with that in untreated AdexlacZ-infected cells. Values are mean±SEM (n=4). *P<0.05 compared with untreated AdexlacZ-infected cells.

To confirm the inhibitory effect of IBN on NF-B activity, we assayed NF-B–induced luciferase activity. As shown in Figure 3B, treatment of AdexlacZ-infected VSMCs with TNF-, IL-1ß, or a combination of TNF- and IL-1ß significantly induced NF-B–driven luciferase activity, and this cytokine-induced activation of NF-B was markedly suppressed by overexpression of IBN. Furthermore, the NF-B–driven luciferase activity in untreated AdexlacZ-infected cells, which showed basal NF-B activity, was abolished by overexpression of IBN (Figure 3B). There were no significant effects of cytokine stimulation on luciferase activity in cells transfected with pTAL-Luc (control vector), suggesting that the cytokine-induced activation of NF-B-Luc reporter activity was dependent on the intact NF-B–binding motifs.

Effect of Overexpression of IBN on TNF-–Dependent Apoptosis
Neither untreated AdexIBN-infected cells nor untreated AdexlacZ-infected cells showed any morphological changes (data not shown). In contrast, TNF- treatment induced a marked increase in indications of cell death in AdexIBN-infected cells but not in AdexlacZ-infected cells. As shown in Figure 4A, 24 hours after TNF- treatment, the AdexIBN-infected cells had a substantial increase in the extent of typical apoptotic chromatin condensation and fragmentation. In AdexIBN-infected cells treated with TNF-, the ratio of apoptotic cells was significantly increased compared with that in AdexlacZ-infected cells (Figure 4B). In addition, at 24 hours, overexpression of IBN significantly induced TNF-–mediated DNA fragmentation compared with mock transfection (Figure 5A). As shown in Figure 5B, a combination of TNF- and IL-1ß also induced DNA fragmentation in AdexIBN-infected cells, but there was no significant difference compared with TNF- alone. Notably, IL-1ß alone had no effect in AdexIBN-infected cells. Thus, infection with AdexIBN resulted in a marked induction of apoptosis in TNF-–stimulated VSMCs, whereas unstimulated VSMCs were barely affected by infection.

View larger version (31K): [in this window] [in a new window]   Figure 4. Morphological effects of overexpression of IBN on TNF-–dependent apoptosis (A) and quantification of apoptosis (B). A, AdexIBN-infected VSMCs were incubated with TNF- (10 ng/mL). Twenty-four hours later, adherent cells were observed under a fluorescence microscope after the nuclei were stained with Hoechst-33258. Original magnification x400. B, On the basis of the results obtained with Hoechst-33258 staining, the ratio of apoptotic cells was calculated as the proportion of nuclei that had undergone apoptosis. Values are mean±SEM of 5 independent experiments. *P<0.05 compared with untreated AdexlacZ-infected cells.

View larger version (33K): [in this window] [in a new window]   Figure 5. Effects of overexpression of IBN on TNF-–dependent DNA fragmentation. A, AdexlacZ-infected and AdexIBN-infected VSMCs were incubated with or without TNF- (1 to 100 ng/mL). Twenty-four hours later, DNA fragmentation was quantified by ELISA. Results were expressed as histone-associated DNA fragmentation relative to that in untreated AdexlacZ-infected cells, normalized to 100% (y axis in Figures 5A and B). Values are mean±SEM (n=4). *P<0.05 compared with untreated AdexlacZ-infected cells. B, AdexlacZ-infected and AdexIBN-infected VSMCs were incubated with TNF- (10 ng/mL), IL-1ß (10 ng/mL), or a combination of TNF- (10 ng/mL) and IL-1ß (10 ng/mL) for 24 hours, and DNA fragmentation was quantified by ELISA. Values are mean±SEM (n=4). *P<0.05 compared with untreated AdexlacZ-infected cells.

We also measured the lactate dehydrogenase activity in the supernatant to investigate the toxicological effect of AdexIBN on VSMCs. In comparison with the untreated control (5.7±2.94 IU/L), neither AdexlacZ-PEI alone (6.8±1.32 IU/L) nor AdexIBN-PEI alone (5.5±1.05 IU/L) caused a significant increase in lactate dehydrogenase activity in the absence of TNF-. This finding suggests that AdexIBN transfection with PEI has little toxicological effect on VSMCs.

Role of Caspase-3 and Caspase-2 Activation in the Effect of Overexpression of IBN on TNF-–Dependent Apoptosis
To investigate the involvement of caspases in TNF--induced apoptosis, we examined the effect of the caspase-3 family inhibitor Z-DEVD-fmk on TNF--induced DNA fragmentation in AdexIBN-infected VSMCs. AdexIBN-infected cells were pretreated with Z-DEVD-fmk for 1 hour before and during treatment with TNF- (10 ng/mL), and DNA fragmentation was detected by ELISA 24 hours later. We found that Z-DEVD-fmk (50 µmol/L) inhibited TNF-–induced DNA fragmentation by 52% in AdexIBN-infected cells. We then examined the effect of overexpression of IBN on TNF-–induced caspase-3–like and caspase-2–like activity. Caspase-3–like activity was detected beginning 8 hours after TNF- treatment. Caspase-3 was not activated by overexpression of AdexlacZ, but overexpression of IBN significantly induced TNF-–mediated caspase-3–like activity in VSMCs (Figure 6A). Overexpression of IBN also significantly induced TNF-–mediated caspase-2–like activity beginning 8 hours after the treatment (Figure 6B).

View larger version (15K): [in this window] [in a new window]   Figure 6. Time course of caspase-3–like and caspase-2–like activity after TNF- (10 ng/mL) treatment. Cytoplasmic proteins were extracted at the times indicated, and caspase-3 (A) or caspase-2 (B) activity was measured. Open circles indicate activity in AdexlacZ-infected cells; solid circles indicate activity in IBN-infected cells. Values are mean±SEM (n=4). *P<0.05 compared with AdexlacZ-infected cells at each time point.

	Discussion

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In this study, we found that inhibition of NF-B by IBN overexpression induced TNF-–dependent apoptosis in human VSMCs and that augmentation of caspase-3 and caspase-2 activity is involved in that induction. NF-B has been proposed to play an essential role in protection against TNF-–induced cell death in different cell types.^{30 31 32} The antiapoptotic genes encoding the inhibitor of apoptosis (IAP) proteins c-IAP1 and c-IAP2 regulated by NF-B have been identified.²⁰ X-chromosome–linked IAP is an NF-B–regulated protein that prevents endothelial cells from undergoing TNF-–induced apoptosis and can protect against apoptosis by inhibiting cell-death caspases.³³ These findings support the possibility that caspases are related to the NF-B signaling pathway. Wang et al²⁰ showed that activation of NF-B in a human fibrosarcoma cell line blocked activation of caspase-8 (which is located at the apex of the caspase pathway) and resulted in inhibition of caspase-3 processing. However, these phenomena may be cell-type specific, and the effect of NF-B on TNF-–induced apoptosis and caspase-3 activation in human VSMCs remains to be clarified. In this study, we found that inhibition of NF-B activation induced TNF-–mediated caspase-3 activity in human VSMCs.

To evaluate the role of NF-B in the regulation of apoptosis, a specific NF-B inhibition system is required. Recombinant adenoviruses have been evaluated extensively for use in such a system because of their relatively high infection efficiency and ability to drive expression of a foreign gene in quiescent cells. We therefore constructed a recombinant adenovirus vector that expresses an NH₂ terminally–deleted form of IB, which is resistant to ubiquitination-based degradation and prevents activation of NF-B. The IBN protein was overexpressed by adenoviral infection with PEI and was not degraded, despite TNF- signal induction. Furthermore, to investigate the inhibitory effect of overexpression of IBN on TNF-–induced NF-B activation, we performed EMSA and assays of NF-B–induced luciferase activity. Our results demonstrate that overexpression of IBN by adenoviral transfer specifically and effectively inhibits TNF-–induced NF-B activation. As shown in Figure 5A, TNF- alone did not increase DNA fragmentation at 24 hours. This finding suggests that inhibition of NF-B activation does not simply accelerate TNF-–dependent apoptosis but turns on a death signal that cannot be activated by TNF- alone. In other words, TNF- itself is not sufficient to trigger apoptosis.

To assess the effectiveness of IBN in the induction of apoptosis after inhibition of NF-B activation in response to other cytokines (used alone or combined) in VSMCs, we examined the effect of IL-1ß, which plays key regulatory roles in response to vascular injury, as well as the effect of TNF- and a combination of TNF- and IL-1ß, under the same experimental conditions. In contrast to TNF-, IL-1ß had no significant effect on DNA fragmentation in AdexIBN-infected VSMCs, although NF-B activation by IL-1ß was significantly suppressed in AdexIBN-infected VSMCs analyzed by EMSA and luciferase assay. Moreover, overexpression of IBN also suppressed NF-B activation and induced DNA fragmentation in VSMCs treated with TNF- in the presence of IL-1ß, as well as in cells treated with TNF- alone. These results suggest that the induction of apoptosis by IBN overexpression may be a phenomenon specific to TNF-.

The presence of an antiapoptotic effect of compounds that inhibit activation of the cysteine protease protein-32/caspase-3 family suggests that apoptosis can be regulated by modification of the caspase cascade. Dimmeler et al³⁴ found that NO inhibits apoptosis by preventing an increase in caspase-3–like activity. In the current study, we showed that DNA fragmentation induced by TNF- was suppressed by an inhibitor of the caspase-3 family and that overexpression of IBN significantly induced the TNF-–mediated caspase-3 and caspase-2 activity. These results suggest that induction of TNF-–mediated caspase-3 or caspase-2 activity is a candidate for the mechanism underlying the sensitization to apoptosis produced by overexpression of IBN.

Our study found that adenovirus-mediated overexpression of a truncated form of IB induced TNF-–dependent apoptosis in human VSMCs, but the in vivo relevance of the data are difficult to ascertain. Sata et al³⁵ reported that Fas-ligand gene transfer to vessel walls suppressed neointimal lesion formation (Fas ligand induces apoptosis in Fas-bearing VSMCs). Selzman et al²⁷ showed that NF-B activation is essential for TNF-–induced VSMC proliferation, which is associated with the release of IL-6. Thus, the induction of VSMC apoptosis may act concurrently with the inhibition of cell proliferation in preventing neointima formation, as has been proposed in studies with experimental models.³⁶ Erl et al³⁷ reported that inhibition of NF-B by adenovirus-mediated overexpression of IB caused a marked increase in cell death at a low cell density but not at a high cell density. Therefore, overexpression of IBN may reduce excessive VSMC proliferation and have therapeutic value in inhibiting neointima formation after angioplasty and arterial injury. During neointima formation, arterial SMCs migrate from the tunica media to the intima, where they proliferate and secrete a variety of extracellular matrix proteins and cytokines that contribute to focal thickening of the intima. In this situation, a high degree of apoptosis may be necessary to limit excessive cell replication and permit high cell turnover in vessels affected by intimal hyperplasia. On the other hand, loss of SMCs in the fibrous cap of atherosclerotic lesions may predispose those lesions to plaque instability and initiate acute coronary artery events.³⁸ Because of these observations, the possible role of the induction of VSMC apoptosis in the pathophysiological mechanisms of atherosclerosis deserves further study.

In summary, we found that adenovirus-mediated overexpression of a truncated form of IB induces TNF-–dependent apoptosis in human VSMCs by means of an efficient and specific suppression of NF-B and upregulation of caspase-3– and caspase-2–like activity. Because inhibition of VSMC apoptosis by inflammatory cytokines plays a key role in the progression of atheromatous lesions, our results may provide a rationale for using adenovirus-mediated IBN gene transfer to treat atherosclerosis or other vascular injury.

	Acknowledgments

 
This study was supported in part by a Grant-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan and a grant from the Japan Foundation of Cardiovascular Research.

	Footnotes

 
Reprint requests to Kenji Matsumoto, MD, Department of Surgery, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan.

Received December 10, 1999; accepted July 18, 2000.

	References

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