Regression of Abdominal Aortic Aneurysms by Simultaneous Inhibition of Nuclear Factor κB and Ets in a Rabbit Model
Because current therapy to treat abdominal aortic aneurysm (AAA), and particularly to manage small AAA, is limited to elective surgical repair, we explored less invasive molecular therapy by simultaneous inhibition of the transcription factors nuclear factor (NF)κB and ets using a decoy strategy. Both NFκB and ets were shown to be markedly activated in human AAA. In addition, NFκB- and ets-positive cells were increased in the aneurysm wall, and a part of the expression of NFκB and ets was detected in migrating macrophages. Thus, we used chimeric decoy oligodeoxynucleotides (ODNs) containing consensus sequences of both NFκB and ets binding sites to treat AAA. Inhibitory effects of chimeric decoy ODNs on matrix metalloproteinase-1 and -9 expression were confirmed by ex vivo experiments using a human aorta organ culture. To examine the regressive effect in a rabbit already-formed AAA model, transfection by wrapping a delivery sheet containing chimeric decoy ODNs around the aneurysm was performed 1 week after incubation with elastase. Importantly, treatment with chimeric decoy ODNs significantly decreased the size of AAA. Interestingly, significant preservation of elastic fibers was observed with chimeric decoy ODN treatment, accompanied by a reduction of matrix metalloproteinase-2 and -9 and induction of macrophage apoptosis. Regression of AAA was also associated with an increase in elastin and collagen type I and III synthesis in the aneurysm wall. Minimally invasive molecular therapy targeted to the inhibition of NFκB and ets is expected to be useful for AAA through the rebalance of matrix synthesis and degradation.
Abdominal aortic aneurysm (AAA) is a common degenerative condition associated with aging and atherosclerosis. It is characterized by aortic wall inflammation, destruction of medial elastin, and expression of matrix metalloproteinases (MMPs).1 Elective surgical repair or stent grafting is an effective approach to prevent death from AAA rupture. However, there is a conspicuous absence of alternative therapeutic strategies for this disease.2 Particularly, despite gradual expansion, small AAAs have a low risk of rupture, and there is currently no well-defined treatment strategy for them. Therefore, the development of a novel therapeutic approach to regress AAA in the clinical setting would be useful.
Aneurysm development involves a complex remodeling process with an imbalance between the synthesis and degradation of connective tissue proteins.2 Elastin and collagens are the major structural components of the aortic wall. Elastic fibers maintain the structure of the vascular wall against hemodynamic stress, resulting in prevention of aortic dilatation. In contrast, collagens are responsible for tensile strength and prevent aneurysm rupture.3 Various extracellular proteinases participate in the process of destruction of the human aortic wall; in particular, MMPs secreted by migrating inflammatory cells are considered to be the predominant proteinases.2,4 Therefore, suppression of MMP activities is thought to be a useful strategy to treat AAA. Indeed, treatment with an MMP inhibitor or antiinflammatory agent is useful to inhibit the progression of AAA5,6 but is not sufficient to achieve the regression of AAA. Not only the suppression of MMP activities but also the production of connective tissue proteins is expected to be necessary for repair of the aneurysm wall.
To develop a new therapeutic approach, we focused on the transcription factors nuclear factor (NF)κB and ets. Both transcription factors exert potent regulatory control of the production and degradation of extracellular matrix. NFκB is well known to mediate inflammatory changes and also to regulate the transcription of MMP-1, MMP-2, MMP-3, and MMP-9.7–9 Furthermore, recent studies have demonstrated that NFκB inhibits transcription of the elastin and collagen genes, leading to suppression of their synthesis.10,11 In contrast, members of the ets family, including MMP-1, MMP-2, and MMP-9, also play important roles in regulating gene expression in response to multiple developmental and mitogenic signals.12–14 From this viewpoint, we used chimeric decoy oligodeoxynucleotides (ODNs) to inhibit both NFκB and ets simultaneously, leading to the inhibition of a wide variety of MMPs, including MMP-1, MMP-2, MMP-3, and MMP-9, which play a pivotal role in human AAA. Indeed, our previous studies successfully inhibited the progression of AAA in rat and rabbit models.15,16 However, for human therapy, treatment should regress the AAA rather than prevent the progression of AAA. To achieve this goal, the present study examined the effects of chimeric decoy ODNs to induce regression of AAA in a rabbit model.
Materials and Methods
An expanded Materials and Methods section is in the online data supplement at http://circres.ahajournals.org.
Synthesis of ODNs and Selection of Target Sequences
The following sequences of phosphorothioate ODNs were used (consensus sequences are underlined):
Chimeric decoy ODNs: 5′-ACCGGAAGTATGAGGGATTTCCCTCC-3′; 3′-TGGCCTTCATACTCCCTAAAGGGAGG-5′
Scrambled chimeric decoy ODNs: 5′-TCGAGCATATACGTGACTGCGCTCAG-3′; 3′-AGCTCGTATATGCACTGACGCGAGTC-5′
NFκB decoy ODNs: 5′-CCTTGAAGGGATTTCCCTCC-3′; 3′-GGAACTTCCCTAAAGGGAGG-5′
ets-1 decoy ODNs: 5′-AATTCACCGGAAGTATTCGA-3′; 3′-TTAAGTGGCCTTCATAAGCT-5′
Ex Vivo Organ Culture
Aortic specimens were obtained from patients with AAA at the time of open surgical repair. Immediately, each specimen was divided into 2-mm2 segments of full-thickness aortic wall, followed by incubation in 20% collagen gel in DMEM containing decoy ODNs for 1 hour at 4°C. Then, they were placed in separate 24-well tissue culture plates with 500 μL of DMEM and incubated for 48 hours. The conditioned medium was collected, and MMP-1 and MMP-9 were measured using ELISA (Amersham). This study was performed with written informed consent.
Electrophoretic Mobility-Shift Assay
Nuclear extracts were prepared from the neck and the most dilated part of human harvested aortic aneurysms, and electrophoretic mobility-shift assay was performed to analyze the expression of NFκB and ets. ODNs containing the NFκB binding site (5′-CCTTGAAGGGATTTCCCTCC-3′; only sense strand is shown) or ets binding site (5′-GTGCCGGGGTAGGAAGTGGGCTGGG-3′) were used as primers.
Transfection of Decoy ODNs After Formation of AAA
Male Japanese white rabbits (Kitayama Labes, Nagano, Japan) were anesthetized, and the infrarenal abdominal aorta was exposed via an extraperitoneal approach. Aortic dilatation was induced by incubation of the abdominal aorta with type I porcine pancreatic elastase for 2 hours. One week after incubation with elastase, dilatation of the aorta was confirmed by ultrasonography. Then, the AAA was carefully isolated again via an extraperitoneal approach, and transfection of decoy ODNs was performed using a delivery sheet containing decoy ODNs, without the use of viral vectors or other delivery techniques.15 This delivery sheet was wrapped around the aneurysm; it immediately changes to a gel in wet conditions to allow incubation with naked ODNs. Animals were divided into 6 groups: (1) control, (2) treatment with scrambled decoy ODNs, (3) treatment with NFκB decoy ODNs (1000 nmol), (4) treatment with ets decoy ODNs (1000 nmol), and treatment with (5) low-dose (200 nmol) and (6) high-dose (1000 nmol) chimeric decoy ODNs. Blood samples were taken before and 4 weeks after incubation, and laboratory parameters were measured with a standard autoanalyzer. This study was performed under the supervision of the Animal Research Committee in accordance with the Guidelines on Animal Experiments of Osaka University Medical School and the Japanese Government Animal Protection and Management Law (No. 105).
Ultrasound and Angiographic Studies
Ultrasonography and angiography were used to assess dilatation of the abdominal aorta.
Histological and Immunohistochemical Studies
Aortic tissue cross-sections of rabbits were stained with elastic van Gieson stain in a standard manner at 4 weeks after operation. Immunohistochemical staining was performed using the immunoperoxidase avidin–biotin complex method. Cross-sections of human AAA tissues were stained with rabbit polyclonal antibodies against NFκB and ets-1 (Santa Cruz Biotechnology). A mouse monoclonal antibody against rabbit macrophages (Dako) was applied to paraffin sections at 1 week after transfection in rabbit experimental AAA. Double-immunofluorescent staining was performed to identify the expression of NFκB and ets in macrophages in human AAA tissues and the expression of caspase-3 (Santa Cruz Biotechnology) in macrophages in rabbit tissues.
In Situ End Labeling of Fragment DNA: TUNEL
To evaluate apoptotic activity of macrophages at 1 week after transfection in the rabbit aneurysm wall, we performed double staining with TUNEL and immunofluorescent staining for macrophages.
MMP expression in the rabbit aneurysm wall was assessed by SDS-PAGE gelatin zymography at 1 week after transfection.
Protein extracts of rabbit AAA tissue were examined by Western blot using mouse monoclonal antibodies against tropoelastin (1:500; Elastin Products Co) and tubulin.
Picrosirius Red Staining
To determine the collagen type I and III content in the rabbit aneurysm wall at 3 weeks after transfection, cross-sections were stained with picrosirius red.
Total RNA was extracted from the rabbit aneurysm wall at 1 week after transfection, and quantitative real-time RT-PCR was performed. Primers based on the published sequences for rabbit collagen I, collagen III, and GAPDH were used. Primers for elastin were selected from gene sequences that were homologous between human and mouse. Primers for rabbit elastin (sense, 5′-CCTGACTCACGACCTCATCA-3′; antisense, 5′-CAGGTGCTTGGGTACCAACT-3′; 425 bp) were designed using Primer3. PCR products were sequenced and confirmed to be highly homologous as compared with selected sequences of the elastin gene.
All values are expressed as means±SEM. For statistical analysis, a paired t test was used for comparison between 2 groups. One-way ANOVA and Tukey–Kramer multiple range test were used for comparisons among multiple groups. Changes in aortic size were also analyzed by repeated-measures ANOVA. P<0.05 was considered significant.
Expression of NFκB and Ets in Human AAA
In considering the feasibility of a chimeric decoy strategy to treat human AAA, it is extremely important to study the expression of NFκB and ets in human aneurysms. First, we examined the expression of NFκB and ets in human AAA samples. Of importance, electrophoretic mobility-shift assay study showed that both NFκB and ets were markedly activated in the neck of human AAA, which is the region showing the most active progression, as compared with the dilated part of the AAA (Figure 1A). In addition, immunohistochemical study demonstrated an increase of NFκB- and ets-positive cells in the outer aneurysm wall (Figure 1B), and, furthermore, a part of the expression of NFκB and ets was detected in migrating macrophages by double-immunofluorescent staining (Figure 1C). These findings suggest that activation of NFκB and/or ets might be among the major factors in the process of aortic dilatation in humans.
Inhibitory Effect of Chimeric Decoy ODNs on MMP Expression in Organ Culture System
Aiming toward human gene therapy, we used an ex vivo organ culture system of human vascular tissue from aneurysms. An excess amount of chimeric decoy ODNs, but not scrambled decoy ODNs, competed with the increased binding of NFκB and ets (data not shown). Among various MMPs, MMP-1 and MMP-9 are considered to be especially important in the pathogenesis of AAA; thus, we examined the effects of chimeric decoy ODNs on the expression of MMP-1 and MMP-9. Secretion of MMP-1 and MMP-9 was significantly decreased by transfection of chimeric decoy ODNs in a dose-dependent manner, as compared with untreated control or transfection of scrambled decoy ODNs (Figure 2).
Regression of AAA by Chimeric Decoy ODNs in a Rabbit Model
To assess the in vivo effects, we investigated the effects of chimeric decoy ODNs to induce regression of AAA in a rabbit model. The elastase-induced AAA model is a very popular model, and an extraperitoneal approach can be used to isolate the aneurysm in rabbits. This approach is suitable for operating twice for transfection of decoy ODNs after the formation of AAA, causing little interference around the aorta. We have previously demonstrated that NFκB and ets activities were significantly increased and continued to be increased for up to 4 weeks in this model.16 In addition, successful transfer of naked decoy ODNs into the aorta using a delivery sheet was confirmed, whereas decoy ODNs were also detected in other tissues beside the aorta, such as vein and fat. Indeed, transfection of chimeric decoy ODNs using this delivery approach significantly inhibited activation of NFκB and ets in the aneurysm wall.16 Using this model and transfection approach, we examined the effects of chimeric decoy ODNs in an already-formed AAA. AAA was already established at 1 week after incubation with elastase, and no significant regression was observed in the natural course. One week after transfection, treatment with chimeric decoy ODNs resulted in a significant reduction in aortic dilatation on ultrasound analysis (Figure 3A and 3B and the online data supplement, Table I). Even 3 weeks after transfection, progressive regression of AAA by application of chimeric decoy ODNs was still observed in a dose-dependent manner as compared with control (nontransfected animal) or scrambled decoy ODN treatment (Figure 3B). Moreover, single transfection of NFκB decoy ODNs or ets decoy ODNs also reduced the size of experimental AAA. However, treatment with high-dose (1000 nmol) single-decoy ODNs showed a similar effect as compared with low-dose (200 nmol) chimeric decoy ODN treatment. In addition, the effect of high-dose chimeric decoy ODNs to regress AAA was significantly greater than that of single transfection of NFκB or ets decoy ODNs (Figure 3B). These results indicated that chimeric decoy ODNs was very potent as compared with single decoy ODNs. Regression of AAA was also readily detected using simple angiographic study (Figure 3C).
Mechanisms of Regression of AAA Using Chimeric Decoy ODNs
We hypothesized that activation of NFκB and ets affects the balance between the synthesis and degradation of structural proteins in the aneurysm wall. Elastic van Gieson staining demonstrated that treatment with chimeric decoy ODNs preserved elastic fibers, whereas scrambled decoy ODN transfer was associated with marked destruction of elastic fibers after elastase incubation (Figure 4A and 4B). Additionally, the effect of chimeric decoy ODNs to induce regression of AAA was also confirmed by the size of the aortic cross-section. (Figure 4C). MMP expression in the aneurysm wall was measured by gelatin zymography. Transfection of chimeric decoy ODNs significantly reduced the production of MMP-2 and MMP-9 and transformation into the active forms as compared with scrambled decoy ODN transfer (Figure 5). In addition, migrating macrophages are considered to be the main source of MMP secretion. Immunohistochemical study demonstrated that recruitment of macrophages was significantly inhibited by transfection of chimeric decoy ODNs, whereas many macrophages migrated into the adventitia and media of aorta transfected with scrambled decoy ODNs (Figure 6A). Furthermore, double staining with TUNEL and an immunofluorescent stain revealed that, following transfection of chimeric decoy ODNs, migrating macrophages induced apoptosis. In contrast, transfection of scrambled decoy ODNs was associated with little apoptotic activity. Additionally, double-immunofluorescent staining also demonstrated the expression of caspase-3 in migrating macrophages by transfection of chimeric decoy ODNs (Figure 6B).
Finally, the regression of AAA was also associated with upregulated elastin and collagen synthesis in the matrix. Treatment with chimeric decoy ODNs resulted in a significant increase in mRNA expression of elastin as compared with sham or scrambled decoy ODN transfer (Figure 7A). Furthermore, a significant increase in tropoelastin, a precursor of elastin, in the aneurysm wall transfected with chimeric decoy ODNs was confirmed by Western blotting (Figure 7B). We also evaluated collagen type I and III synthesis in the aneurysm wall. Picrosirius red staining, which stains collagen type I and III, demonstrated that treatment with chimeric decoy ODNs significantly increased collagen accumulation in the adventitia (Figure 8A). In addition, type I and III collagen gene expression was also significantly increased within the aneurysm wall as compared with scrambled decoy ODN transfer (Figure 8B). Given these data, the regression of AAA by chimeric decoy ODNs might be mediated by not only inhibition of degradation of connective tissue proteins but also an increase of elastin and collagen synthesis.
To consider the clinical utility of chimeric decoy ODNs, laboratory parameters were examined before and 3 weeks after transfection. There was no significant difference in body weight between rabbits treated with chimeric decoy ODNs and scrambled decoy ODNs at 4 weeks after surgery (data not shown). After transfection, there were no significant changes in liver and renal function as compared with the data before transfection (supplemental Table II). In addition, no histological changes or increases of apoptotic activity were detected in the heart and periaortic lymphatic tissue (supplemental Figure I).
Development of AAA is a complex remodeling process of the aortic wall as a result of progressive imbalance between the synthesis and degradation of structural proteins of the extracellular matrix, particularly elastin and collagen.2 Destruction of the matrix is dependent on the expression of extracellular proteinases. MMP-2 and MMP-9 have attracted the most interest because MMP-9 expression was correlated with increasing aneurysm diameter17 and MMP-2 level was elevated in the vasculature remote from the human aneurysm wall.18 Also, inflammation appears to play a fundamental role in MMP production and AAA development. In human histological studies, increasing aneurysm diameter was associated with a higher density of inflammatory cells in the adventitia,19 and MMPs were actively expressed by infiltrating macrophages located in the outer aneurysm wall.20 NFκB and ets are the center of interest in these pathological conditions of inflammation and matrix degradation. Indeed, this study demonstrated that NFκB and ets were markedly activated in the human aneurysm wall, and a part of the activity was detected in migrating macrophages, although the comparison between normal aorta and AAA would be necessary in future studies. Therefore, the simultaneous inhibition of both NFκB and ets using chimeric decoy ODNs could inhibit a wide variety of MMP expression and inflammation in the process of human AAA development. Importantly, a dose-dependent inhibitory effect of chimeric decoy ODNs on MMP-1 and MMP-9 expression was confirmed by ex vivo experiments using human aorta organ culture. These findings strongly support the feasibility of a chimeric decoy strategy to treat human AAA.
To assess further therapeutic effects of chimeric decoy ODNs, we examined the effects of chimeric decoy ODNs to induce regression of AAA in a rabbit model. The present study clearly demonstrated that chimeric decoy ODNs significantly reduced the size of AAA in a dose-dependent manner. In addition, chimeric decoy ODNs was very potent as compared with single transfection of NFκB or ets decoy ODNs, as a result of the concomitant blockade of both transcription factors. The specificity of the therapeutic effects of chimeric decoy ODNs was associated with alteration of extracellular matrix turnover. Treatment with chimeric decoy ODNs inhibited the expression and activation of MMP-2 and MMP-9, associated with direct inhibition of MMP gene expression driven by either the NFκB or ets binding site. Moreover, the recruitment of macrophages, which are considered to be the main source of MMP secretion, was also reduced in the aneurysm wall. In addition to the inhibition of adhesion molecule expression, treatment with chimeric decoy ODNs induced apoptosis of migrating macrophages. Apoptosis is a potential mechanism for the reduction in migrating tissue macrophages before transfection. For macrophage survival, activation of NFκB is necessary for the expression of antiapoptotic proteins, such as Bcl-2, A1, and IAP.21,22 Therefore, it is suggested that suppression of antiapoptotic proteins by chimeric decoy ODNs induced macrophage apoptosis in the aneurysm wall.
Reconstruction of the aneurysm wall may require insertion of newly synthesized elastin and collagen into damaged fibers as well as inhibition of matrix degeneration. Upregulation of elastin and collagen synthesis by chimeric decoy ODNs might be more important to treat AAA. In adults, elastin turnover is slow and its production is almost absent. To maintain a steady state of synthesis, various factors participate in the downregulation of elastin synthesis, but recent studies revealed that elastogenic cells begin to synthesize elastin in some pathologic conditions.23,24 Interestingly, the present study demonstrated that chimeric decoy ODNs shifted the balance toward elastin synthesis. In addition, elastic fibers treated with chimeric decoy ODNs did not show a constructive abnormality on histological study. Our observations are supported by several reports that showed an association between NFκB and elastin synthesis. Activation of NFκB induced by interleukin-1β decreased elastin promoter activity and elastin mRNA level.10 NFκB may also affect elastin synthesis indirectly through decreased tumor necrosis factor α expression. Tumor necrosis factor α is regulated by NFκB and has been found to markedly suppress the elastin mRNA level and transcriptional activity of the elastin gene in cultured human skin fibroblasts and rat aortic vascular smooth muscle cells.25
Another principal structural component in the aortic wall is collagens, the degradation of which makes aneurysms prone to rupture. To repair the aneurysm wall, increased collagen synthesis is essential, as well as elastin synthesis. The major fibrillar collagens in the aortic wall are type I and III, which undergo continual metabolic turnover. In human aneurysm tissues, the amount of collagen is contentious, but assessment of amino-terminal propeptide of type I and III procollagen, a marker of type I collagen synthesis and type III collagen turnover, revealed that the turnover of type III collagen was enhanced in the serum and aneurysm wall tissues of patients with aneurysms.26 However, the newly synthesized type III collagen was mainly present in the media and resulted in impaired fibril formation of the aneurysm wall.27 In contrast, production of procollagen type I was maintained at a low rate in serum and the aneurysm wall.26,27 These results may explain the decreased total collagen content, because collagen type I is abundant in the aortic wall. NFκB is known to bind the murine α1 (I) and α2 (I) and human α2 (I) collagen promoter, and binding of this site results in inhibition of promoter activity and gene expression.11,28,29 Ets factors are also associated with collagen synthesis. ets1 strongly suppresses transforming growth factor β induction of the collagen type I gene in human fibroblasts.30 Our present study demonstrated that type I and III collagen gene expression and protein accumulation were significantly increased in the aneurysm wall by treatment with chimeric decoy ODNs, whereas treatment with scrambled decoy ODNs was associated with significantly decreased collagen content. Importantly, increased collagen accumulation in the media led to fibrosis and weakness of the aortic wall, but treatment with chimeric decoy ODNs resulted in more intense deposition in the adventitia than in the media. This phenomenon has a beneficial effect to stabilize the aortic wall against tensile strength, leading to prevention of aneurysm rupture.
We have previously reported the preventive effect of decoy ODNs against NFκB and/or ets in rat and rabbit AAA models.15,16,31 However, the previous studies did not assess the effects of decoy ODNs to regress AAA. It is obviously important to show the regression, rather than prevention, of AAA to consider the clinical utility of decoy ODNs. The present study clearly demonstrated regression of AAA using chimeric decoy ODNs. In addition to the previous observation, an increase in matrix protein synthesis and induction of macrophage apoptosis might also contribute to repair of the aneurysm wall. However, the influence of species differences on regression of AAA by chimeric decoy ODN treatment is not clear. It might be necessary to perform studies in other models, such as an angiotensin II–infused apolipoprotein E–deficient mouse model.
Recently, pharmacologic inhibition of c-Jun N-terminal kinase (JNK) has been reported to regress AAA in an animal model.32 JNK activates the transcription factor activator protein-1, which acts with NFκB independently or cooperatively to regulate expression of target genes in various physiological processes. Therefore, a part of the effect of chimeric decoy ODNs might be dependent on the same mechanism of inhibition of JNK. However, NFκB mediates the induction of various genes without an association with activator protein-1 and negatively regulates JNK activation in the process of tumor necrosis factor α–induced apoptosis.33 Furthermore, chimeric decoy ODNs control the expression of ets-induced genes. This evidence indicates that the effect of chimeric decoy ODNs was, in large part, associated with different signal transduction pathways from that of JNK to repair the aneurysm wall.
Regression of AAA by chimeric decoy ODNs could be mediated by the following pathways: (1) direct inhibition of MMP gene expression driven by either the NFκB or ets binding site, (2) indirect inhibition of MMP secretion through inhibition of macrophage accumulation, (3) induction of apoptosis of migrating macrophages, and (4) upregulation of elastin and collagen synthesis in the matrix. More importantly, the present study demonstrated activation of NFκB and ets in human AAA, and a significant decrease in MMP-1 and MMP-9 by chimeric decoy ODNs was documented in ex vivo cultured human blood vessels. Furthermore, it could be possible to use chimeric decoy ODNs with traditional treatment for the management of large AAA. Application of chimeric decoy ODNs at the time of open surgical repair or decoy eluting–stent grafting would be useful to prevent anastomotic aneurysm and dilatation of the remaining aorta and iliac arteries after operation. Given the successful regression of AAA using chimeric decoy ODNs in a rabbit model, gene therapy or molecular therapy targeted to inhibit NFκB and ets simultaneously might provide a new therapeutic strategy to treat AAA.
Sources of Funding
This work was supported in part by a grant-in-aid from the Organization for Pharmaceutical Safety and Research, a grant-in-aid from the Ministry of Public Health and Welfare, a grant-in-aid from Japan Promotion of Science, and Special Coordination Funds of the Ministry of Education, Culture, Sports, Science and Technology, Japan.
R.M. owns stock in AnGes MG and serves as a board member of AnGes MG, which developed the decoy ODNs.
Original received January 16, 2007; revision received August 29, 2007; accepted September 12, 2007.
Shah PK. Inflammation, metalloproteinases, and increased proteolysis: an emerging pathophysiological paradigm in aortic aneurysm. Circulation. 1997; 96: 2115–2117.
Takeshita H, Yoshizaki T, Miller WE, Sato H, Furukawa M, Pagano JS, Raab-Traub N. Matrix metalloproteinase 9 expression is induced by Epstein-Barr virus latent membrane protein 1 C-terminal activation regions 1 and 2. J Virol. 1999; 73: 5548–5555.
Kuang PP, Berk JL, Rishikof DC, Foster JA, Humphries DE, Ricupero DA, Goldstein RH. NF-kappaB induced by IL-1beta inhibits elastin transcription and myofibroblast phenotype. Am J Physiol Cell Physiol. 2002; 283: C58–C65.
Kouba DJ, Chung KY, Nishiyama T, Vindevoghel L, Kon A, Klement JF, Uitto J, Mauviel A. Nuclear factor-kappa B mediates TNF-alpha inhibitory effect on alpha 2(I) collagen (COL1A2) gene transcription in human dermal fibroblasts. J Immunol. 1999; 162: 4226–4234.
Gum R, Lengyel E, Juarez J, Chen JH, Sato H, Seiki M, Boyd D. Stimulation of 92-kDa gelatinase B promoter activity by ras is mitogen-activated protein kinase kinase 1-independent and requires multiple transcription factor binding sites including closely spaced PEA3/ets and AP-1 sequences. J Biol Chem. 1996; 271: 10672–10680.
Watabe T, Yoshida K, Shindoh M, Kaya M, Fujikawa K, Sato H, Seiki M, Ishii S, Fujinaga K. The Ets-1 and Ets-2 transcription factors activate the promoters for invasion-associated urokinase and collagenase genes in response to epidermal growth factor. Int J Cancer. 1998; 77: 128–137.
Nakashima H, Aoki M, Miyake T, Kawasaki T, Iwai M, Jo N, Oishi M, Kataoka K, Ohgi S, Ogihara T, Kaneda Y, Morishita R. Inhibition of experimental abdominal aortic aneurysm in the rat by use of decoy oligodeoxynucleotides suppressing activity of NFκB and ets transcription factors. Circulation. 2004; 109: 132–138.
Miyake T, Aoki M, Nakashima H, Kawasaki T, Oishi M, Kataoka K, Tanemoto K, Ogihara T, Kaneda Y, Morishita R. Prevention of abdominal aortic aneurysms by simultaneous inhibition of NFkappaB and ets using chimeric decoy oligonucleotides in a rabbit model. Gene Ther. 2006; 13: 695–704.
McMillan WD, Tamarina NA, Cipollone M, Johnson DA, Parker MA, Pearce WH. Size matters: the relationship between MMP-9 expression and aortic diameter. Circulation. 1997; 96: 2228–2232.
Goodall S, Crowther M, Hemingway DM, Bell PR, Thompson MM. Ubiquitous elevation of matrix metalloproteinase-2 expression in the vasculature of patients with abdominal aneurysms. Circulation. 2001; 104: 304–309.
Freestone T, Turner RJ, Coady A, Higman DJ, Greenhalgh RM, Powell JT. Inflammation and matrix metalloproteinases in the enlarging abdominal aortic aneurysm. Arterioscler Thromb Vasc Biol. 1995; 15: 1145–1151.
Thompson RW, Holmes DR, Mertens RA, Liao S, Botney MD, Mecham RP, Welgus HG, Parks WC. Production and localization of 92-kilodalton gelatinase in abdominal aortic aneurysms. An elastolytic metalloproteinase expressed by aneurysm-infiltrating macrophages. J Clin Invest. 1995; 96: 318–326.
Chu ZL, McKinsey TA, Liu L, Gentry JJ, Malim MH, Ballard DW. Suppression of tumor necrosis factor-induced cell death by inhibitor of apoptosis c-IAP2 is under NF-κB control. Proc Natl Acad Sci U S A. 1997; 94: 10057–10062.
Pagliari LJ, Perlman H, Liu H, Pope RM. Macrophages require constitutive NF-kappaB activation to maintain A1 expression and mitochondrial homeostasis. Mol Cell Biol. 2000; 20: 8855–8865.
Poiani GJ, Tozzi CA, Yohn SE, Pierce RA, Belsky SA, Berg RA, Yu SY, Deak SB, Riley DJ. Collagen and elastin metabolism in hypertensive pulmonary arteries of rats. Circ Res. 1990; 66: 968–978.
Kahari VM, Chen YQ, Bashir MM, Rosenbloom J, Uitto J. Tumor necrosis factor-alpha downregulates human elastin gene expression. Evidence for the role of AP-1 in the suppression of promoter activity. J Biol Chem. 1992; 267: 26134–26141.
Satta J, Juvonen T, Haukipuro K, Juvonen M, Kairaluoma MI. Increased turnover of collagen in abdominal aortic aneurysms, demonstrated by measuring the concentration of the aminoterminal propeptide of type III procollagen in peripheral and aortal blood samples. J Vasc Surg. 1995; 22: 155–160.
Rippe RA, Schrum LW, Stefanovic B, Solis Herruzo JA, Brenner DA. NF-kappaB inhibits expression of the alpha1(I) collagen gene. DNA Cell Biol. 1999; 10: 751–761.
Novitskiy G, Potter JJ, Rennie-Tankersley L, Mezey E. Identification of a novel NF-kappaB-binding site with regulation of the murine alpha2(I) collagen promoter. J Biol Chem. 2004; 279: 15639–15644.
Czuwara-Ladykowska J, Sementchenko VI, Watson DK, Trojanowska M. Ets1 is an effector of the transforming growth factor beta (TGF-beta) signaling pathway and an antagonist of the profibrotic effects of TGF-beta. J Biol Chem. 2002; 277: 20399–20408.
Miwa K, Nakashima H, Aoki M, Miyake T, Kawasaki T, Iwai M, Oishi M, Kataoka K, Ohgi S, Ogihara T, Kaneda Y, Morishita R. Inhibition of ets, an essential transcription factor for angiogenesis, to prevent the development of abdominal aortic aneurysm in a rat model. Gene Ther. 2005; 12: 1109–1118.