Inhibition of ets, an essential transcription factor for angiogenesis, to prevent the development of abdominal aortic aneurysm in a rat model

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The pathophysiology of abdominal aortic aneurysms (AAA) is considered to be complicated. As matrix degradation contributes to the progression of AAA, the destruction and degradation of elastin fibers caused by an increase in matrix metalloproteinases (MMPs) plays a pivotal role in the development of AAA. Although ets, an essential transcription factor for angiogenesis, regulates MMPs, the role of ets in the development of AAA has not yet been clarified. Thus, we evaluated the role of ets in a rat AAA model using a decoy strategy. Transfection of ODN into AAA was performed by transient aortic perfusion of elastase and by wrapping the AAA in a delivery sheet containing decoy ODN. The inhibitory effect of ets decoy ODN on ets binding activity was confirmed by gel mobility shift assay. MMPs expression was decreased in the aorta transfected with ets decoy ODN as compared to scrambled decoy ODN. Also, ultrasound study demonstrated that elastase-induced aneurismal dilation was significantly suppressed by transfection of ets decoy ODN at 4 weeks after treatment as compared to scrambled decoy ODN. Moreover, the destruction of elastin fibers was inhibited in the aorta transfected with ets decoy ODN, accompanied by a reduction of MMPs expression. An inhibitory effect of decoy ODN on MMP expression was confirmed by ex vivo experiments showing that transfection of decoy ODN into an organ culture of human aorta resulted in significant inhibition of the secretion of both MMP-1 and MMP-9. Here, we demonstrated that ets may play a pivotal role in the progression of AAA through the activation of MMPs in a rat model. Ets might be a potential target to develop pharmacotherapy/gene therapy to treat AAA through the inhibition of MMPs.


Members of the ets family play important roles in regulating gene expression in response to multiple developmental and mitogenic signals.1, 2, 3, 4 The ets family activates the transcription of genes encoding matrix metalloproteinase (MMP)-1, MMP- 3, MMP-7, MMP-9, MMP-13 and urokinase plasminogen activator, which are proteases involved in extracellular matrix degradation.5, 6, 7, 8, 9, 10 From these properties, we consider that the ets family plays an important role in matrix regulation. To clarify this issue, we focused on the pathophysiology of abdominal aortic aneurysm (AAA). AAA is a common and life-threatening disorder. Inflammation, which is temporally and spatially associated with disruption of the orderly lamellar structure of the aortic media followed by adventitial remodeling and fibrosis, appears to play a fundamental role in the development and progression of AAA.11, 12 The destruction of normally long-lived matrix macromolecules such as elastin has been ascribed to a family of endopeptidases, the MMPs. Pathological vascular remodeling is considered to be mediated by MMPs secreted by invasive macrophages, vascular smooth muscle cells and endothelial cells.13, 14, 15 It has been reported that the mRNA and protein levels of MMP-1 (collagenase-1), MMP-2 (72-kDa gelatinase), MMP-3 (stromelysin-1), MMP-9 (92-kDa gelatinase) and MMP-12 (macrophage elastase) are significantly increased in harvested human aneurysms.13, 14, 15, 16, 17, 18 These findings suggest that MMPs are strongly associated with the activity of AAA. From this viewpoint, a pharmacological strategy to inhibit MMP activity might prevent the progression from asymptomatic AAA to critical large AAA, resulting in a delay or avoidance of surgical repair. Some researchers have reported that MMP inhibitors suppressed the progression of experimental AAA.19, 20, 21 MMPs are also promising surrogate markers of AAA evolution.22 Although the close relationship between ets and MMPs is well known, no study has exclusively examined the role of ets in the pathogenesis of AAA. Thus, in this study, we demonstrated that ets plays a pivotal role in the development of AAA in a rat model.


Prevention of aneurysmal dilation by ets decoy ODN in rat AAA model

Initially, we examined the feasibility of transfection of decoy ODN using a delivery sheet wrapped around the aortic wall in a rat AAA model. Our previous paper has already indicated the successful ODN transfer by an ODN delivery sheet.23 Consistent with the previous paper, fluorescence was readily detected mainly in the adventitia and part of the media at 7 days after transfection, whereas no fluorescence was detected in untransfected artery. Moreover, as shown in Figure 1a, fluorescence due to FITC-labeled ODN could be detected mainly in the adventitia even at 4 weeks after transfection. Successful ODN transfer was also confirmed by electrophoretic mobility shift assay (EMSA) study (Figure 1b–d). Of importance, the binding activity of ets, as assessed by gel mobility shift assay, was markedly increased in the aorta after perfusion with elastase as compared to sham-operated rats (Figure 1b). In addition, in vivo transfection of ets decoy ODN by a delivery sheet significantly suppressed activation of ets induced by elastase as compared to scrambled decoy ODN (Figure 1c and d, P<0.01). In contrast, the increased binding of ets was not inhibited by scrambled decoy ODN. We therefore examined the inhibitory effect of decoy ODN on aortic dilatation, using a delivery sheet. As shown in Figure 2a and b, ultrasound analysis demonstrated that treatment with ets decoy ODN prevented the progression of aortic dilatation after elastase perfusion (68% inhibition as compared to scrambled decoy ODN). Even 4 weeks after transfection, the progression of AAA was still inhibited by ets decoy ODN. The inhibitory effect of ets decoy ODN on aortic dilatation was also confirmed by histological studies (Figure 2c). Elastase perfusion resulted in a marked increase in both the outer diameter and inner diameter, and the thickness of the vascular adventitia. Also, breaks in the media were observed. Interestingly, treatment with ets decoy ODN attenuated the progression of dilatation and destruction of the vascular media. In contrast, scrambled decoy ODN against ets failed to prevent aortic dilatation (data not shown).

Figure 1

(a) Typical photograph of fluorescence in aorta of rat transfected with FITC-labeled ODN using delivery sheet at 4 weeks after transfection. Upper: Sham operation (× 200); lower: transfection of FITC-labeled decoy ODN (× 200). (b) Gel-mobility shift assay for ets binding site at 1 week after transfection. Elastase=nuclear extract (30 μg) from aorta with elastase perfusion; sham=nuclear extract (30 μg) from aorta from sham-operated rats without elastase perfusion. (c) Gel-mobility shift assay for ets binding site at 1 week after transfection. (d) Percent decrease in ets activity by ets decoy ODN (**P<0.01 versus SD). N=6/group. Elastase=nuclear extract (30 μg) from aorta with elastase perfusion; sham=nuclear extract (30 μg) from aorta from sham-operated rats without elastase perfusion (only saline perfusion), SD=nuclear extract (30 μg) from aorta of rat AAA model transfected with scrambled decoy ODN; ets=nuclear extract (30 μg) from aorta of rat AAA model transfected with ets decoy ODN; and cold=nonlabeled ets probe (× 100 excess).

Figure 2

(a) Representative ultrasound showing aortic dilatation. (b) Time course of aortic area after elastase perfusion, as assessed by ultrasound. N=8/group. (c) Representative cross-sections of HE staining (× 40) at 4 weeks after transfection. (d) Representative histological sections of rat aorta stained with Miller's elastin and van Gieson's stain (EVG) at 4 weeks after transfection (× 400). Sham=aorta from sham-operated rats without elastase perfusion; untreated=aorta with elastase perfusion only; SD=AAA model transfected with scrambled decoy ODN; and ets=AAA model transfected with ets decoy ODN.

One of the major pathogenetic mechanisms of AAA is considered to be degradation of extracellular matrix. As elastic fibers maintain the structure of the vascular wall against hemodynamic stress, resulting the in prevention of aortic dilatation, we, therefore, evaluated the effect of ets decoy ODN on the destruction of elastic fibers. As shown in Figure 2d, treatment with scrambled decoy ODN resulted in significant degradation of elastic fibers as compared to those in sham-operated rats (without elastase perfusion), whereas treatment with ets decoy ODN markedly inhibited the proteolysis of elastin as compared to scrambled decoy ODN. Van Gieson's (EVG) staining suggested that transfection of ets decoy ODN inhibited the destruction of elastic fibers. Inhibition of degradation of matrix was also confirmed by an experiment in which transfection of ets decoy ODN markedly abolished the decrease in elastin in an AAA model (Figure 3a, P<0.05). However, transfection of ets decoy ODN did not completely restore elastic fiber degradation, since the inhibitory effect of ets decoy ODN was significant, but only partial (Figure 3b, P<0.05). Among various MMPs, MMP-3 and MMP-9 are considered to be especially important in the pathogenesis of this rat AAA model. To confirm the inhibitory effects of ets decoy ODN, we measured the expression of MMP-3 and MMP-9 using quantitative RT-PCR. As shown in Figure 4, the expression of mRNA of both MMP-3 and MMP-9 was increased in rats with elastase perfusion only (untreated) and rats transfected with scrambled decoy ODN, whereas there was no significant difference between untreated rats and those treated with scrambled decoy ODN. Interestingly, transfection of ets decoy ODN resulted in significant inhibition of MMP-3 and MMP-9 mRNA (P<0.05). Moreover, in situ zymography showed that the expression of MMPs in the adventitia was markedly reduced in the aorta of an AAA model transfected with ets decoy ODN, while MMP expression was markedly increased in the AAA model transfected with scrambled decoy ODN (Figure 5). Alternatively, infiltration of macrophages into the aortic wall, followed by vascular inflammation, is also considered to be a key mechanism in the progression of AAA in animal models as well as humans,24, 25, 26, 27 since macrophages are major cells that secrete MMPs. However, since ets does not influence the regulation of adhesion molecules and MCP-1, it was not expected that transfection of ets decoy ODN would affect the recruitment of inflammatory cells into the vascular wall. Actually, immunohistochemical study demonstrated that macrophage infiltration in the adventitia and media was not inhibited by transfection of ets decoy ODN (ets decoy: 440±41/mm2; scrambled decoy: 394±39/mm2; untreated: 43±21/mm2; sham: 32±11/mm2). The effect of ets decoy ODN might be mainly mediated by inhibition of MMP expression. Since we observed macrophages that secrete MMPs mainly in the adventitia23 and our in situ zymography data showed that expression of MMPs were observed mainly in the adventitia (Figure 5), the present approach to transfer decoy ODN in in vivo experiments seems to be ideal.

Figure 3

Western blotting of elastin in rat AAA model at 4 weeks after transfection. (a) Typical example of Western blotting and (b) quantitative analysis of elastin protein. Sham=aorta from sham-operated rats without elastase perfusion; SD=AAA model transfected with scrambled decoy ODN; and ets=AAA model transfected with ets decoy ODN. *P<0.05, **P<0.01 versus sham. N=6/group.

Figure 4

(a) Representative example of RT-PCR. (b) Gene expression of MMP-3 and MMP-9 in rat AAA model transfected with decoy ODN at 4 weeks after transfection. Sham=aorta from sham-operated rats without elastase perfusion; untreated=aorta with elastase perfusion only; SD=AAA model transfected with scrambled decoy ODN; and ets=AAA model transfected with ets decoy ODN. **P<0.01, ##P<0.01 versus sham. N=6/group.

Figure 5

Representative cross-sections of in situ gelatin zymography (× 200). Gelatin beneath the adventitia of vessels was degraded by the induction of MMP in the rat elastase-infusion aneurysm model treated with scrambled decoy ODN. However, ets decoy ODN prevented gelatin degradation.

Inhibitory effect of ets decoy ODN on MMP-1 and MMP-9 expression in organ culture system of human AAA

To further explore the role of ets in human AAA, we examined the expression of ets in human aneurysms. Importantly, transcription activity of ets was significantly increased in the neck of human AAA, which is the region showing most active progression, as compared to the existing dilated part of human AAA (Figure 6, P<0.01). These findings suggest that activation of ets might be one of the major factors regulating MMP and the process of aortic dilatation. Since MMP-1 and MMP-9 are considered to play a pivotal role in the pathogenesis in human AAA,27, 28, 29 we examined the effect of ets decoy ODN on the expression of MMP-1 and MMP-9. To further explore the role of ets in human AAA, we employed an ex vivo organ culture system of human vascular tissue from aneurysms. MMP-1 as well as MMP-9 was readily detected in the conditioned medium from the organ culture system. Importantly, secretion of MMP-1 and MMP-9 from harvested human aortic aneurysms in organ culture was significantly decreased by transfection of ets decoy ODN as compared to untreated control or transfected scrambled decoy ODN, as shown in Figure 7.

Figure 6

Transcriptional activity of ets in human harvested AAA, assessed by gel-mobility shift assay. (a) Gel-mobility shift assay for ets binding site and (b) percent change in ets activity in dilated part of AAA. N=nuclear extracts (30 μg) from the neck of human harvested AAA; D=nuclear extracts (30 μg) from the most dilated part of human harvested AAA; and C=nonlabeled probe (× 100 excess). *P<0.01 versus N. N=6/group.

Figure 7

Inhibitory effect of ets decoy ODN on secretion of MMP-1 (a) and MMP-9 (b) in organ culture systems. Untreated=untreated aortic specimens; SD=aortic specimens transfected with scrambled decoy ODN; and ets=aortic specimens transfected with ets decoy ODN. *P< 0.05 versus untreated. N=8/group.


AAA is a common degenerative condition that is enhanced by genetic and environmental factors.30 and is characterized by structural alterations of the aortic wall, resulting in part from degradation of the macromolecules, collagen and elastin. These changes are associated with an inflammatory infiltrate and excessive production of MMPs, which are assumed to orchestrate the widespread matrix destruction.22 Although elective surgical repair is an effective approach to prevent deaths from AAA rupture, there is a conspicuous absence of alternative therapeutic strategies for this disease.31 As human aneurysm tissues are characterized by destructive remodeling of the elastic media and outer aortic wall, recent investigations have emphasized disease mechanisms involving chronic aortic wall inflammation and the progressive degradation of fibrillar matrix proteins.31, 32, 33 The dissolution of elastic fibers requires the presence of specific proteinases, and several elastolytic MMPs such as MMP-2, MMP-9, MMP-12 and MT1-MMP are thought to contribute to aneurysm development.16, 17, 27, 34, 35, 36, 37, 38, 39, 40, 41 MMP-9 has attracted particular interest, because it is the most abundant elastolytic proteinase secreted by human AAA tissue explants in vitro and is actively expressed by aneurysm-infiltrating macrophages located at the site of tissue damage in situ.42 MMP-9 expression also correlates with increasing aneurysm diameter,18, 43 and is elevated in the circulating plasma of patients with AAA.44 Further knowledge of how these enzymes play an etiologic role in the process may lead to prospects for pharmacologic, rather than surgical treatment of AAA such as graft replacement. These observations have led to speculation that either MMP-1 or MMP-9 might be necessary for aneurysmal degeneration, thereby providing targets for pharmacologic therapy.18, 19, 20

Ets-1 is encoded by the c-ets-1 proto-oncogene, the founding member of the novel ets family of transcription regulators.45, 46 Combined cotransfection assays and deletion studies of promoters have shown that ets-1 is required for the transactivation of several genes encoding proteases involved in matrix degradation such as collagenase 1 (MMP-1), gelatinase B (MMP-9) and stromelysin I (MMP-3), as well as the urokinase-type plasminogen activator, which activates these enzymes by proteolytic cleavage.47, 48 In the pathogenesis of cancer, it is well known that all of these proteases are necessary for matrix degradation, which is required during both tumor invasion and new blood vessel formation.48 Interestingly, several of these enzymes were coexpressed with ets-1 within invasive human carcinomas, whereas noninvasive lesions were negative, suggesting an important role of ets in degradation of matrix.49, 50 From these findings, it is suggested that ets plays an important role in the degradation of collagen and elastin, leading to remodeling of the aortic wall structure. Also, a recent report demonstrated that vascular endothelial growth factor (VEGF) was induced in the aortic wall in vascular diseases such as atherosclerotic abdominal aortic aneurysm, inflammatory AAA and aortic occlusive disease.51 Since ets is also known to be induced by VEGF,52 these findings support our hypothesis. Using a decoy strategy, the present study clearly demonstrated that ets plays a pivotal role in the progression of AAA. As the promoter region of the elastase gene contains two ets family consensus sequences,53 positive feedback between elastase and ets might exist.

Specificity of the inhibitory effect of ets decoy ODN on the progression of AAA is supported by several lines of evidence: (1) ex vivo organ culture experiments using aortic extracts from patients with AAA have documented that ets decoy ODN selectively inhibited the expression of targeted MMP genes (MMP-1 and MMP-9), accompanied by inhibition of ets binding activity. (2) In vivo administration of ets decoy ODN, but not scrambled ODN, by a peripheral wrapping sheet, significantly inhibited dilatation of the aorta, accompanied by inhibition of both ets binding activity and the expression of MMP-3 and MMP-9 mRNA. Our data demonstrated that ets binding activity assessed by EMSA and the expression of MMP-3 and MMP-9 mRNA assessed by real-time RT-PCR were parallel in the ets decoy ODN, sham and scrambled decoy ODN groups. (3) A decrease in matrix degradation activity in adventitia transfected with ets decoy ODN was associated with a decrease in aortic diameter. However, ets decoy ODN did not inhibit macrophage recruitment, which is one of the major mechanisms of aortic dilation, although macrophage invasion was significantly increased in the aorta treated with elastase perfusion as compared to that of sham (untreated: 432±21/mm2; sham: 32±11/mm2, P<0.05). Also, as the experimental AAA model induced by elastase infusion might be far from human AAA, further experiments are necessary. In this study, we did not evaluate the affect of TIMPs, which are considered to play an important role in the pathogenesis of AAA. Other factors such as TIMPs and NFkB might be involved in the pathogenesis of human AAA.54, 55

Overall, the present study demonstrated that ets plays a pivotal role in the progression of AAA through the activation of MMPs in a rat model. Ets might be a potential target to develop pharmacotherapy/gene therapy to treat AAA through the inhibition of MMPs.

Materials and methods

Synthesis of ODN and selection of target sequences

Sequences of the phosphorothioate ODN utilized were as follows:

  • ets decoy ODN (consensus sequences are underlined): IndexTerm5′-AATTCACCGGAAGTATTCGA-3′ and IndexTerm3′-TTAAGTGGCCTTCATAAGCT -5′.

  • Scrambled ets decoy ODN: IndexTerm5′-ATACTACGAGCATATGCATG -3′ and IndexTerm3′-TATGATGCTCGTATACGTAC-5′.

Ets decoy ODN have been shown to bind the ets transcription factor, consistent with previous reports.14 Synthetic ODN were washed with 70% ethanol, dried and dissolved in sterile Tris-EDTA buffer (10 μM Tris, 1 μM EDTA). The supernatant was purified over a NAP 10 column (Pharmacia, Piscataway, NJ, USA) and quantified by spectrophotometry.

Procedure of AAA model

Male Wistar rats (400–500 g; Charles River Breeding Laboratories) were anesthetized, and underwent laparotomy. Briefly, the abdominal aorta was isolated from the level of the left renal vein to the bifurcation. The right femoral artery was exposed and a PE-10 polyethylene tube (Baxter Healthcare Corp., McGraw Park, IL, USA) was introduced through the femoral artery into the distal aorta. The aorta was clamped above the level of the tip of the PE tube and ligated with a silk suture near the aortic bifurcation (approximately 15 mm in length), followed by perfusion with 0.2 ml saline containing 5 U (25 U/ml) type I porcine pancreatic elastase (Sigma Chemical, St Louis, MO, USA). Aortic perfusion with 2 ml saline containing elastase (25 U/ml) was performed for 30 min at 100 mmHg.23 After perfusion, the clamp and ligatures were removed and the PE tube was withdrawn.23 Transfection of decoy ODN was performed by wrapping a delivery sheet around the abdominal aorta at the same time as surgery. Then, 73 mg hydroxypropyl cellulose (Wako, Osaka) and 7.3 mg polyethylene glycol 400 (Wako, Osaka) were dissolved in 70% ethanol, and 400 nmol decoy ODN was mixed into this solution. After mixing overnight at 4°C, the solution was put into the sheet box (4 cm2). Natural drying overnight or 2 days to remove ethanol resulted in a 4-cm2 thin sheet containing 100 nmol decoy ODN/cm2. This delivery sheet is easily converted to a gel in wet conditions. When the sheet is wrapped around the aorta, it immediately changes to a gel to allow incubation of ODN around the aorta for at least a week. 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).

Electrophoretic mobility shift assay

Rats were killed 1 week after the operation, and nuclear extracts were prepared from transfected or untransfected aortic aneurysms, as described previously.56, 57 In brief, rat aortas were homogenized with a Potte–Elvehjem homogenizer in four volumes of ice-cold homogenization buffer (10 mM Hepes (pH 7.5), 0.5 M sucrose, 0.5 mM spermidine, 0.15 mM spermin, 5 mM EDTA, 0.25 M EGTA, 7 mM beta-mercaptoethanol and 1 mM phenylmethylsulfonylfluoride). After centrifugation at 12 000 g for 30 min at 4°C, each pellet was lysed in one volume of ice-cold homogenization buffer containing 0.1% NP-40 by homogenization in a Dounce homogenizer. Then, it was centrifuged at 12 000 g for 30 min at 4°C, and the pellet nucleus was washed twice with ice-cold buffer containing 0.35 M sucrose. After washing, the nucleus was preextracted with one volume of ice-cold homogenization buffer containing 0.05 M NaCl and 10% glycerol for 15 min at 4°C. The nucleus was then extracted with homogenization buffer containing 0.3 M NaCl and 10% glycerol for 1 h at 4°C. Subsequently, the concentration of DNA was adjusted to 1 mg/ml. After pelleting the extracted nucleus at 12 000 g for 30 min at 4°C, 45% (NH4)2SO4 was added to the supernatant. Then, the mixture was stirred for 30 min at 4°C. The precipitated protein was collected at 17 000 g for 30 min, resuspended in homogenization buffer containing 0.35 M sucrose and stored in aliquots at −70°C.

ODN containing the ets binding site (IndexTerm5′-AATTCACCGGAAGTATTCGA-3′; only the sense strand is shown) were labeled as a primer at the 3′-end using a 3′-end-labeling kit (Clontech Inc., Palo Alto, CA, USA). After end labeling, 32P-labeled ODN was purified by application to a Nick column (Pharmacia). Binding mixtures (10 μl) including 32P-labeled primers (0.5–1 ng, 10 000–15 000 c.p.m.) and 1 μg polydeoxyinosinic-deoxycytidic acid (Sigma Chemical) were incubated with 10 μg nuclear extract for 30 min at room temperature, and then loaded onto 5% polyacrylamide gel. The gels were subjected to electrophoresis, dried and preincubated with parallel samples for 10 min before the addition of the labeled probe. As a control, samples were incubated with an excess (100 ×) of nonlabeled ODN, which completely abolished binding. Gels were analyzed by autoradiography.


Ultrasonography was used to demonstrate dilatation of the AAA. A cardiovascular ultrasound system (Power Vision 6000, Toshiba) and a linear transducer (15 MHz) were used to image the abdominal aorta noninvasively in anesthetized rats. Rats were scanned transversely to obtain images for the measurement of the luminal diameter and the area of the lumen of the aneurysm at the segment with maximum diameter. The aortic size was measured before and after laparotomy once a week up to 4 weeks after operation.

Histological and immunohistochemical studies

Rats were killed 4 weeks after the operation. The excised aorta was fixed in 10% neutral-buffered formalin, and processed for routine paraffin embedding. Aortic tissue cross-sections (6 μm) were stained with both hematoxylin and eosin (HE), and Miller's elastin and van Gieson's stain (EVG) in a standard manner. Mouse monoclonal antibody against rabbit macrophages (RAM11, Dako, CA, USA) was also used to analyze macrophage recruitment in rat experimental AAA. For negative control experiments, the primary antibody was omitted. Immunohistochemical staining was performed using the immunoperoxidase avidin–biotin complex system. Then, 5-μm sections were deparaffinized, rehydrated before blocking endogenous peroxidase activity with 3% hydrogen peroxidase and preincubated with 5% normal horse serum in sodium phosphate-buffered saline (PBS) for 30 min. Diluted primary antibodies (RAM11) (1:50) were then applied to the sections, and these sections were incubated overnight at 4°C. With intervening washing in PBS, they were serially incubated with biotinylated anti-mouse IgG (Vector Laboratories, Burlingame, CA, USA) in PBS for 30 min and avidin-biotinylated horseradish peroxidase complex in PBS for 30 min, according to the manufacturer’s specifications (Vectastain Elite ABC kit, Vector Laboratories). Immune complexes were localized using 0.05% 3,3′-diaminobenzidine (Vector Laboratories) and slides were counterstained with hematoxylin.

Western blotting

Frozen aortic tissues were resuspended in 200 μl lysis buffer buffer (50 mM Tris-HCl, pH 8.0, 20 mM EDTA, 1% SDS, 100 mM NaCl) containing a protease inhibitor cocktail (Sigma), homogenized and collected by centrifugation at 1200 g for 5 min. Samples (20 μg) were electrophoresed in SDS-PAGE acrylamide gels, transferred onto nitrocellulose membranes (Hybond ECLTM, Amersham), and incubated for 24 h in PBS, 5% nonfat milk and 0.2% Tween 20 at 4°C. Membranes were then incubated for 24 h at 4°C with anti-elastin polyclonal antibody (Cosmobio, Japan; 1:2000 dilution), washed in PBS and 0.1% Tween 20, incubated for 2 h at room temperature with peroxidase-conjugated donkey anti-rabbit IgG secondary antibody (Amersham Biosciences; 1:2000) and visualized using an ECLplus chemiluminescent kit (Amersham Biosciences) following the manufacturer’s instructions and exposed to XAR-5 X-ray film (Eastman Kodak Co.). To quantity and compare the levels of proteins, the density of each band was measured by densitometry (Shimazu, Kyoto, Japan).

RNA extraction

Rat aorta was homogenized in 700 μl ISOGEN (Nippon Gene, Japan) and total RNA was extracted according to the manufacturer’s instructions. RNA was reversetranscribed with using a ProSTARTMHF Single-Tube RT-PCR System (High Fidelity) (Stratagene, USA). Thereafter, PCR was performed in 50 μl reactions using primer pairs (100 ng) with the following sequences:



Conditions for these reactions were 30 s at 95°C for denaturation, 30 s at 60°C for annealing and 1 min at 68°C for extension.

Real-time PCR

Total RNA was isolated with the use of ISOGEN, and 500 ng of RNA and mRNA of TagMan rodent GAPDH control reagent (Applied Biosystems, USA) were reverse transcribed to cDNA using oligo(dT) as primers and a ThermoScriptTM RT-PCR System (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's instructions. Real-time PCR was performed by monitoring in real time the increase in fluorescence of SYBR Green dye on an ABI PRISM 7700 Sequence Detector System (Applied). The reaction was carried out in a final volume of 25 μl with 2 × SYBR Green PCR master mix, 10 μM each primer and 50 ng cDNA for 40 cycles (20 s at 94°C for denaturation, 20 s at 60°C for annealing and 30 s at 72°C for extension). Relative expression level of the target gene was plotted as fold change compared with the control. Each RT-PCR assay was performed twice using triplicate samples. Primer sequences were as follows:



Results were analyzed using comparative Ct Methods, where Ct is the number of cycles required to reach an arbitrary threshold.58 Ct was determined for each sample and normalized to Ct of β-actin in the same sample. These values were compared to control levels and expressed as fold difference.

In situ zymography

Gelatinolytic activity in the rat aorta was analyzed with gelatin-coated film (Fuji Photo Film Co., Ltd, Tokyo, Japan) by methods reported previously.59 Aneurysms of rats were excised 1 week after the operation, and frozen sections (5 μm) of tissue samples were placed on this film. Films with specimens were incubated in a humidified chamber at 37°C overnight. Then, the film was stained with 1% Amido Black 10B (Wako Inc., Tokyo, Japan) in 70% methanol and 10% acetic acid for 15 min. After destaining with a solution of distilled water, 70% methanol and 10% acetic acid, and lysis of the substrate was observed by light microscopy.

Organ cultures of human AAA specimens

Aortic specimens were obtained from patients with AAA at the time of open surgical repair. All cases of open surgical repair were performed as scheduled procedures. We used eight samples of aorta obtained from three patients (age: 65±5 years; size of aneurysm: 5.0±0.3 cm). These patients did not take any medication that could influence MMP activity such as statins, and no patient had any inherited disorder such as Marfan syndrome. The harvested aortic specimens were transported to a sterile tissue culture hood in cold Dulbecco's modified Eagle's medium (DMEM) supplemented with 1% bovine serum albumin and antibiotics. Each specimen was immediately divided into 2 mm2 segments of full-thickness aortic wall, followed by incubation in 20% collagen gel in DMEM containing each decoy ODN (100 μM) for 1 h at 4°C. Then, they were placed in separate 24-well tissue culture plates with 500 μl DMEM, and incubated at 37°C in a humidified 5% CO2 atmosphere. After 48 h, the conditioned medium was collected and stored at −20°C until used for ELISA (enzyme-linked immunosorbent assays) of MMP-1 and MMP-9. Concentrations of MMP-1 and MMP-9 were measured using ELISA from Amersham Biotech (Biotrak, Buckinghamshire, UK). Intra-assay coefficients of variance were less than 10% for all assays.

Statistical analysis

Samples were coded so that analysis was performed without the knowledge of which treatment each group had received. The observer was blinded to other data concerning the samples. All values are expressed as mean±s.e.m. Analysis of variance with ANOVA was used to determine the significance of differences in multiple comparisons. P<0.05 was considered significant.


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This work was partially supported 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 through Special Coordination Funds of the Ministry of Education, Culture, Sports, Science and Technology, the Japanese Government.

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Correspondence to R Morishita.

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Miwa, K., Nakashima, H., Aoki, M. et al. Inhibition of ets, an essential transcription factor for angiogenesis, to prevent the development of abdominal aortic aneurysm in a rat model. Gene Ther 12, 1109–1118 (2005) doi:10.1038/

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  • aneurysms
  • ets
  • decoy
  • matrix metalloproteinase

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