Chimeric Decoy Oligodeoxynucleotides Research Articles

The Effects of Synthetic SREBP-1 and PPAR-γ Decoy Oligodeoxynucleotide on Acne-like Disease In Vivo and In Vitro via Lipogenic Regulation.

Acne vulgaris has a pathogenesis that involves increased sebum production and perifollicular inflammation. Sterol regulatory element-binding protein-1 (SREBP-1) and peroxisome proliferator activated receptor-γ (PPAR-γ) are transcription factors that regulate numerous genes involved in lipid biosynthesis. To improve a new therapeutic approach, we designed the SREBP/PPAR decoy oligodeoxynucleotide (ODN), a synthetic short DNA containing complementary sequences for the SREBP and PPAR transcription factors. We aim to investigate the beneficial functions and the molecular mechanisms of the synthetic SREBP/PPAR decoy ODN in lipogenic models. C. acnes was intradermally injected with a 1.0 × 107 colony forming unit/20 μL. The synthetic SREBP/PPAR decoy ODN or scrambled decoy ODN (10 μg) was transferred via the mouse tail vein injection. SZ95 cells were transfected with 2 μg of synthetic ODNs. After transfection, the SZ95 cells were cultured in serum-free medium containing 20 ng/μL of insulin-like growth factor-1 (IGF)-1 for 24 h. To investigate the expression of gene and signaling pathways, we performed Western blotting. The distribution of the chimeric decoy ODN was confirmed by EMSA. Lipid levels were assessed by Nile red and Oil Red O staining. The cytokine levels were measured by ELISA kit. This study showed that C. acnes-injected mice and IGF-1-stimulated SZ95 cells exhibited increased expression of SREBP-1 and PPAR-γ compared to the normal controls. In contrast, the administration of the SREBP/PPAR chimeric decoy ODN significantly suppressed the upregulation of lipogenic genes. Furthermore, the SREBP/PPAR decoy ODN decreased the plasma cytokines and cytokine levels of total protein. These results suggested that the SREBP/PPAR decoy ODN exerts its anti-lipogenic effects by regulating lipid metabolism and by inhibiting lipogenesis through the inactivation of the SREBP and PPAR pathways. Therefore, the synthetic SREBP/PPAR ODN demonstrates substantial therapeutic feasibility for the treatment of acne vulgaris.

Prevention of Asthma Exacerbation in a Mouse Model by Simultaneous Inhibition of NF-κB and STAT6 Activation Using a Chimeric Decoy Strategy.

Transactivation of inflammatory and immune mediators in asthma is tightly regulated by nuclear factor κB (NF-κB) and signal transducer and activator of transcription 6 (STAT6). Therefore, we investigated the efficacy of simultaneous inhibition of NF-κB and STAT6 using a chimeric decoy strategy to prevent asthma exacerbation. The effects of decoy oligodeoxynucleotides were evaluated using an ovalbumin-induced mouse asthma model. Ovalbumin-sensitized mice received intratracheal administration of decoy oligodeoxynucleotides 3 days before ovalbumin challenge. Fluorescent-dye-labeled decoy oligodeoxynucleotides could be detected in lymphocytes and macrophages in the lung, and activation of NF-κB and STAT6 was inhibited by chimeric decoy oligodeoxynucleotide transfer. Consequently, treatment with chimeric or NF-κB decoy oligodeoxynucleotides protected against methacholine-induced airway hyperresponsiveness, whereas the effect of chimeric decoy oligodeoxynucleotides was significantly greater than that of NF-κB decoy oligodeoxynucleotides. Treatment with chimeric decoy oligodeoxynucleotides suppressed airway inflammation through inhibition of overexpression of interleukin-4 (IL-4), IL-5, and IL-13 and inflammatory infiltrates. Histamine levels in the lung were reduced via suppression of mast cell accumulation. A significant reduction in mucin secretion was observed due to suppression of MUC5AC gene expression. Interestingly, the inhibitory effects on IL-5, IL-13, and histamine secretion were achieved by transfer of chimeric decoy oligodeoxynucleotides only. This novel therapeutic approach could be useful to treat patients with various types of asthma.

580. Prevention of Airway Inflammation by Simultaneous Inhibition of NFkB and STST6 Using Chimeric Decoy Oligonucleotides in a Mouse Model of Asthma

Allergic asthma is a common inflammatory disease of the airways characterized by reversible airflow obstruction and airway hyperresponsiveness (AHR) to bronchoconstrictor stimuli. The pathophysiology in asthma mainly occurs as a consequence of overexpression of the TH2 cytokines. These inflammatory factors are tightly regulated by a transcriptional network system. Nuclear factor kappaB (NFkB) and STAT6 are well known to regulate a set of gene associated with inflammatory and immune responses, and is thought to play an important role in the induction of allergic asthma. Therefore, we focused on the simultaneous inhibition of these important transcription factors using a decoy strategy to develop a novel therapeutic approach for treating asthma. For increased efficacy of decoy oligodeoxynucleotides (ODN) in vivo, we employed chimeric decoy ODN containing consensus sequences of both NFkB and STAT6 binding sites. In addition, two strands of decoy ODN were combined by the chemical spacer to increase its resistance to endonuclease for intratracheal administration. The therapeutic effect of chimeric decoy ODN was investigated using OVA-induced experimental asthma in mice. Mice were sensitized by intraperitoneal injections of 20 ug of ovalbumin (OVA) and 2 mg aluminum hydroxide constituted in 0.1 ml of saline on days 0 and 14. On days 21, 22 and 23, mice were challenged with 1% OVA aerosol for 20 min. At 24 hours after the last challenge, AHR was measured by methacholine-induced airflow obstruction. Intratracheal administration of decoy ODN was performed in OVA-sensitized mice at 3 days before the first challenge with aerosolized OVA. FITC-labeled chimeric decoy ODN could be detected in macrophages and monocytes migrating into the lung and airway, and NFkB and STAT6 activity were simultaneously inhibited by chimeric decoy ODN. Twenty-four hours after the last OVA challenge, treatment with chimeric or single transfection of NFkB decoy ODN was protected from methacholine-induced AHR, while mice treated with scrambled decoy ODN or saline developed a significantly increase in airway reactivity to methacholine. Importantly, this inhibitory effect of chimeric decoy ODN on airway hyperresponsiveness was significantly greater than that of NFkB decoy ODN. Treatment with chimeric decoy ODN markedly suppressed airway inflammation after OVA sensitization and challenge as compared with control and scrambled decoy ODN treatment. Inflammatory infiltrate, such as macrophage, was significantly inhibited by chimeric decoy ODN through suppression of ICAM-1 and eotaxin expression. In addition, secretion of Th2 cytokines including IL-4, IL-5 and IL-13 in BALF, and histamine in the whole lung were reduced by chimeric decoy ODN. Furthermore, a significant reduction of mucin secretion was observed by chimeric decoy ODN treatment accompanied by a suppression of MUC5AC gene expression. However, intratracheal administration of chimeric decoy ODN did not affected IgE synthesis. The present study provides a novel strategy for treating bronchial asthma by the simultaneous inhibition of both NFkB and STAT6 using chimeric decoy ODN. Further modification of chimeric decoy ODN would be useful to treat asthma as a decoy-based therapy.

Antifibrotic effect of synthetic Smad/Sp1 chimeric decoy oligodeoxynucleotide through the regulation of epithelial mesenchymal transition in unilateral ureteral obstruction model of mice

Renal tubulointerstitial fibrosis is considered to be a common final pathway related to the progressive loss of renal function in chronic kidney disease. It is characterized by the excessive accumulation of extracellular matrix through the pivotal role of epithelial–mesenchymal transition. Transforming growth factor-β1 is postulated to play a central role in renal fibrosis via a downstream pathway such as Smad. Specificity protein 1 (Sp1), which is another transcription factor, is also involved in the basal expression of extracellular matrix. In this study, we investigate the effect of Smad decoy oligodeoxynucleotides (ODN) and Sp1 decoy ODN in unilateral ureteral obstruction induced renal fibrosis in mice. Furthermore, the effectiveness of the newly designed chimeric decoy ODN, which contains both Smad and Sp1 binding sequences in one decoy molecule (Smad/Sp1 chi decoy ODN), was demonstrated. The expression of fibrosis and inflammatory related cytokines and products of fibrosis were ameliorated in the Smad, Sp1 and chimeric decoy ODN treated groups compared with the scrambled decoy ODN treated group. Epithelial–mesenchymal transition was suppressed by the Smad, Sp1 and Smad/Sp1 chi decoy ODN. Immunohistochemistry and Western-blot analysis revealed that Smad/Sp1 chi decoy ODN showed a more significant inhibitory effect on fibrosis and EMT compared with Smad and Sp1 decoy ODNs. These results support the efficacy of Smad/Sp1 chi decoy compared with a single Smad or Sp1 decoy ODNs in preventing renal fibrosis induced by unilateral ureteral obstruction.

Erratum to: The inhibitory effect of chimeric decoy oligodeoxynucleotide against NF-κB and Sp1 in renal interstitial fibrosis

Erratum to: The inhibitory effect of chimeric decoy oligodeoxynucleotide against NF-κB and Sp1 in renal interstitial fibrosis

Effects of Chimeric Decoy Oligodeoxynucleotide in the Regulation of Transcription Factors NF‐κB and Sp1 in an Animal Model of Atherosclerosis

Atherosclerosis is a multifactorial and progressive disease in which the inflammatory reaction and inflammation-related factors play important roles at all stages. Modulation of NF-κB and Sp1 expression may be important targets for the prevention and treatment of atherosclerotic vascular disease. To develop a novel therapeutic approach in atherosclerosis, we examined the simultaneous suppression of the transcription factors NF-κB and Sp1 which regulate inflammation. We employed chimeric decoy oligodeoxynucleotide (ODN) containing the consensus of NF-κB and Sp1-binding sites to suppress these transcription factors simultaneously and to test chimeric decoy for anti-atherogenic effects in an atherogenic diet-induced atherosclerotic mouse model with inflammatory stimulation. C57BL/6 mice were fed with an atherogenic diet (15% fat, 1.25% cholesterol and 0.5% cholic acid) for 12 weeks to induce atherosclerosis; lipopolysaccharide (LPS, 2 mg/kg) was intraperitoneally injected in the first week of study to simulate underlying infectious burden during development of atherosclerosis. Decoy ODNs were injected into tail vein at 2, 4, 6, 8, 10 and 12 weeks after only three LPS injections in mice fed the atherogenic diet. Chimeric decoy ODN alleviated atherosclerotic changes and reduced serum cholesterol and inflammatory cytokines. In accordance with these results, the expressions of atherosclerotic markers were inhibited by chimeric decoy ODN. Chimeric decoy ODN modulates multiple pathogenic aspects of an atherogenic diet-induced atherosclerosis with inflammatory stimulation: hypercholesterolaemia and inflammation. Therefore, this study demonstrates the efficacy of chimeric decoy ODN on atherosclerosis with immunological complication.

The inhibitory effect of chimeric decoy oligodeoxynucleotide against NF-κB and Sp1 in renal interstitial fibrosis

The pathophysiology of chronic renal disease is characterized by a progressive loss of renal function and deposition of the extracellular matrix, leading to widespread tissue fibrosis. Much of the matrix in chronic renal disease is synthesized by interstitial myofibroblasts, recruited from resident fibroblasts and circulating precursors. These changes are believed to be derived from epithelial-mesenchymal transition (EMT) of tubuloepithelial cells. To develop a novel therapeutic approach for treating renal fibrosis, we examined the simultaneous inhibition of the transcription factors NF-κB and Sp1 in a mouse model of unilateral ureteral obstruction (UUO). To simultaneously inhibit both NF-κB and Sp1, we developed chimeric (Chi) decoy oligodeoxynucleotide (ODN) which contained binding sequences for both NF-κB and Sp1 in a single decoy molecule to enhance the effective use of decoy ODN strategy. Chi decoy ODN significantly attenuated tubulointerstitial fibrosis in a mouse model of UUO compared to scrambled decoy ODN, as demonstrated by the reduced interstitial volume, macrophage infiltration, and fibrosis-related gene expression. Interestingly, Chi decoy ODN also regulated EMT-related gene expression, leading to the inhibition of renal fibrotic changes in vivo and in vitro. The present study demonstrates the feasibility of Chi decoy ODN treatment for preventing renal fibrosis and EMT processes. This strategy might be useful to improve the clinical outcome after chronic renal disease.

Regression of Intracranial Aneurysms by Simultaneous Inhibition of Nuclear Factor-kB and Ets With Chimeric Decoy Oligodeoxynucleotide Treatment

Regression of Intracranial Aneurysms by Simultaneous Inhibition of Nuclear Factor-kB and Ets With Chimeric Decoy Oligodeoxynucleotide Treatment

Regression of Intracranial Aneurysms by Simultaneous Inhibition of Nuclear Factor-κB and Ets With Chimeric Decoy Oligodeoxynucleotide Treatment

Despite a high mortality and morbidity of subarachnoid hemorrhage due to an intracranial aneurysm (IA), there is no effective medical treatment to prevent the rupture of IAs. Recent studies have revealed the involvement of the transactivation of proinflammatory genes by nuclear factor-κB (NF-κB) and Ets-1 in the pathogenesis of IA formation and enlargement. To examine the regressive effect of chimeric decoy oligodeoxynucleotides (ODNs), which simultaneously inhibit NF-κB and Ets-1, on IA development in the rat model. One month after IA induction, rats were treated with NF-κB decoy ODNs or chimeric decoy ODNs. Size, media thickness, macrophage infiltration, and collagen biosynthesis in IA walls were analyzed in both groups. The treatment with chimeric decoy ODNs decreased IA size and thickened IA walls of preexisting IAs induced in the rat model, although the treatment with NF-κB decoy ODNs failed to regress preexisting IAs. Chimeric decoy ODN-treated rats exhibited decreased expression of monocyte chemotactic protein-1 and macrophage infiltration in IA walls. In addition, decreased collagen biosynthesis in IA walls was ameliorated in the chimeric decoy ODN-treated group. The results suggest the possibility of a minimally invasive molecular therapy targeting the inhibition of NF-κB and ets-1 for IAs in humans.

The inhibitory effect of chimeric decoy oligodeoxynucleotide in the regulation of transcription factors NF‐¥êB and Sp1 in an animal model of liver cirrhosis

Liver fibrosis is a process of healing and scarring in response to chronic liver injury. Following injury an acute inflammation response takes place resulting in moderate cell necrosis and extracellular matrix damage. To develop a novel therapeutic approach in hepatic fibrogenesis, we examined the simultaneous suppression of the transcription factor NF‐κB and Sp1, which regulate acute inflammation and continuous deposition of extracellular matrix in liver fibrosis. In this study, we employed chimeric decoy oligodeoxynucleotide containing the consensus sequences of both NF‐κB and Sp1 binding sites, to suppress these transcription factors simultaneously. Treatment of chimeric decoy oligodeoxynucleotide reduced the activity of hepatic stellate cells in vitro, and decreased the expression of fibrotic and proinflammatory gene responses in a mouse model of liver fibrosis. These results suggest that chimeric decoy oligodeoxynucleotide strategy might be the potential therapeutic application to prevent liver fibrosis.

간경화 동물모델에서 Chimeric decoy oligodeoxynucleotide로 억제되는 NF-κB와 Sp1 전사인자 발현 억제 효과에 대한 연구

간섬유화는 지속적인 간세포 손상에 대한 수복현상으로 일어나며, 급성 염증반응과 같은 손상이 주어진 후에는 간세포의 괴사 및 세포외기질의 축적이 일어나게 된다. 간섬유화에 대한 새로운 치료방법을 모색하기 위하여 본 연구에서는 간섬유화 과정에서 염증 반응과 관련된 NF-<TEX>$\kappa$</TEX>B와 세포외기질의 축적과 관련된 Sp1전사인자를 동시에 조절하여 간섬유화 억제효과를 관찰하고자 하였다. 전사인자인 Sp1과 NF-<TEX>$\kappa$</TEX>B를 동시에 억제하기 위하여 한 분자 내에 Sp1과 NF-<TEX>$\kappa$</TEX>B의 전사인자와 결합하는 부위를 가지는 Chimeric (Chi) decoy oligodeoxynucleotide (ODN)을 제작하였다. Chi decoy ODN은 활성화된 간성상세포에서 간섬유화 와 관련된 유전자 발현을 억제시켰으며, 섬유화 동물모델에서도 간 조직의 염증 반응 및 섬유화 관련 인자의 발현을 현저히 억제시켰다. 따라서 Chi decoy ODN은 간섬유화 및 활성화된 간성상세포의 활성을 억제할 수 있는 유전자 치료제로 고려될 수 있을 것으로 사료된다. Liver fibrosis is a process of healing and scarring in response to chronic liver injury. Following injury, an acute inflammation response takes place resulting in moderate cell necrosis and extracellular matrix damage. To develop a novel therapeutic approach in hepatic fibrogenesis, we examined the simultaneous suppression of the transcription factors NF-<TEX>$\kappa$</TEX>B and Sp1, which regulate acute inflammation and continuous deposition of extracellular matrix in liver fibrosis. We employed chimeric decoy oligodeoxynucleotide containing the consensus sequences of both NF-<TEX>$\kappa$</TEX>B and Sp1 binding sites, to suppress these transcription factors simultaneously. Treatment of chimeric decoy oligodeoxynucleotide reduced the activity of hepatic stellate cells in vitro, and decreased the expression of fibrotic and proinflammatory gene responses in a mouse model of liver fibrosis. These results suggest that chimeric decoy oligodeoxynucleotide strategy can be a potential therapeutic application to prevent liver fibrosis.

Abstract 430: Inhibition of Aneurysm Development by Intra-peritoneal Application of Ribbon-type Decoy Oligodeoxynucleotide against Nuclear Factor-κB and Ets in a Rat Model

As elective surgical or endovascular repair is not effective to improve survival of small abdominal aortic aneurysm (AAA), the development of a novel therapeutic approach for treating small AAA is awaited. We have previously reported the regressive effect of chimeric decoy oligodeoxynucleotides (ODN) against NFκB and ets in an experimental AAA model. However, local application of conventional phosphorothioated chimeric decoy ODN (PC-ODN) was necessary due to easily degradation. Therefore, we developed a novel chimeric decoy ODN with a ribbon-shaped circular structure (RC-ODN) to stabilize decoy ODN against nucleases. Transfection of RC-ODN inhibited MMP-2 expression in VSMC, but not MMP-9 expression. In contrast, in cultured THP-1 cells, MMP-9 expression was suppressed by RC-ODN, whereas MMP-2 expression was not altered. Interestingly, these inhibitory effects of RC-ODN were more potent than that of PC-ODN (n=8, P&lt;0.05). Next, intra-peritoneal application of RC-ODN was performed in an elastase-induced rat AAA model. One week after injection, FITC-labeled RC-ODN were detected in migrating macrophages into aneurysm wall. In addition, both NFκB and ets activity were suppressed by RC-ODN. Four weeks after treatment, RC-ODN treatment inhibited the aortic dilatation as compared to control or scrambled decoy ODN transfer (n=6, P&lt;0.05), while PC-ODN treatment failed to prevent aneurysm formation. Interestingly, a significant preservation of elastic fibers was observed with RC-ODN treatment, accompanied by a reduction of expression of MMP-9, MMP-12, cathepsin B and cathepsin K (P&lt;0.05), while the recruitment of macrophages was not inhibited. Finally, to consider the clinical application, we investigated the effect of intravenous injection of RC-ODN. Four weeks after infusion of angiotensin II in apolipoprotein E-deficient mice, intravenous injection of RC-ODN (1 mg/Kg) was performed 3 times per week. RC-ODN treatment showed a tendency to decrease the size of AAA, but not significant. In conclusion, the present study provided a novel strategy to treat AAA using intra-peritoneal application of RC-ODN in a rat model. Further development of this strategy would be useful to treat human AAA, as a minimally invasive molecular therapy.

Inhibition of anastomotic intimal hyperplasia using a chimeric decoy strategy against NF B and E2F in a rabbit model

Neointimal formation remains a major limitation after arterial reconstruction. To overcome this problem, we focused on two important transcription factors, nuclear factor-kappaB (NFkappaB) and E2F. The purpose of this study was to determine the effects of simultaneous inhibition of these transcription factors on the formation of neointimal hyperplasia. We employed chimeric decoy oligodeoxynucleotides (ODN) to inhibit both NFkappaB and E2F simultaneously, and examined the effects of chimeric decoy ODN on the proliferation and migration of cultured vascular cells and on the formation of neointimal hyperplasia using prosthetic graft placement in a rabbit hypercholesterolemia model. Our in vitro study demonstrated that transfection of chimeric decoy ODN inhibited platelet-derived growth factor (PDGF)-induced proliferation and migration of vascular smooth muscle cells, whereas endothelial cell proliferation was not inhibited. In an in vivo study, treatment with chimeric decoy ODN significantly inhibited proximal and distal anastomotic intimal hyperplasia, and accelerated re-endothelialization. alpha-Smooth muscle actin (alpha-SMA)-positive cell proliferation was inhibited at the anastomotic sites. Expression of PDGF-BB and PDGF receptor-beta was also suppressed by chimeric decoy ODN, resulting in a reduction of alpha-SMA-positive cell accumulation. In addition, chimeric decoy ODN treatment inhibited macrophage accumulation, which was accompanied by a reduction of vascular cell adhesion molecule-1 and monocyte chemoattractant protein-1 gene expression. The present study demonstrates the feasibility of chimeric decoy ODN treatment for preventing neointimal formation. This strategy might be useful to improve the clinical outcome after arterial reconstruction.

Abstract 483: Prevention of Aneurysm Formation using Ribbon-type Decoy Oligodeoxynucleotides against NFκB and Ets in a Rat Model

Current therapy to treat abdominal aortic aneurysm (AAA) is limited to elective surgical repair. Especially, there is no well-defined treatment strategy for small AAAs. Therefore, the development of a novel therapeutic approach to treat AAA is awaited. We focused on the simultaneous inhibition of the transcription factors, NFκB and ets. Indeed, we previously reported the preventive and regressive effects of chimeric decoy oligodeoxynucleotides (ODN) against NFκB and ets. However, local application of ODN is necessary due to easily degradation. To overcome this limitation, we developed novel chimeric decoy ODN with a ribbon-shaped circular structure (ribbon-type chimeric decoy ODN, RC-ODN) to stabilize decoy ODN against nucleases. First, we examined the stability of RC-ODN. RC-ODN was more stable than the conventional phosphorothioated chimeric decoy ODN (PC-ODN) after incubation in the presence of exonuclease or serum. In addition, the inhibitory effect of RC-ODN on MMP-9 expression in cultured THP-1 cells was more potent than that of PC-ODN (n=8, P&lt;0.005). To achieve a minimally invasive molecular therapy, intra-peritoneal application of RC-ODN was performed in a rat AAA model. One week after injection of FITC-labeled RC-ODN, FITC-positive macrophages, migrated from peritoneal space, could be detected in the aneurismal wall. In addition, EMSA showed that both NFκB and ets activity were inhibited by RC-ODN. 4 weeks after treatment, transfection of RC-ODN inhibited the aortic dilatation as compared to control or ribbon-type scrambled decoy ODN transfer (n=6 per group, RC-ODN 5.66±1.08 mm 2 , Scrambled 13.89±3.55 mm 2 , P&lt;0.05). Interestingly, a significant preservation of elastic fibers was observed with RC-ODN treatment, accompanied by a reduction of MMP-9 activity (P&lt;0.0005) and MMP-12 expression (P&lt;0.0005), while the recruitment of macrophages was not inhibited. The expression of cysteine proteases, cathepsin B and K, was also suppressed by RC-ODN treatment (P&lt;0.05). Here, the present study provided a novel strategy to treat AAA by the simultaneous inhibition of NFκB and ets using intra-peritoneal application of RC-ODN in a rat model. Minimally invasive molecular therapy using this strategy is expected to be useful for treating AAA.

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)kappaB and ets using a decoy strategy. Both NFkappaB and ets were shown to be markedly activated in human AAA. In addition, NFkappaB- and ets-positive cells were increased in the aneurysm wall, and a part of the expression of NFkappaB and ets was detected in migrating macrophages. Thus, we used chimeric decoy oligodeoxynucleotides (ODNs) containing consensus sequences of both NFkappaB 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 NFkappaB and ets is expected to be useful for AAA through the rebalance of matrix synthesis and degradation.

Hypertension accelerated experimental abdominal aortic aneurysm through upregulation of nuclear factor κB and Ets

Conclusion: Hypertension accelerates progression of experimental abdominal aortic aneurysms (AAAs) by upregulation of Ets and nuclear factor κB (NF κB). Summary: The pathogenesis of AAA is complex but appears to involve upregulation of certain matrix metalloproteinases (MMPs). Experimental AAAs are characterized by upregulation of transcription factors, NF κB, which regulate transcription, and Ets, which regulates expression of MMPs. The role of hypertension in pathogenesis of AAA is controversial, with some studies suggesting it contributes to aneurysm formation and other studies suggesting it is not an independent risk factor for aneurysm formation. In this study, the authors used an experimental model of AAA to investigate the effects of hypertension on transcription factors NF κB and Ets. They also investigated the effects of hypertension on progression of AAA. Experimental AAAs were produced by elastase perfusion in both normotensive and hypertensive rats. Compared with normotensive rats, the size of the AAAs increased more rapidly in the hypertensive rats. Western blot analysis indicated the expression of MMP-2, MMP-3, MMP-9, and MMP-12, as well as that of intercellular adhesion molecule, was increased in hypertensive rats. This was accompanied by upregulation of NFκB and Ets. There was also increased activity of MMPs in the aortas of hypertensive rats vs those of normotensive rats. Chimeric decoy oligodeoxynucleotide (ODN) inhibits expression of NFκB and Ets. Transfection of the ODN resulted in significant inhibition of aortic dilatation in both hypertensive and normotensive rats. Transfection with chimeric decoy ODN resulted in significant inhibition of degeneration of elastic fibers in aortas of both normotensive and hypertensive rats. Expression of MMP-2, MMP-3, MMP-9, and MMP-12 and intercellular adhesion molecule was decreased by chimeric decoy ODN. This was accompanied by inhibition of macrophage migration. Comment: The study indicates hypertension accelerates progression of AAA in an experimental rat model. Perhaps inhibition of NF κB and Ets may someday be used as a method of decreasing aneurysm expansion in both normotensive and hypertensive patients.

Hypertension Accelerated Experimental Abdominal Aortic Aneurysm Through Upregulation of Nuclear Factor κB and Ets

In this study, we focused on the effect of hypertension on the transcription factors nuclear factor kappaB (NFkappaB) and ets in the mechanisms of abdominal aortic aneurysm (AAA), and we investigated how hypertension affects the progression of AAA. AAA was produced by elastase perfusion in hypertensive rats and normotensive rats. The size of AAA rapidly increased in hypertensive rats as compared with normotensive rats. Western blot analysis demonstrated that the expression of matrix metalloproteinase (MMP)-2, -3 , -9, and -12, as well as intercellular adhesion molecule, was increased in hypertensive AAA rats, accompanied by upregulation of NFkappaB and ets. Moreover, in situ zymography showed that the activity of MMPs was increased in the aorta of a hypertensive AAA model as compared with that in a normotensive AAA model. Interestingly, transfection of chimeric decoy oligodeoxynucleotide (ODN) resulted in significant inhibition of aortic dilatation both in normotensive and hypertensive rats at 4 weeks after transfection. Destruction of elastic fibers was also significantly inhibited by transfection of chimeric decoy ODN in both hypertensive rats and normotensive rats. The expression of MMP-2, -3, -9, and -12, as well as intercellular adhesion molecule, was significantly attenuated by the chimeric decoy ODN, accompanied by inhibition of the migration of macrophages. Also, the effect of chimeric decoy ODN was confirmed in an organ culture. The present study demonstrated that hypertension accelerated the progression of experimental AAA through upregulation of NFkappaB and ets. Inhibition of NFkappaB and ets could be a novel therapeutic strategy to treat AAA in hypertensive patients.

Prevention of abdominal aortic aneurysms by simultaneous inhibition of NFκB and ets using chimeric decoy oligonucleotides in a rabbit model

Abdominal aortic aneurysm (AAA) is one of the major vascular diseases caused by atherosclerosis. Because treatment for AAA mainly consists of surgery to prevent deaths from AAA rupture and there is a conspicuous absence of alternative therapeutic strategies, the development of minimally invasive treatment is needed. To develop a novel therapeutic approach, we examined the simultaneous inhibition of the transcription factors NFkappaB and ets, which regulate inflammation and matrix degradation, in a rabbit AAA model. In this study, we employed chimeric decoy oligodeoxynucleotides (ODN), containing the consensus sequences of both the NFkappaB- and ets-binding sites, to inhibit both the transcription factors simultaneously. Using a delivery sheet, we examined the inhibitory effect of chimeric decoy ODN on aortic dilatation. Ultrasound and angiographic analysis demonstrated that treatment with chimeric decoy ODN significantly prevented the progression of elastase-induced aortic dilatation. The inhibitory effect of chimeric decoy ODN on aortic dilatation was also confirmed by histological studies. Treatment with chimeric decoy ODN reduced the activities of matrix metalloproteinase (MMP)-2 and MMP-9 and markedly inhibited the proteolysis of elastin as compared to scrambled decoy ODN. Interestingly, treatment with chimeric decoy ODN also suppressed VCAM-1 and MCP-1 gene expression, leading to inhibition of macrophage infiltration in the adventitia and media. The present study in a rabbit model provides a novel strategy to treat AAA by the simultaneous inhibition of both NFkappaB and ets using chimeric decoy ODN. Further modification of chimeric decoy ODN would be useful to treat AAA as a decoy-based therapy.

Inhibition of experimental abdominal aortic aneurysm in the rat by use of decoy oligodeoxynucleotides suppressing activity of nuclear factor kappaB and ets transcription factors.

Two phenomena, inflammation and matrix degradation, contribute to the progression of abdominal aortic aneurysm (AAA). Importantly, the inflammation is regulated by the transcription factor nuclear factor (NF)-kappaB, whereas the destruction and degradation of elastin fibers by matrix metalloproteinases (MMP) are regulated by ets. Thus, we developed a novel strategy to treat AAA by simultaneous inhibition of both NF-kappaB and ets by using chimeric decoy oligodeoxynucleotides (ODN). AAA was induced in rats by transient aortic perfusion with elastase, whereas transfection of decoy ODN was performed by wrapping a delivery sheet containing decoy ODN around the aorta. Gel-mobility shift assay at 7 days after treatment demonstrated that both NF-kappaB and ets binding activity were simultaneously inhibited by chimeric decoy ODN. Transfection of chimeric decoy ODN resulted in significant inhibition of the progression of AAA such as aneurysmal dilation at 4 weeks after treatment as compared with control, accompanied by a reduction of MMP expression. Moreover, the destruction of elastin fibers was inhibited in the aorta transfected with chimeric decoy ODN. Importantly, transfection of chimeric decoy ODN demonstrated potent inhibition of aneurysmal dilatation compared with NF-kappaB decoy ODN alone, whereas scrambled decoy ODN had no effects. Interestingly, the migration of macrophages was significantly inhibited by chimeric decoy ODN. We demonstrated that inhibition of the progression of AAA was achieved by a novel strategy with chimeric decoy ODN used against NF-kappaB and ets in rat model. NF-kappaB and ets are considered to play an important role in the pathogenesis of AAA.

Oxidant Stress as a Marker for Cardiovascular Events

During the past decade, numerous experimental and clinical studies have demonstrated that many common conditions predisposing to atherosclerosis, such as hypercholesterolemia, hypertension, diabetes, and smoking, are associated with a reduced vascular availability of nitric oxide (NO•). Nitric oxide not only produces vasodilation but also has potent antiatherogenic properties. These properties include inhibition of platelet aggregation, prevention of smooth muscle cell proliferation, reduction of lipid peroxidation, and inhibition of adhesion molecule expression. Thus, the loss of NO• observed in these various conditions not only alters vascular tone but also may explain in part why these conditions are risk factors for atherosclerosis. See p 2673 Given this apparent link between loss of nitric oxide and atherosclerosis, several groups have been interested in the concept that endothelium-dependent vasodilation, a surrogate for NO• bioavailability, may predict cardiovascular events. Indeed, Suwaidi et al1 followed 157 patients with mildly diseased coronary arteries for an average of 28 months and observed cardiac events only in the patients with the lowest tertile of coronary vasodilation to acetylcholine. Similarly, in a study of 147 patients, Schachinger et al2 used 3 different stimuli for endothelial release of NO: acetylcholine, cold pressor testing, and increased blood flow. The authors showed that responses to each of these stimuli were independent predictors of cardiovascular events during a follow-up period of ≈8 years. Perticone et al3 also demonstrated that endothelial dysfunction in the forearm circulation predicts cardiovascular events in hypertensive subjects. There have been several explanations for why the various risk factors impair endothelial function. One that has received substantial attention is increased production of reactive oxygen species within the vessel.4 In particular, superoxide (O2•-) reacts rapidly with NO•, resulting in the formation of the peroxynitrite anion and loss of NO• …