NF-kappaB Decoy Research Articles

Inhibition of NFkappaB activation using cis-element 'decoy' of NFkappaB binding site reduces neointimal formation in porcine balloon-injured coronary artery model.

Application of DNA technology to regulate the transcription of disease-related genes has important therapeutic potential. The transcription factor NFkappaB plays a pivotal role in the transactivation of inflammatory and adhesion molecule genes, leading to vascular lesion formation. Double-stranded DNA with high affinity for NFkappaB may be introduced as 'decoy' cis elements to bind NFkappaB and block the activation of genes mediating inflammation, resulting in effective drugs for treating intimal hyperplasia. In this study, we tested the feasibility of NFkappaB decoy therapy to treat neointimal formation in a porcine coronary artery balloon injury model as a pre-clinical study. An angioplasty catheter was introduced into the left anterior descending coronary artery of the pig to cause vascular injury. First, we tested the feasibility of transfection of FITC-labeled NFkappaB decoy ODN using a hydrogel balloon catheter. Fluorescence due to NFkappaB decoy ODN could be detected throughout the medial layer. Therefore, we transfected NFkappaB decoy ODN into the balloon-injured LAD using a hydrogel catheter. Histological evaluation demonstrated that the neointimal area in the balloon-injured artery was significantly reduced by NFkappaB decoy ODN as compared to scrambled decoy ODN at 1 week after single transfection, accompanied by a significant reduction in PCNA-positive stained cells (P < 0.01). Interestingly, the reduction of ICAM-positive staining was observed, accompanied by the inhibition of migration of macrophages. Of importance, intravascular ultrasound (IVUS) confirmed that neointimal area in the balloon-injured artery was significantly reduced by NFkappaB decoy ODN at 4 weeks after transfection (P < 0.01). Interestingly, the inhibition of neointimal area was only limited to the lesion transfected with NFkappaB decoy ODN, while other lesions without NFkappaB decoy ODN demonstrated a marked increase in neointimal formation. Here, we report the successful in vivo transfer of NFkappaB decoy ODN using a hydrogel catheter to inhibit vascular lesion formation in balloon-injured porcine coronary artery.

Cytoplasmic deposition of NFkappaB decoy oligonucleotides is insufficient to inhibit bleomycin-induced pulmonary inflammation.

Lung inflammation leads to severe tissue destruction and ultimately organ failure in a number of diseases, including cystic fibrosis (CF). The transcription factor nuclear factor kappa B (NFkappaB) regulates expression of many pro-inflammatory mediators. We have assessed the effect of topical administration of NFkappaB decoys in a bleomycin model of acute lung inflammation. Using fluorescein-labelled decoy oligonucleotides (ODN) (80 microg/mouse) we have shown that lipid-complexed and 'naked' ODN transfect conducting airway epithelium in a comparable manner (approximately 65% of cells). However, the ODN were detectable in the cytoplasm, but not in the nucleus of transfected cells. An increase of ODN dose to 500 microg/mouse did not increase nuclear transfection significantly. We determined the effect of cytoplasmic NFkappaB decoys on bleomycin-induced inflammation. We transfected mice with 'naked' decoy and scrambled ODN (500 microg) 1 h before intratracheal administration of bleomycin. We measured IL6 secretion in BALF and lung homogenates and total and differential cell counts in BALF 5 days after bleomycin administration. We did not detect a difference between NFkappaB decoy and scrambled ODN-treated animals in any of the parameters tested. We suggest that access of ODN to the nucleus of airway epithelial cells is a key problem, limiting the efficacy of such decoy strategies, as well as attempts at gene repair.

Targeted gene therapy for rat glomerulonephritis using HVJ‐immunoliposomes

Kidney targeted gene transfer has been attempted by many researchers over the last 10 years; however, unfortunately, no reliable technique for gene transfer to the kidney has been established. At experimental level several in vivo gene transfer methods have been reported. We were the first to report successful in vivo gene transfer into the kidney using the HVJ-liposome method. Since then, this method has been modified to achieve highly efficient gene transfer. In this study, we have developed a renal glomerulus-specific gene transfer method using HVJ-liposomes with anti-Thy 1 antibody, OX-7. Following systemic delivery of fluoroisothiocyanate (FITC)-labeled oligodeoxynucleotides (ODN) by HVJ-liposomes coupled with OX-7, we observed fluorescence in renal glomeruli from 2 h post-administration. To examine the efficacy of this delivery system, NF-kappaB or scrambled (SD) decoy ODN was administered by HVJ-liposomes coupled with OX-7 into a crescent glomerulonephritis, anti-glomerular basement membrane (GBM) model. Animals given SD decoy ODN developed severe glomerulonephritis by day 7 with heavy albuminuria, glomerular crescent formation and up-regulated renal expression of IL-1beta and ICAM-1. In contrast, NF-kappaB decoy ODN treatment substantially inhibited the disease with a reduction in alubuminuria, histological damage and the renal expression of inflammatory cytokines. This study has demonstrated that systemic delivery of HVJ-liposomes coupled with OX-7 results in efficient ODN transfer in rat glomeruli. NF-kappaB, but not SD decoy ODN administered systemically via HVJ-liposomes complexed with OX-7 showed clear therapeutic potential for glomerulonephritis. This novel ODN transfer method combined with decoy strategy has the potential to lead to the establishment of a new therapeutic approach to glomerular diseases.

Retroinfusion of NFkappaB decoy oligonucleotide extends cardioprotection achieved by CD18 inhibition in a preclinical study of myocardial ischemia and retroinfusion in pigs.

Myocardial reperfusion injury is partially mediated by postischemic inflammation. Beyond acute PMN recruitment, postischemic inflammation comprises subacute PMN adhesion, eg via NFkappaB activation. In a pig model of 60-min LAD occlusion by PTCA ballon inflation and 1 to 7 days of reperfusion, we investigated the impact of targeted NFkappaB decoy oligonucleotide (ODN) transfection in the area at risk (AAR) on infarct size and regional myocardial function. After 55 min of LAD occlusion, liposomes containing NFkappaB ODN were selectively retroinfused into the anterior interventricular vein for 5 min. Then, retroinfusion was stopped and reperfusion was initiated. Where indicated, CD18 antibody IB4 was infused systemically at 30 min of ischemia. Methylen blue and tetrazolium-red staining were used for quantification of the infarct size. Subendocardial segment shortening (SES) by sonomicrometric crystals in infarct area and AAR was assessed under pacing (expressed as % of control region). NFkappaB decoy ODN retroinfusion reduced infarct size (36 +/- 4% versus 49 +/- 5% in control hearts at day 7), whereas functional reserve of the AAR (SES 73 +/- 17% versus 46 +/- 18% at 180/min) tended to improve. Similar effects were observed after IB4 infusion (38 +/- 5% infarct size, 85 +/- 7% SES at 180/min). A combination of NFkappaB decoy ODN retroinfusion and IB4 infusion further decreased infarct size (26 +/- 2%) and improved functional reserve (SES 94 +/- 6% at 180/min). We conclude that NFkappaB decoy ODN transfection by retroinfusion is feasible in pig hearts and provides postischemic cardioprotection in addition to CD18 blockade.

The structure of PEG-modified poly(ethylene imines) influences biodistribution and pharmacokinetics of their complexes with NF-kappaB decoy in mice.

To study the relationship between structure of poly(ethylene imine-co-ethylene glycol), PEI-PEG, copolymers and physicochemical properties as well as in vivo behavior of their complexes with NF-kappaB decoy. A variety of copolymers of PEG grafted onto PEI as well as PEI grafted onto PEG were synthesized and their complexes with a double stranded 20mer oligonucleotide were examined regarding size, surface charge, biodistribution and pharmacokinetics. Polyplexes of copolymers were smaller compared to polyplexes formed by non-PEGylated PEI 25 kDa (58 - 334 nm vs. 437 nm for a nitrogen/phosphate ratio of 3.5 and 85 - 308 nm vs. 408 nm for N/P 6.0) and showed reduced zeta potential (-2.5 - 6.4 mV vs. 14.5 mV for N/P 6.0). IV injection into mice revealed liver (35-76% of injected dose), kidney (3 - 22%) and spleen (2 - 16%) to be the main target organs for all injected complexes. Complexes formed by copolymers with few PEG blocks of higher molecular weight (5 kDa and 20 kDa) grafted onto PEI 25 kDa did not show different blood levels from PEI 25 kDa. In contrast, a copolymer with more short PEG blocks (550 Da) grafted onto PEI showed elevated blood levels with an increase in AUC of 62 %. A sufficiently high density of PEG molecules is necessary to effectively prevent opsonization and thereby rapid clearance from blood stream.

Nuclear factor-kappaB and TNF-alpha mediate gastric ulceration induced by phorbol myristate acetate.

Phorbol esters induce inflammation in rodents by activating protein kinase C. We determined whether nuclear factor-kappaB (NF-kappaB) and tumor necrosis factor-alpha (TNF-alpha) play role in the formation of gastric ulcer induced by phorbol-12-myristate-13-alphacetate (PMA) in rats. Subserosally injected PMA dose-dependently induced gastric mucosal ulcer. Activation of NF-kappaB in the gastric mucosa corresponding to the PMA injection sites was observed before the ulcers became obvious as assessed by an in situ fluorescence DNA binding assay and electrophoretic mobility shift assay. The NF-kappaB activation and subsequent ulcer formation were significantly inhibited by injection of pyrrolidine dithiocarbamate, proteasome inhibitor (MG132), or NF-kappaB decoy. Antibody against TNF-alpha significantly inhibited ulcer formation without attenuating NF-kappaB activation. These results suggest that both NF-kappaB activation followed by TNF-alpha release contribute to tissue damage in PMA-induced gastric ulcer formation.

The Role of Nitric Oxide and Cyclooxygenase-2 in Attenuating Apoptosis

The production of nitric oxide (NO) is an essential determinant in auto- and paracrine signaling. NO is generated under inflammatory conditions and may serve as a cytotoxic molecule to produce cell demise along an apoptotic or necrotic pathway. NO also gained attention as a regulator of immune function and a death inhibitor. Cytotoxicity because of substantial NO-formation is established to initiate apoptosis, characterized by upregulation of the tumor suppressor p53, changes in the expression of pro- and antiapoptotic Bcl-2 family members, cytochrome c relocation, activation of caspases, and DNA fragmentation. However, NO-toxicity is not a constant value and NO may protect several cell types from entering programmed cell death. Preactivation of macrophages with a nontoxic dose of S-nitrosoglutathione (200 microM) or lipopolysaccharide/interferon-gamma/N(G)-monomethyl-L-arginine for 15 hours attenuated death in response to various agonists, suppressed p53 accumulation, and abrogated caspase activation. Prestimulation of macrophages with cytokines or low-level NO activated the transcription factor NF-kappaB as well as AP-1 and promoted immediate early gene expression of cyclooxygenase-2 (COX-2). NF-kappaB activation comprised p50/p65-heterodimer formation, IkappaB degradation, and activation of a luciferase reporter construct, that contained four copies of the NF-kappaB-site derived from the murine COX-2 promoter. A NF-kappaB decoy approach (oligonucleotides directed against NF-kappaB) or transfection of a dominant-negative c-Jun mutant (TAM67) abrogated not only the COX-2 expression but also the inducible protection. Blocking NO- or cytokine-mediated inducible protection at the level of NF-kappaB and/or AP-1 restored the occurrence of apoptotic features. Our experiments underscore the role of COX-2 in attenuating natural occurring cell death (i.e., apoptosis).

NF-kappa B decoy suppresses cytokine expression and thermal hyperalgesia in a rat neuropathic pain model.

Pro-inflammatory cytokines have been shown to be involved in the genesis, persistence, and severity of neuropathic pain following nerve injury. The transcription factor, nuclear factor-kappa B (NF-kappaB), plays a pivotal role in regulating pro-inflammatory cytokine gene expression. To elucidate the role of NF-kappaB in the pathogenesis of neuropathic pain, using a gene-based approach of NF-kappaB decoy, we tested whether the activated NF-kappaB affected pain behavior via the expression of inflammatory mediators. Single endoneurial injections of NF-kappaB decoy, at the site of nerve lesion, significantly alleviated thermal hyperalgesia for up to 2 weeks and suppressed the expression of mRNA of the inflammatory cytokines, iNOS, and adhesion molecules at the site of nerve injury. This finding suggests that a perineural inflammatory cascade, that involves NF-kappaB, is involved in the pathogenesis of neuropathic pain.

Zinc-induced activation of the human cytomegalovirus major immediate-early promoter is mediated by metallothionein and nuclear factor-kappaB.

We previously reported that major immediate-early promoter (MIEP) activity was regulated by intercellular zinc levels. In this report, we elucidate the mechanisms involved in this phenomenon. In luciferase reporter assays, zinc-induced activation of MIEP (-735/+62) was decreased with deletion of the promoter in stages, and MIEP (-117/+62) did not respond to zinc. The time course of the activity of MIEP responding to diethylenetriamine pentaacetic acid and zinc was not parallel with metallothionein (MT) promoter, which contains metal responsive elements. SV40 promoter that contains AP-1 binding sites, a candidate for the zinc-responsive motif in the MIEP, was not affected by zinc under our conditions. The activation of MIEP (-735/+62) by zinc was prevented with NF-kappaB decoy. When three kappaB motifs from the enhancer in the MIEP were inserted in the front of the zinc-nonresponsive MIEP (-117/+62), it became responsive to zinc. Moreover, overexpression of MT up-regulates the DNA binding of NF-kappaB and NF-kappaB-induced activation of transcription. These findings strongly suggest that MT and NF-kappaB act as mediator/regulator in zinc-induced activation of MIEP.

Intravenous injection of oligodeoxynucleotides to the NF-kappaB binding site inhibits hepatic metastasis of M5076 reticulosarcoma in mice.

We have developed synthetic double-stranded oligodeoxynucleotides (ODN) as 'decoy' cis elements that block the binding of nuclear factors to promoter regions of targeted genes, resulting in the inhibition of gene transactivation in vivo. In the present study, we employed decoy ODN targeting the transcription factor nuclear factor-kappaB (NF-kappaB) binding cis-elements to hepatic metastasis of murine reticulosarcoma M5076 in mice. Intravenous inoculation of M5076 into mice caused a marked increase in gene expression of interleukin-1beta, tumor necrosis factor-alpha and intercellular adhesion molecule-1 in the liver, whereas intravenous treatment with NF-kappaB decoy ODN reduced M5076-induced transactivation of these genes. Treatment with NF-kappaB decoy ODN, but not scrambled decoy ODN, significantly inhibited hepatic metastasis of M5076 in mice, and furthermore the combined treatment of NF-kappaB decoy ODN with an anti-cancer drug resulted in complete inhibition of hepatic metastasis in half of the mice, without affecting myelosuppression induced by the anti-cancer drug. Here, NF-kappaB decoy ODN inhibited hepatic metastasis of M5076 in mice possibly through a decrease in transactivation of important NF-kappaB-driven genes and also potentiated the anti-metastatic effect of an anti-cancer drug, demonstrating the first successful in vivo therapy for cancer metastasis using NF-kappaB decoy ODN as a novel molecular decoy approach.

Nuclear Factor-κB Mediates Simultaneous Induction of Inducible Nitric-Oxide Synthase and Up-Regulation of the Cationic Amino Acid Transporter CAT-2B in Rat Alveolar Macrophages

The connection between the regulation of L-arginine transport and nitric oxide (NO) synthesis was studied in rat alveolar macrophages. Lipopolysaccharides (LPSs) and interferon-gamma stimulated in the same concentration- and time-dependent manner NO synthesis (measured by nitrite accumulation) and L-[(3)H]arginine uptake. This correlated with an increased mRNA expression for iNOS and the cationic amino acid transporter CAT-2B (analyzed by reverse transcription-polymerase chain reaction), with the same kinetics observed for the up-regulation of both mRNAs. Because nuclear factor-kappaB (NF-kappaB) is essential for induction of iNOS its role for the regulation of CAT-2B expression and L-arginine transport was investigated. The NF-kappaB inhibitors pyrrolidine dithiocarbamate and N(alpha)-p-tosyl-L-lysine chloromethyl ketone abrogated LPS- and interferon-gamma-induced increase of nitrite accumulation and L-[(3)H]arginine uptake as well as up-regulation of iNOS and CAT-2B mRNA. LPS-induced increase in iNOS and CAT-2B mRNA was also suppressed by specific NF-kappaB decoy oligodesoxynucleotides, confirming the essential role of NF-kappaB for iNOS and CAT-2B expression. Dexamethasone did not affect the initial (5 h) LPS-induced increase of iNOS and CAT-2B mRNA, but down-regulated both mRNAs after prolonged (20 h) exposure and this was accompanied by partial inhibition of LPS-stimulated nitrite accumulation and L-[(3)H]arginine uptake. These findings demonstrate parallel regulation of the expression of iNOS and CAT-2B, and of NO synthesis and L-arginine uptake in rat alveolar macrophages. NF-kappaB is an essential transcription factor not only for the induction of iNOS, but also for the up-regulation of CAT-2B. The simultaneous up-regulation of CAT-2B with iNOS is considered as a mechanism to ensure a high substrate supply for iNOS.

Decoy against nuclear factor-kappa B attenuates myocardial cell infiltration and arterial neointimal formation in murine cardiac allografts.

Acute rejection and graft arteriopathy in cardiac transplantation limit the long-term survival of recipients; these processes are enhanced by several cytokines and adhesion molecules. Nuclear factor-kappa B (NFkappaB) is critical in the transcription of multiple genes involved in inflammation and cell proliferation. To test the hypothesis that NFkappaB decoy can attenuate acute rejection and arteriopathy, we performed single intraluminal delivery of NFkappaB decoy into murine cardiac allografts using a hemagglutinating virus of Japan (HVJ)-artificial viral envelope (AVE)-liposome method. No decoy or scrambled decoy transfer was performed for control. Hearts were heterotopically transplanted from BALB/c to C3H/He mice (major mismatch group) and from DBA/2 to B10.D2 mice (minor mismatch group). Nontreated or scrambled decoy transfected allografts of the major mismatch group were acutely rejected, while NFkappaB decoy prolonged their survival. While severe cell infiltration and intimal thickening with enhancement of inflammatory factors were observed in untreated or scrambled decoy-treated allografts of minor mismatch group at day 28, NFkappaB decoy attenuated these changes. We conclude that NFkappaB is critically involved in the development of acute as well as chronic rejection of the transplanted hearts. NFkappaB decoy attenuates both acute rejection and graft arteriopathy by blocking the activation of several genes.

Transcription factor decoy for NFkappaB inhibits TNF-alpha-induced cytokine and adhesion molecule expression in vivo.

The expression of several cytokines and adhesion molecules is regulated by the transcription factor NFkappaB, which is activated by tumor necrosis factor alpha (TNF-alpha). In this study, we employed a mouse model of nephritis induced by TNF-alpha to examine whether inhibition of NFkappaB activity using transcription factor decoy oligonucleotides (ODN) blocks cytokine and adhesion molecule expression and attenuates the renal inflammatory response. First, we developed a method for delivering FITC-ODN in vivo into mouse kidney glomeruli by employing HVJ-liposome. Then, in order to study the feasibility of decoy strategy in vivo, the reporter gene chloramphenicol acetyltransferase (CAT) driven by three tandemly repeated NFkappaB binding sequences was transfected into the kidney. Intrapenetorial injection of TNF-alpha stimulated CAT expression in vivo, and the increase in CAT expression was completely abolished by NFkappaB decoy ODN, but not scrambled ODN. Therefore, we examined the effect of NFkappaB decoy ODN transfection on TNF-alpha-induced endogenous interleukin (IL)-1alpha, IL-1beta, IL-6, ICAM-1 and VCAM-1 gene expression as assessed by RT-PCR and Northern blotting. Our present data showed that NFkappaB decoy, but not scrambled, ODN abolished TNF-alpha induced gene expression in vivo, as well as glomerular inflammation as assessed by CD45 staining. Taken together, our results suggest the potential utility of NFkappaB decoy strategy for molecular therapy to glomerular inflammatory diseases.

Transcription factor decoy for NFkappaB inhibits cytokine and adhesion molecule expressions in synovial cells derived from rheumatoid arthritis.

Numerous cytokines are expressed in lesions of synovial hyperplasia of patients with rheumatoid arthritis (RA), and their pathophysiological contributions have been the subject of speculation. These genes are regulated by the transcription factor NFkappaB which in turn is activated by tumour necrosis factor-alpha (TNF-alpha) and cytokines. In this study we examined the inhibition of the production of pro-inflammatory cytokines, adhesion molecule and matrix metalloproteinase (MMP) from synovial tissue of patients with RA by the introduction of synthetic double-stranded DNA with high affinity for the NFkappaB binding site. NFkappaB decoy oligonucleotides (ODN) were introduced with the aid of the haemagglutinating virus of Japan (HVJ)-liposome method into synovial tissue or synovial cells derived from patients with RA. The levels of interleukin-1beta (IL-1beta), IL-6, TNF-alpha, intercellular adhesion molecule-1 (ICAM-1) and MMP-1 were determined by means of enzyme-linked immunosorbent assay (ELISA) and Northern blotting analysis. A cell counting kit was used to study the effect of NFkappaB decoy ODN on synovial cell proliferation. The production of these mediators was significantly inhibited by the introduction of NFkappaB decoy ODN compared with the effect of scrambled decoy ODN. Transfection of NFkappaB decoy ODN resulted in a significant inhibition of synovial cell proliferation as compared with that of scrambled decoy ODN. The results demonstrated in this study suggest the potential usefulness of NFkappaB decoy ODN for gene therapy of inflammatory synovitis of RA.

A role for NF-kappaB-dependent gene transactivation in sunburn.

Exposure of skin to ultraviolet (UV) radiation is known to induce NF-kappaB activation, but the functional role for this pathway in UV-induced cutaneous inflammation remains uncertain. In this study, we examined whether experimentally induced sunburn reactions in mice could be prevented by blocking UV-induced, NF-kappaB-dependent gene transactivation with oligodeoxynucleotides (ODNs) containing the NF-kappaB cis element (NF-kappaB decoy ODNs). UV-induced secretion of IL-1, IL-6, TNF-alpha, and VEGF by skin-derived cell lines was inhibited by the decoy ODNs, but not by the scrambled control ODNs. Systemic or local injection of NF-kappaB decoy ODNs also inhibited cutaneous swelling responses to UV irradiation. Moreover, local UV-induced inflammatory changes (swelling, leukocyte infiltration, epidermal hyperplasia, and accumulation of proinflammatory cytokines) were all inhibited specifically by topically applied decoy ODNs. Importantly, these ODNs had no effect on alternative types of cutaneous inflammation caused by irritant or allergic chemicals. These results indicate that sunburn reactions culminate from inflammatory events that are triggered by UV-activated transcription of NF-kappaB target genes, rather than from nonspecific changes associated with tissue damage.

Hypoxia-induced endothelial apoptosis through nuclear factor-kappaB (NF-kappaB)-mediated bcl-2 suppression: in vivo evidence of the importance of NF-kappaB in endothelial cell regulation.

The transcription factor nuclear factor-kappaB (NF-kappaB) plays a pivotal role in the coordinated transactivation of cytokine and adhesion molecule genes involved in endothelial activation. Although recent reports have documented the contribution of NF-kappaB to apoptosis, it is still controversial. Especially, the role of NF-kappaB in endothelial apoptosis is largely unknown. Hypoxia significantly induced human aortic endothelial cell death and apoptosis in a time-dependent manner (P<0.01), accompanied by NF-kappaB activation. Decrease in total cell number and increase in apoptotic cells induced by hypoxia were significantly attenuated by NF-kappaB decoy, but not by scrambled decoy, oligodeoxynucleotides (ODNs) (P<0.01). Increase in DNA fragmentation induced by hypoxia was also significantly inhibited by NF-kappaB decoy ODNs as compared with scrambled decoy ODNs (P<0.01). Moreover, transfection of NF-kappaB decoy ODNs resulted in a significant decrease in caspase-3-like activity, which is a common pathway for apoptosis, compared with scrambled decoy ODNs. Importantly, transfection of NF-kappaB decoy ODNs significantly increased protein of bcl-2, an inhibitor of apoptosis, and did not alter bax, a promoter of apoptosis, thereby resulting in a significant increase in the ratio of bcl-2 to bax (P<0.01). bcl-2 mRNA was also decreased by hypoxia, whereas transfection of NF-kappaB decoy ODNs significantly attenuated decrease in bcl-2 mRNA. These results demonstrate that activation of NF-kappaB by hypoxia induced endothelial apoptosis in a bcl-2-dependent manner. The importance of NF-kappaB in endothelial apoptosis was confirmed by the observation that pyrrolidine dithiocarbamate, a potent NF-kappaB inhibitor, prevented endothelial apoptosis, caspase 3-like activity, and bcl-2 downregulation induced by hypoxia. To test this hypothesis in vivo, we transfected NF-kappaB decoy ODNs into rat intact carotid artery after reperfusion injury. Reperfusion injury was associated with a significant increase in endothelial apoptosis at 24 hours, whereas NF-kappaB decoy ODN treatment markedly decreased terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL)-positive endothelial cells at 24 hours after reperfusion (P<0.01). Here, using synthetic double-stranded DNA with high affinity for NF-kappaB as a decoy approach, we demonstrated that activation of NF-kappaB by hypoxia caused aortic endothelial cell death and apoptosis through the suppression of bcl-2. NF-kappaB-mediated endothelial apoptosis induced by hypoxia may be involved in the pathogenesis of endothelial dysfunction observed in cardiovascular ischemic diseases.

Evidence that de novo protein synthesis is dispensable for anti-apoptotic effects of NF-κB

The transcription factor NF-kappaB is a positive transcription factor for a number of genes and has been recognized as an anti-apoptotic regulator. However, the mechanism by which NF-kappaB blocks apoptosis is still controversial. Here, we demonstrate the evidence that NF-kappaB could attenuate the TNF-alpha-induced apoptosis without de novo protein synthesis using human pancreatic cancer cell lines, MIA PaCa-2 and Capan-2. The TNF-alpha-induced apoptosis was blocked by IL-1beta, a potent inducer of NF-kappaB activation. This inhibitory effect of IL-1beta was evident when cells were treated with protein synthesis inhibitors such as cycloheximide (CHX). Moreover, NF-kappaB decoy oligonucleotides could not block the anti-apoptotic effect of IL-1beta at doses sufficient to block the NF-kappaB-dependent transcription induced by IL-1beta. To confirm the role of NF-kappaB in blocking apoptosis, we generated stable cell lines expressing IkappaBdeltaN, a highly stable form of IkappaBalpha, a cytoplasmic inhibitor of NF-kappaB. In these stable transfectants, the antiapoptotic effect of IL-1beta was totally abolished, indicating that the anti-apoptotic action of IL-1beta could be ascribed to the NF-kappaB action. These findings show that de novo protein synthesis is dispensable for anti-apoptotic effects of NF-kappaB and support the possibility that NF-kappaB could exert its anti-apoptotic action through protein-protein interaction.

NFkappaB decoy oligodeoxynucleotides reduce monocyte infiltration in renal allografts.

Monocyte influx secondary to ischemia-reperfusion conditions the renal allograft to rejection by presentation of antigens and production of cytokines. Monocyte influx depends on NFkappaB-dependent transcription of genes encoding adhesion molecules and chemokines. Here we demonstrate that cationic liposomes containing phosphorothioated oligodeoxynucleotides (ODN) with the kappaB binding site serving as competitive binding decoy, can prevent TNF-alpha-induced NFkappaB activity in endothelial cells in vitro. In an allogenic rat kidney transplantation model (BN to LEW), we show that perfusing the renal allograft with this decoy prior to transplantation abolishes nuclear NFkappaB activity in vivo and inhibits VCAM-1 expression in the donor endothelium (P<0.05). At 24 h postreperfusion, periarterial infiltration of monocytes/macrophages was significantly reduced in decoy ODN-treated allografts compared to control allografts (3.7+/-0.7 vs. 9.2+/-1.2 macrophages/vessel; P<0.01). At 72 h, there was a reduction of tubulointerstitial macrophage infiltration in decoy ODN-treated kidneys compared to controls (75.6+/-13.9 vs. 120.0+/-11.2 macrophages/tubulointerstitial area; P<0.05). In conclusion, perfusion of the renal allograft with NFkappaB decoy ODN prior to transplantation decreases the initial inflammatory response in a stringent, nonimmunosuppressed allogenic transplantation model. Therefore, the NFkappaB decoy approach may be useful to explore the role of endothelium and macrophages in graft rejection and may be developed into a graft-specific immunosuppressive strategy allowing reduction of systemic immunosuppression on organ transplantation.

Antioxidants inhibit TNFalpha-induced motility and invasion of human osteosarcoma cells: possible involvement of NFkappaB activation.

Osteosarcoma is the most frequent malignant bone tumor in children. It is highly invasive, however, the mechanisms behind osteosarcoma cell invasion are as yet still unknown. In the present study, treatment with TNFalpha enhanced the invasiveness of two human osteosarcoma cell lines, OST and MNNG. TNFalpha treatment also induced tumor cell motility, adhesion to laminin, the expression of matrix metalloproteinase 9 (MMP9), and the nuclear translocation of nuclear factor kappaB (NFkappaB) in the osteosarcoma cells. Moreover, antioxidants inhibited TNFalpha-induced osteosarcoma cell invasion, motility and NFkappaB nuclear translocation, but not adhesion to laminin or MMP9 expression. NFkappaB decoy, another NFkappaB inhibitor, also inhibited TNFalpha-induced osteosarcoma cell invasion and motility. Therefore, motility and NFkappaB activation were possibly related to TNFalpha-induced osteosarcoma cell invasion. However, adhesion to laminin or MMP did not demonstrate any correlation with TNFalpha-induced osteosarcoma cell invasion. Although NFkappaB is known to regulate TNFalpha-induced phenotypes, it may influence only motility and invasion, but not the MMP or laminin-mediated adhesion of these osteosarcoma cells.

Suppressed severity of collagen-induced arthritis by in vivo transfection of nuclear factor kappaB decoy oligodeoxynucleotides as a gene therapy.

In both rheumatoid arthritis and collagen-induced arthritis (CIA), the nuclear factor kappaB (NF-kappaB) transcription factor plays a pivotal role in the coordinated transactivation of many cytokines related to pathogenesis. This study investigated whether synthetic double-stranded DNA that show a high affinity for NF-kappaB could be introduced in vivo as "decoy" cis elements to bind the transcription factor and block the activation of such proinflammatory cytokine genes as interleukin-1 (IL-1) and tumor necrosis factor alpha (TNFalpha), and thus suppress the severity of joint destruction. NF-kappaB decoy oligonucleotides (ODN) were introduced by an intraarticular injection into the bilateral hind ankle joints of CIA rats using the hemagglutinating virus of Japan (HVJ)-liposome method. Joint destruction was evaluated by histology and radiography. IL-1 and TNFalpha levels were assessed by enzyme-linked immunosorbent assay and Northern blot analysis. Using the HVJ-liposome method, the presence of fluorescein isothiocyanate-labeled ODN in the synovium was confirmed until 28 days after intraarticular injection. In vivo transfection of NF-kappaB decoy ODN by an intraarticular injection into CIA rats decreased the severity of hind-paw swelling. Histologic and radiographic studies showed a marked suppression of joint destruction treated by NF-kappaB decoy ODN transfection. This treatment method also suppressed the production of IL-1 and TNFalpha in the synovium of arthritic joints. The present results demonstrate that administration of NF-kappaB decoy ODN in arthritic joints of rats with CIA led to an amelioration of arthritis. These findings suggest that intraarticular transfection of NF-kappaB decoy ODN may provide a useful therapeutic approach for the treatment of inflammatory arthritis.