Transfection Of Decoy Oligodeoxynucleotides Research Articles

NF-κB Decoy ODN-Loaded Poly(Lactic-co-glycolic Acid) Nanospheres Inhibit Alveolar Ridge Resorption.

Residual ridge resorption combined with dimensional loss resulting from tooth extraction has a prolonged correlation with early excessive inflammation. Nuclear factor-kappa B (NF-κB) decoy oligodeoxynucleotides (ODNs) are double-stranded DNA sequences capable of downregulating the expression of downstream genes of the NF-κB pathway, which is recognized for regulating prototypical proinflammatory signals, physiological bone metabolism, pathologic bone destruction, and bone regeneration. The aim of this study was to investigate the therapeutic effect of NF-κB decoy ODNs on the extraction sockets of Wistar/ST rats when delivered by poly(lactic-co-glycolic acid) (PLGA) nanospheres. Microcomputed tomography and trabecular bone analysis following treatment with NF-κB decoy ODN-loaded PLGA nanospheres (PLGA-NfDs) demonstrated inhibition of vertical alveolar bone loss with increased bone volume, smoother trabecular bone surface, thicker trabecular bone, larger trabecular number and separation, and fewer bone porosities. Histomorphometric and reverse transcription-quantitative polymerase chain reaction analysis revealed reduced tartrate-resistant acid phosphatase-expressing osteoclasts, interleukin-1β, tumor necrosis factor-α, receptor activator of NF-κB ligand, turnover rate, and increased transforming growth factor-β1 immunopositive reactions and relative gene expression. These data demonstrate that local NF-κB decoy ODN transfection via PLGA-NfD can be used to effectively suppress inflammation in a tooth-extraction socket during the healing process, with the potential to accelerate new bone formation.

Beneficial effect of STAT3 decoy oligodeoxynucleotide transfection on organ injury and mortality in mice with cecal ligation and puncture-induced sepsis

Sepsis is a major clinical challenge with unacceptably high mortality. The signal transducers and activators of transcription (STAT) family of transcription factors is known to activate critical mediators of cytokine responses, and, among this family, STAT3 is implicated to be a key transcription factor in both immunity and inflammatory pathways. We investigated whether in vivo introduction of synthetic double-stranded STAT3 decoy oligodeoxynucleotides (ODNs) can provide benefits for reducing organ injury and mortality in mice with cecal ligation and puncture (CLP)-induced polymicrobial sepsis. We found that STAT3 was rapidly activated in major end-organ tissues following CLP, which was accompanied by activation of the upstream kinase JAK2. Transfection of STAT3 decoy ODNs downregulated pro-inflammatory cytokine/chemokine overproduction in CLP mice. Moreover, STAT3 decoy ODN transfection significantly reduced the increases in tissue mRNAs and proteins of high mobility group box 1 (HMGB1) and strongly suppressed the excessive elevation in serum HMGB1 levels in CLP mice. Finally, STAT3 decoy ODN administration minimized the development of sepsis-driven major end-organ injury and led to a significant survival advantage in mice after CLP. Our results suggest a critical role of STAT3 in the sepsis pathophysiology and the potential usefulness of STAT3 decoy ODNs for sepsis gene therapy.

STAT3 decoy oligodeoxynucleotides improve end‐organ tissue injury and survival in septic mice

STAT3 serves as a key transcription factor in both immunity and inflammatory pathways. We examined whether in vivo introduction of STAT3 decoy oligodeoxynucleotides (ODNs) is beneficial in reducing septic end‐organ injury. Polymicrobial sepsis was induced by cecal ligation and puncture (CLP) in mice. STAT3 was rapidly activated in major end‐organ tissues following CLP, which was accompanied by activation of the upstream kinase JAK2. Transfection of STAT3 decoy ODNs significantly improved histopathological changes in lung, liver, kidney, and heart tissues. STAT3 decoy ODN transfection downregulated pro‐inflammatory cytokine/chemokine overproduction and reduced the increases in tissue and serum levels of HMGB1 in CLP mice. These beneficial effects of STAT3 decoy ODNs led to s significant survival advantage in mice after CLP. Our results indicate a detrimental role of STAT3 in the sepsis pathophysiology and the potential usefulness of STAT3 decoy ODNs for sepsis therapy.Support or Funding InformationGrants‐in‐Aid for Scientific Research from Japan Society for Promotion of Science (17K08586, 19H03757)

ClassC1 decoy oligodeoxynucleotide inhibits profibrotic genes expression in rat hepatic stellate cells.

The aim of the present study was to investigate whether class C1 decoy oligodeoxynucleotides (ODNs) can inhibit the expression of pro-fibrotic genes associated with rat hepatic stellate cell (HSC) activation and hepatic fibrosis. Luciferase reporter assays were performed to test the promoter activities of transforming growth factor (TGF)-β and its downstream target genes following transfection of decoy ODNs and plasmids into HSC-T6 cells, and western blot assays were performed to measure the protein expression of those genes following decoy ODN transfection. Class C1 decoy ODNs were confirmed to inhibit the promoter activity of TGF-β and its downstream target genes, such as type 1 collagen (COLI)α1, tissue inhibitor of metalloproteinases (TIMP)1 and α-smooth muscle actin by Gaussia luciferase reporter assay, and to further downregulate the expression of TGF-β, SMAD3, COLIα1 and TIMP1 by western blotting in activated HSC-T6 cells. In conclusion, class C1 decoy ODNs inhibited pro-fibrotic gene expression in rat HSCS by downregulating TGF-β signaling.

Inhibition of transcription factor T-cell factor 3 (TCF3) using the oligodeoxynucleotide strategy increases embryonic stem cell stemness: possible application in regenerative medicine.

The transcription factor T-cell factor 3 (TCF3), one component of the Wnt pathway, is known as a cell-intrinsic inhibitor of many pluripotency genes in embryonic stem cells (ESCs) that influences the balance between pluripotency and differentiation. In this study, the effects of inhibition of TCF3 transcription factor on the stemness of mouse ESCs (mESCs) were investigated using the decoy oligodeoxynucleotides (ODNs) strategy. The TCF3 decoy and its scramble ODNs were designed and synthesized. The interaction specificity of the TCF3 decoy with the TCF3 transcription factor was evaluated by the electrophoretic mobility shift assay. Subcellular localization was carried out using fluorescence and confocal microscopy. Self-renewal and pluripotency of mESCs were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), cell cycle and apoptosis, alkaline phosphatase (ALP), embryoid body (EB) formation, and real-time assays. All experiments were performed in triplicate. The results showed that knockdown of TCF3 by decoy ODNs transfection in mESCs led to an increase in the cell proliferation, ALP enzyme activity, and master regulatory stemness genes and a decrease in the number and diameter of EBs. These results supported TCF3 as a potential target to maintain the pluripotency and self-renewal capacity of mESCs. Knockdown of the TCF3 transcription factor using decoy ODNs can be a promising method to maintain the stemness of stem cells in regenerative medicine and cell therapy researches.

The beneficial effect of STAT3 decoy oligodeoxynucleotide transfection on multiple organ injury in mice with cecal ligation and puncture-induced sepsis

The beneficial effect of STAT3 decoy oligodeoxynucleotide transfection on multiple organ injury in mice with cecal ligation and puncture-induced sepsis

Activator Protein-1 Decoy Oligodeoxynucleotide Transfection Is Beneficial in Reducing Organ Injury and Mortality in Septic Mice.

Inflammation and apoptosis are decisive mechanisms for the development of end-organ injury in sepsis. Activator protein-1 may play a key role in regulating expression of harmful genes responsible for the pathophysiology of septic end-organ injury along with the major transcription factor nuclear factor-κB. We investigated whether in vivo introduction of circular dumbbell activator protein-1 decoy oligodeoxynucleotides can provide benefits for reducing septic end-organ injury. Laboratory and animal/cell research. University research laboratory. Male BALB/c mice (8-10 wk old). Activator protein-1 decoy oligodeoxynucleotides were effectively delivered into tissues of septic mice in vivo by preparing into a complex with atelocollagen given 1 hour after surgery. Polymicrobial sepsis was induced by cecal ligation and puncture in mice. Activator protein-1 decoy oligodeoxynucleotide transfection inhibited abnormal production of proinflammatory and chemotactic cytokines after cecal ligation and puncture. Histopathologic changes in lung, liver, and kidney tissues after cecal ligation and puncture were improved by activator protein-1 decoy oligodeoxynucleotide administration. When activator protein-1 decoy oligodeoxynucleotides were given, apoptosis induction was strikingly suppressed in lungs, livers, kidneys, and spleens of cecal ligation and puncture mice. These beneficial effects of activator protein-1 decoy oligodeoxynucleotides led to a significant survival advantage in mice after cecal ligation and puncture. Apoptotic gene profiling indicated that activator protein-1 activation was involved in the up-regulation of many of proapoptotic and antiapoptotic genes in cecal ligation and puncture-induced sepsis. Our results indicate a detrimental role of activator protein-1 in the sepsis pathophysiology and the potential usefulness of activator protein-1 decoy oligodeoxynucleotides for the prevention and treatment of septic end-organ failure.

Influence of pentylenetetrazol and NF-κB decoy oligodeoxynucleotides on p38 expression in neuron-like cells.

The aim of this study was to investigate the effects of pentylenetetrazol (PTZ) and nuclear factor κ B (NF-κB) decoy oligodeoxynucleotides (ODNs) on p38 expression in neuron-like PC12 cells. In addition, the role of NF-κB activation in the pathogenesis of epilepsy was explored. p38 expression levels in control and PTZ-treated neuron-like PC12 cells were examined using western blotting. NF-κB decoy ODNs were transfected into the neuron-like PC12 cells using Lipofectamine 2000. NF-κB activation was investigated by confocal laser scanning microscopy (CLSM), and p38 expression levels were assessed using western blotting prior to and following transfection of decoy ODNs. Western blot analysis revealed that p38 levels in PTZ-treated neuron-like PC12 cells were significantly higher than those in control cells. CLSM demonstrated that the decoy ODNs inhibited NF-κB activation in neuron-like PC12 cells, and western blotting indicated that the decoy ODNs did not reduce p38 levels. The results of this study indicate that PTZ enhances p38 expression levels and activates NF-κB in PC12 cells. However, NF-κB does not modulate p38 expression levels.

Transfection of hypoxia‐inducible factor‐1 decoy oligodeoxynucleotides suppresses expression of vascular endothelial growth factor in oral squamous cell carcinoma cells

Vasculature development is thought to be an important aspect in the growth and metastasis of solid tumors. Among the many angiogenic factors produced by tumor cells, vascular endothelial growth factor (VEGF) is considered to play a key role in angiogenic processes. VEGF synthesis is modulated by hypoxia-inducible factor-1 (HIF-1) function within the hypoxic microenvironment of growing cancer tissue. To inhibit HIF-1 activation, oligodeoxynucleotides (ODNs) were synthesized and transferred with either the consensus sequence for HIF-1 binding or a mutated form of this sequence. If we could transfer a large number of ODNs into the cancer cell nucleus, activated HIF-1 might bind to the ODNs, resulting in inhibition of hypoxia-induced VEGF synthesis. We transferred these ODNs into cultured oral squamous cell carcinoma cells (SAS cells) using the hemagglutinating virus of Japan (HVJ)-liposome method. Hypoxia-mediated expression of VEGF by cancer cells was suppressed by transfection of HIF-1 decoy ODNs, but not by mutated HIF-1 decoy ODNs. HIF-1 decoy ODN transfection also inhibited VEGF protein synthesis. These results suggest that transfection with HIF-1 decoy ODNs is effective for regulating tumor growth by reducing VEGF.

Off-target response to decoy oligodeoxynucleotide treatment

Abstract *Background:* The transcription factor PAX2 regulates key developmental processes, including mediation of resistance to apoptosis. Inappropriate PAX2 expression has been implicated in facilitating tumour survival, and we have previously shown that siRNA-mediated blockade of PAX2 signalling at the transcript level in EJ bladder carcinoma cells promotes cell death. In this study, we attempted to disrupt PAX2 transcriptional activity in EJ cells by using a decoy oligodeoxynucleotide (ODN). *Results:* We could not show an interaction between PAX2 and our PAX2 decoy ODN, and in both PAX2-positive EJ and PAX2-negative HEK293 control cells, decoy and control ODN transfection resulted in a marked retardation of cell growth, irrespective of sequence, but not in COS7 and NZM4 melanoma cells. *Conclusions:* Our data indicate that decoy ODN transfection had off-target effects that inhibited cell growth in a cell line-dependent manner, and we suggest caution is required to determine the specificity of decoy ODN sequences before considering their application as a potential therapeutic agent.

Long‐term follow up of initial clinical cases with NF‐κB decoy oligodeoxynucleotide transfection at the site of coronary stenting

An essential transcription factor for inflammation, nuclear factor-kappa B (NF-kappaB), plays a pivotal role for restenosis after percutaneous coronary intervention (PCI). To evaluate the safety and effectiveness of NF-kappaB decoy oligodeoxynucleotides (ODN) to prevent restenosis, we initiated an open-label phase I/IIa clinical trial. Seventeen patients who were suffering from angina with organic coronary stenosis were treated with NF-kappaB decoy ODN after PCI using bare metal stents. Although the average coronary stenosis was 90.8 +/- 7.0% before the stent implantation, the stenosis improved to 1.4 +/- 5.9% after the intervention. Serum MCP-1 levels were significantly suppressed in NF-kappaB decoy ODN treated patients compared to those in non-treated patients on day 3 after the PCI. Ticlopidine treatment was employed, since clopidogrel was not launched in Japan. Six months after the PCI and decoy ODN transfection, significant restenosis was found in only 1 of the 17 patients, and the average restenosis rate was 39.6 +/- 22.3%. No in-stent thrombosis was found and no significant systemic adverse effect occurred in any of the patients in this observation period. These results suggest the clinical usefulness and safety of the NF-kappaB decoy ODN transfer after PCI, although further placebo control trials are necessary.

Nuclear Factor-κB Decoy Oligodeoxynucleotides Prevent Acute Lung Injury in Mice with Cecal Ligation and Puncture-Induced Sepsis

The transcription factor nuclear factor-kappaB (NF-kappaB) plays a key role in expression of many inflammatory genes responsible for the pathophysiology of sepsis-induced acute lung injury. We investigated whether the introduction of synthetic double-stranded oligodeoxynucleotides (ODNs) with consensus NF-kappaB sequence as transcription factor decoy can prevent acute lung injury with suppression of pulmonary expression of multiple genes involved in its pathological process in a cecal ligation and puncture septic mouse model. NF-kappaB decoy ODNs were introduced with the aid of the hemagglutinating virus of Japan-envelope vector method. Northern blot analysis indicated that transfection of NF-kappaB decoy ODN, but not of its scrambled form, resulted in a significant inhibition of sepsis-induced gene overexpression of inducible nitric-oxide synthase (iNOS), cyclooxygenase-2, histamine H(1)-receptor, platelet-activating factor receptor, and bradykinin B(1) and B(2) receptors in lung Histological damage in lungs tissues. (wall thickening, inflammatory infiltrate, and hemorrhage), increased pulmonary vascular permeability, and blood gas exchange impairment were clearly documented in mice after cecal ligation and puncture. These changes were strongly eliminated by the introduction of NF-kappaB decoy but not of scrambled ODN. The effects of the iNOS inhibitor FR260330 on these histological and functional derangements compared unfavorably with those of NF-kappaB decoy ODN transfection. Our results suggest that ODN decoy, acting as in vivo competitor for the transcription factor's ability to bind to cognate recognition sequence, may represent an effective strategy in the treatment of septic acute lung injury.

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.

Inhibitory effects of novel E2F decoy oligodeoxynucleotides on mesangial cell proliferation by coexpression of E2F/DP

Proliferation of glomerular mesangial cells (MCs) is an important feature of several forms of glomerulonephritis. The transcription factor E2F coordinately regulates expression of genes required for cell proliferation, thereby mediating cell growth control. Here we investigated the role of E2F1 and E2F4 expression, with or without co-expression of DP1 or DP2, on cell proliferation in transiently transfected primary rat MCs. In transfected cells, cell proliferation induced by over-expression of E2F was significantly enhanced by co-expression of DP proteins. Previous studies showed that the transfection of decoy oligodeoxynucleotides (ODNs) corresponding to E2F binding sites inhibits cell proliferation. Here we have developed a Ring-E2F (R-E2F) decoy ODN with a circular dumbbell structure and compared its effects with those of a phosphorothioated E2F decoy (PS-E2F decoy) ODN. The R-E2F decoy ODN showed enhanced stability in the presence of nucleases and sera, and inhibited E2F/DP-dependent promoter activity of cell cycle genes more effectively than the PS-E2F decoy ODN. Transfection of R-E2F decoy ODN resulted in strong inhibition of cell cycle gene expression and MC proliferation. Our data suggest that E2F/DP complexes play a critical role in the MC proliferation and that the R-E2F decoy ODN may be a powerful tool for inhibiting cell proliferation.

Therapeutic potential of oligonucleotide-based therapy in cardiovascular disease.

Molecular therapy is emerging as a potential strategy for the treatment of cardiovascular disease such as restenosis after angioplasty, vascular bypass graft occlusion and transplant coronary vasculopathy, for which no known effective therapy exists. One strategy for combating disease has been to target the transcriptional and translational processes. Three approaches have been used to accomplish this. One approach is the use of antisense oligodeoxynucleotides (ODN) that are complimentary to the messenger RNA (mRNA) of interest. The second approach is the use of ribozymes, a unique class of RNA molecules that not only store information but also possess catalytic activity. Ribozymes are known to catalytically cleave specific target RNA species, leading to their degradation, whereas antisense molecules inhibit translation by binding to mRNA sequences on a stoichiometric basis. Thus, theoretically, ribozymes are more effective in inhibiting target-gene expression. A third approach is the transfection of cis-element double-stranded decoy ODN. Transfection of decoy ODN will result in attenuation of authentic cis-trans interaction, leading to the removal of trans-factors from the endogenous cis-elements, with subsequent modulation of gene expression. This novel decoy ODN strategy not only promises therapeutic potential but also represents a powerful tool for the study of endogenous gene regulation in vivo as well as in vitro. In particular, early work relating to graft restenosis appears promising. The application of DNA-based therapies to regulate the transcription of disease-related genes in vivo has important therapeutic potential.

Increased activity of nuclear factor-kappaB participates in cardiovascular remodeling induced by chronic inhibition of nitric oxide synthesis in rats.

Chronic inhibition of endothelial nitric oxide (NO) synthesis by the administration of N(omega)-nitro-L-arginine methyl ester (L-NAME) to rats induces early vascular inflammatory changes [monocyte infiltration into coronary vessels, nuclear factor-kappaB (NF-kappaB) activation, and monocyte chemoattractant protein-1 expression] as well as subsequent arteriosclerosis (medial thickening and perivascular fibrosis) and cardiac fibrosis. However, no direct evidence for the importance of NF-kappaB in this process is known. We examined the effect of a cis element decoy strategy to address the functional importance of NF-kappaB in the pathogenesis of cardiovascular remodeling. We found here that in vivo transfection of cis element decoy oligodeoxynucleotides against NF-kappaB to hearts prevented the L-NAME-induced early inflammation and subsequent coronary vascular medial thickening. In contrast, NF-kappaB decoy oligodeoxynucleotide transfection did not decrease the development of fibrosis, the expression of transforming growth factor-beta(1) mRNA, or systolic pressure overload induced by L-NAME administration. The NF-kappaB system participates importantly in the development of early vascular inflammation and subsequent medial thickening but not in fibrogenesis in this model. The present study may provide a new aspect of how endothelium-derived NO contributes to anti-inflammatory and/or antiarteriosclerotic properties of the vascular endothelium in vivo.

An oligonucleotide decoy for transcription factor E2F inhibits mesangial cell proliferation in vitro.

The transcription factor E2F controls expression of several genes involved in cell proliferation including c-myc, c-myb, proliferating cell nuclear antigen (PCNA), and cdk2 kinase. Having established that both PCNA and cdk2 kinase are induced in rat mesangial cells (MC) by serum stimulation, we attempted to inhibit MC proliferation in vitro by transfecting these cells with cationic liposomes containing a synthetic double-stranded oligodeoxynucleotide (ODN) with high affinity for E2F. Using a gel mobility shift assay, we detected increased specific binding of E2F in MC following serum stimulation. This binding was completely inhibited by preincubation of MC nuclear extracts with the double-stranded ODN with high affinity for E2F but not by preincubation with a missense ODN containing two point mutations. MC were also transfected with a luciferase reporter gene construct containing three E2F binding sites. Luciferase activity was enhanced by serum stimulation of MC, and this effect was specifically abolished by cotransfection of MC with E2F decoy ODN. Furthermore, RT-PCR analysis revealed that serum-induced upregulation of PCNA and cdk2 kinase gene expression was inhibited by E2F decoy ODN transfection but not by transfection of missense ODN. These changes in gene expression were paralleled by a reduction in PCNA and cdk2 kinase protein expression in E2F decoy ODN transfected cells. MC number increased following serum stimulation. This effect was blunted by transfection with E2F decoy ODN but not by transfection of missense ODN. These data suggest that the transcription factor E2F plays a crucial role in the regulation of MC proliferation and that this factor can be successfully targeted to inhibit MC cell cycle progression.

A thiol antioxidant regulates IgE isotype switching by inhibiting activation of nuclear factor-κB

The binding site for nuclear factor-κB (NF-κB) is present at the promoter region of the germline Cϵ gene, but there is little information on whether this factor is involved in regulating IgE synthesis by human B cells. Accordingly, we studied the role of NF-κB in germline Cϵ transcription by using two human Burkitt's lymphoma B cell lines, DND39 and DG75. In both cell lines, n-acetyl-l-cysteine (NAC), a potent thiol antioxidant, inhibited the triggering of the nuclear expression of NF-κB by IL-4 and by anti-CD40 monoclonal antibody. Although IL-4 activated signal transducers and activators of transcription (STAT) 6 in addition to NF-κB, NAC treatment or the transfection of decoy oligodeoxynucleotides for NF-κB or STAT6 only partly blocked IL-4–induced germline Cϵ transcription. However, these two decoy oligodeoxynucleotides together almost completely abrogated IL-4–induced germline Cϵ transcription. Of note, CD40-mediated enhancement of IL-4–driven germline Cϵ transcription was markedly decreased by NAC or by a decoy oligodeoxynucleotide for NF-κB. The effect of NAC was also examined on deletional switch recombination underlying the isotype switch to IgE. NAC inhibited the generation of Sμ/Sϵ switch fragments in normal human B cells costimulated with IL-4 and anti-CD40 monoclonal antibody. It also abolished IL-4–induced upregulation of CD40 but promoted upregulation of CD23. These results suggest that coordination of NF-κB and STAT6 may be required for induction of germline Cϵ transcription by IL-4, and that CD40-mediated NF-κB activation may be important in regulating both enhancement of germline Cϵ transcription and class switching to IgE. (J Allergy Clin Immunol 1997;100:S33-8.)

Role of transcriptional cis-elements, angiotensinogen gene-activating elements, of angiotensinogen gene in blood pressure regulation.

Results of recent genetic studies suggest that the angiotensinogen gene is a possible determinant of hypertension. Using antisense technology, we demonstrated that generation of circulating angiotensinogen is a rate-limiting step in blood pressure regulation. In the present study, we examined how the angiotensinogen gene is regulated in vivo. The transcriptional cis-elements, angiotensinogen gene-activating elements (AGE) 2 and 3, have been reported to regulate angiotensinogen production in human hepatocytes in vitro. To determine the critical transcriptional regulator of angiotensinogen production in vivo, we used synthetic double-stranded oligodeoxynucleotides (ODN) as "decoy" cis-elements to block the binding of nuclear factors to promoter regions of the targeted gene, resulting in the inhibition of gene transactivation. Here we examined whether AGE 2 and AGE 3 in the promoter region of the angiotensinogen gene have a pivotal role in hepatic angiotensinogen production in vivo. Hepatic angiotensinogen mRNA was decreased by the transfection of AGE 2 but not mismatched decoy ODN. Transfection of decoy but not mismatched ODN against AGE 2 resulted in a transient decrease in blood pressure of spontaneously hypertensive rats (SHR), accompanied by a reduction in plasma angiotensinogen and angiotensin II levels. In contrast, transfection of AGE 3 decoy ODN had little effect on blood pressure. Overall, our results demonstrate that transfection of decoy ODN against AGE 2, but not against AGE 3, of the angiotensinogen gene resulted in a transient decrease in high blood pressure of SHR, suggesting that the transcriptional cis-element AGE 2, rather than AGE 3, has an important role in blood pressure regulation through the control of circulating angiotensinogen.