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(Circulation Research. 2001;89:850.)
© 2001 American Heart Association, Inc.

Editorials

Nuclear Factor-B Decoy

Infiltrating the Heart of the Matter in Inflammatory Heart Disease

Peter P. Liu, Jenny Le, Min Nian

From the Heart & Stroke/Richard Lewar Centre of Excellence, University of Toronto; and Division of Cardiology, Toronto General Hospital, University Health Network, Toronto, Canada.

Correspondence to Dr Peter Liu, Heart & Stroke/RL Centre of Excellence, EN12-324, Toronto General Hospital, 200 Elizabeth St, Toronto, Ontario, M5G 2C4, Canada. E-mail peter.liu{at}utoronto.ca

See related article, pages 899–906

Key Words: myocarditis • inflammation • nuclear factor-B • transcription factor • molecular decoy

Inflammation is part of the host response repertoire in defense against injury. Inflammatory processes play an important role in all the common cardiovascular diseases such as atherosclerosis, myocardial infarction, and heart failure. However, when the inflammatory process becomes uncontrolled, it can become part of the disease. This situation is best exemplified by inflammatory heart diseases such as viral or autoimmune myocarditis, where the immune system can be triggered by either external antigens such as a virus, or internal antigens such as myosin. The activated immune cells then undergo exuberant signal amplification, leading to clonal expansion of T lymphocytes and elaboration of cytokines, resulting in cell and matrix disruption and the phenotype of dilated cardiomyopathy. ^1,2

Giant cell myocarditis is an example of disproportionate immune response, including coalescence of macrophages into giant cells mixed with lymphocytes, with associated high mortality and no specific treatment. To understand the pathogenesis of giant cell myocarditis, an experimental model of autoimmune myocarditis (EAM) induced by myosin antigen mixed with adjuvant in the murine model has been previously developed. ^3,4 The myosin molecule contains a very specific epitope of approximately 8 amino acids in size that can also be mimicked by infectious agents, which is capable of triggering immune cell activation in an MHC-restricted context, leading to T cell–mediated destruction of the myocardium (Figure 1). This has been definitively demonstrated by humanized murine models in which the mouse MHC system has been substituted with the human homologues, and the disease can be perfectly reproduced with immunogens containing human myosin.⁵

View larger version (57K): [in this window] [in a new window]   Figure 1. Participation of the immune system in inflammatory heart disease. The viral exogenous antigen (Ag) or autoimmune myosin antigen can both trigger a T-cell response repertoire in an MHC-restricted manner leading to myocardial cellular and matrix disruptions. This is also facilitated by the antigen-presenting cells including macrophages and dendritic cells, which in turn can also activate antibody-producing B cells. Much of the immune targeting is also mediated by cytokines, which in turn are dependent on inflammatory signaling involving transcription factors such as NF-B. TNF indicates tumor necrosis factor; IFN, interferon; IL, interleukin; TCR, T-cell receptor; MHC, major histocompatibility complex; and Co-stim, costimulatory ligands.

The agents of destruction in this process include cytokines such as tumor necrosis factor (TNF)⁶ and nitric oxide produced in large quantities by the inducible nitric oxide synthase (iNOS).⁷ However, the immune system, being critical for host survival, is extremely redundant in its response. The question is whether there are additional central modulators of immune response that may be targets for therapeutic manipulation.

Potential Role of NF-B as Immune Regulator

The activation of inflammatory response requires coordinated expression of a large number of effector systems to respond to external stress. The external stimuli, including infection, cytokine or neurohormone receptor activation, oxidative stress, matrix alterations, integrin binding, and cytoskeletal deformation, all converge through intracellular signaling pathways upon specific families of nuclear transcription factors, a prominent member being nuclear factor-B (NF-B).⁸

NF-B is a dimeric transcription factor consisting of homodimers or heterodimers of Rel-related proteins. It normally resides in the cytoplasm, complexed with an inhibitor, IB, and is in an inactive form. Upon activation by external stimuli, the inflammatory signals converge on a set of regulatory control IB kinases known as the IKK complex. ⁹ The IKK complexes can phosphorylate IB, leading to its ubiquitination and degradation by the proteosome (Figure 2). The liberated NF-B then enters the nucleus, interacts with B elements in the promoter region of many inflammatory response genes, and activates their transcription. NF-B is thus one of the central regulators of inflammatory response.¹⁰ Additional signaling events, including phosphorylation of NF-B, hyperphosphorylation of IKK, and transcriptional regulation of IB and NF-B, all modulate the level and duration of NF-B activity. The classic antiinflammatory agent aspirin acts by binding IKKß, thereby preventing the activation of NF-B, similar to specific peptide strategies of NF-B inhibition.¹¹

View larger version (46K): [in this window] [in a new window]   Figure 2. The normal activation of NF-B nuclear transcription complex and its potential inhibition through the administration of cis element decoys in binding to the NF-B complex. NF-B normally exists in the cytoplasm as a dimer inhibited by IB. The latter can be dissociated by phosphorylation control via IKK complexes. The NF-B liberated is thus free to enter the nucleus to initiate transcriptional activity but can be "neutralized," in this case by a decoy binding onto the dimer, thus rendering it ineffective in binding onto DNA. AT1 indicates angiotensin receptor 1; Ub, ubiquitin.

The question is whether NF-B also plays a central role in inflammatory heart disease, and whether it could represent a target for therapeutic modulation to alter the outcomes of the disease.

Role of NF-B in Autoimmune Myocarditis: Insight Through Decoys

To test this strategy, Yokoseki and colleagues,¹² in this issue of Circulation Research, have designed an NF-B cis element decoy oligonucleotide (ODN), which was delivered in vivo using an HVJ-AVE-liposome system. The administered decoy ODN competes for the cis NF-B binding site in the promoter region of several cytokine genes as demonstrated by gel-shift assays. The decoy ODN led to the reduced expression and production of ICAM-1, IL-2, TNF, and iNOS in the heart as demonstrated by real-time PCR and Western blotting. This treatment resulted in the reduction in the severity of EAM compared with the control mice by histopathology, heart to body weight ratios, and clinical outcome.

The decoy ODN competes with endogenous B sites for binding to NF-B (Figure 2). This decoy thus mimics, to a certain extent, the natural IB inhibitors by complexing with the NF-B transcription factor. However, it appears to work in the nucleus rather than the cytoplasm, as demonstrated by the nuclear localization of the administered complex. In turn, this strategy served as an important investigative tool to illustrate the central regulatory role played by nuclear factors such as NF-B and its effector systems in the development of autoimmune myocarditis. Furthermore, this seems to be a more effective means of simultaneously modulating several immune response repertoires without having to completely disable a single immune response pathway.

NF-B activation is likely very important for host survival, even though excessive activation can lead to exuberant inflammatory response that paradoxically induces host injury. In the setting of cytokine-induced intracellular activation where there is activation of both prosurvival signals and proapoptotic signals, NF-B is responsible for most of the host survival factors. Thus, modulating NF-B response rather than eliminating it will be an important part of the consideration in its targeting for therapeutic purposes.

The Potential of Molecular Decoys as Tools and Therapies

This decoy strategy of modulating the immune responses also opens the door for future targeted therapeutic manipulation of host immune response repertoire and has been reviewed previously in this journal.¹³ This approach has the advantage that it simultaneously modulated several effector pathways without singling out a particular pathway for complete elimination. This may help to preserve the overall immune response capability while reducing the overall intensity of inflammatory response that is responsible for the immune destruction of the myocardium in autoimmune myocarditis. NF-B decoy has already shown potential benefit in models of tumor metastasis, cytokine-mediated apoptosis, and immune-mediated neural damage.

This strategy may also be applicable in other inflammation-related cardiovascular disease, including viral myocarditis, sepsis-induced cardiac depression, and even generic heart failure or atherosclerosis. However, whether NF-B is the right target in these instances and how much and how long to treat for these conditions are yet to be determined.

It is important to keep in mind that the molecular decoys can sometimes have nonspecific effects in the target cells. Therefore having adequate controls, including scrambled ODNs, meticulous examination of changes in the nuclear transcription factors, and downstream gene transcription targets, will be critical to ensure that a "smart bomb" rather than a "shotgun" approach is achieved.

Acknowledgments

This work was supported in part by grants from the Heart and Stroke Foundation of Ontario and the Canadian Institutes of Health Research (CIHR). Dr Liu is the Heart & Stroke/Polo Chair Professor of Medicine at the University of Toronto.

Footnotes

The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.

References

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