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(Circulation. 2003;108:1164.)
© 2003 American Heart Association, Inc.

Focused Perspective

Cyclic AMP Response Element-Binding Protein in the Vessel Wall

Good or Bad?

Jane E.B. Reusch, MD; Dwight J. Klemm, PhD

From the Denver VA Medical Center and University of Colorado Health Sciences Center.

Correspondence to Jane E.B. Reusch, MD, Denver VA Medical Center Endo 111-H, 1055 Clermont St, Denver, CO 80220. E-mail jane.reusch{at}uchsc.edu

Key Words: Editorials • protein, DNA-binding, cyclic AMP–responsive • atherosclerosis • restenosis • cells, smooth muscle, vascular

	Introduction

Top Introduction CREB in Vascular Tissues So, Is CREB Good... Conclusions References

 
Atherosclerosis and postangioplasty restenosis are leading causes of death in the Western world. Recent advances in our understanding of vascular biology would suggest that targeting specific signals in the post–balloon injury vessel or in persons prone to development of atherosclerosis could decrease lesion formation and in the long term decrease cardiovascular events. In light of this, numerous laboratories around the world are investigating the functional activities of the various cellular components of the atherosclerotic plaque, including vascular smooth muscle cells (SMCs), endothelial cells (ECs), and monocyte/macrophages.

See p 1246

One of the strategies for examining vascular wall response to injury in animal models has been the use of the balloon injury model. In this model, a balloon catheter is used to cause an injury and denuding of the endothelium. The animals are monitored serially over 7, 14, and 28 days for narrowing of the blood vessel lumen after injury. This model traditionally has been used to examine activation of SMCs and their proliferative response. In response to balloon injury, vascular SMCs (which are normally contractile and quiescent [nonproliferative]) are released into a proliferation phase by the injury and loss of the endothelium.

Immunostaining studies have suggested that the majority of the cells that cause luminal narrowing are SMCs.¹ However, recent studies have also demonstrated the presence of inflammatory cells in this model.² Macrophages are observed both in the balloon injury model and in balloon injury/stent models. Thus, interfering with either SMC function or macrophage activation would be expected to have an impact on restenosis.

Agents that can be delivered either pharmacologically or directly at the time of angioplasty to decrease neointimal proliferation are considered to have therapeutic promise. The most useful are agents that decrease SMC proliferation. In the present issue of Circulation, Tokunou et al³ present a series of experiments that indicate that delivery of a DNA-binding dominant negative isoform of the transcription factor CREB (cyclic AMP response element-binding protein) at the time of balloon injury leads to a decrease in neointimal thickening. At first glance, this study may appear to contradict our recent reports that CREB restrains mitogen-stimulated SMC proliferation and migration.⁴ In the present commentary, we will review the available data on CREB in macrophages and SMCs and offer our view of the studies by Tokonou et al.³

	CREB in Vascular Tissues

Top Introduction CREB in Vascular Tissues So, Is CREB Good... Conclusions References

 
CREB is a 43-kDa nuclear transcription factor in the beta leucine zipper family of transcription factors, and it has been shown to have important functions in differentiation of numerous target tissues, including adipocytes, vascular SMCs, neurons, and cardiac myocytes. One additional function of CREB is the prevention of apoptosis.⁵ This has been carefully outlined in neuronal cells, fat cells, and most recently, beta cells.^6–8 Additional unpublished studies suggest that CREB may also play a pivotal role in terminal macrophage differentiation (C.K. Glass, MD, Professor of Medicine, University of California San Diego, personal communication, 2003). No published studies have examined CREB in ECs. Given the potent impact of CREB on numerous vascular cell types, CREB would be expected to have pleiotropic effects on the vessel wall.

SMC proliferation is the primary end point for most investigators studying the balloon injury/restenosis model. The question is what the expected impact of CREB on vascular SMC proliferation and migration would be. Our laboratory has demonstrated both in vivo and in vitro that CREB content correlates negatively with proliferation.⁴ In addition, expression of active CREB decreases mitogen-stimulated proliferative capacity and migratory capacity. Expression of dominant negative CREB augments the ability of platelet-derived growth factor to stimulate proliferation and migration in vitro. CREB decreases expression of numerous cell cycle–regulatory proteins, as well as expression of the growth factor receptors endothelin, endothelin 1 receptor, and platelet-derived growth factor receptor .^4,9 In the study by Tokunou et al³ in the present issue of Circulation, the authors demonstrate in vitro that adenoviral delivery of dominant negative CREB into SMCs in culture leads to increased smooth muscle apoptosis and no appreciable change in entry through cell cycle.

So how do we resolve these divergent and paradoxical results? First, we should consider the noncontroversial aspects of this work. The fact that interference with CREB activity through a dominant negative isoform of CREB would augment apoptosis is an expected result, consistent with studies conducted in our laboratory (J.E.B. Reusch, MD, and P.A. Watson, PhD, unpublished data, 2003) and in numerous other cell types and cell culture systems. The second issue, the apparent discrepancy between acute CREB activation of prothrombotic genes (thrombin) and restraint of mitogen-stimulated proliferation, could be interpreted as follows. Loss of CREB function in unstimulated SMCs in culture does not affect proliferation in a robust way (observed by both groups). In contrast, in Figure 5 of their article, Tokunou et al³ demonstrate a modest decrease in BrdU labeling in the intimal layer and a robust increase in apoptotic cell death in vessels treated with the dominant negative CREB. The authors interpret this decrease in proliferative index as evidence that dominant negative CREB decreases SMC proliferation. There is no clear histological evidence that the cells infected with the dominant negative CREB are nonproliferative (ie, double staining). Another explanation for decreased intimal proliferation could be loss of the stimulatory effects of macrophages and ECs. Interference with macrophage function decreases neointimal lesion formation in other animal models.^1,2,10,11 If CREB is a key determinant of macrophage terminal differentiation, then you might expect to see less macrophage accumulation in these lesions and therefore less mitogen stimulation of SMC proliferation. The impact of CREB on ECs is unknown. In the balloon injury model, a large amount of virus is delivered to the endothelium. Dominant negative CREB could easily induce apoptosis in the ECs and thereby decrease EC cytokine production. This would be a reasonable interpretation of the studies presented, in light of the fact that this particular phosphorylation-deficient CREB mutant, CREBM1, has normal DNA binding and does not appear to have an impact on proliferative capacity in the basal state (Figure 1).

View larger version (14K): [in this window] [in a new window]   Figure 1. Regulation of CREB in vascular SMCs. In the healthy vessel wall (left), transient extracellular signals stimulate phosphorylation of CREB, which increases its transcriptional activity. Subsequent elevation of CREB-dependent gene expression promotes SMC differentiation, maintenance of the mature SMC phenotype, immediate early gene (IEG) expression, and cell survival. However, chronic extracellular signaling in the diseased vessel wall (right) decreases total CREB and phosphorylated (activated) CREB levels in SMCs, with concomitant reductions in CREB-dependent gene expression. These events promote SMC dedifferentiation and apoptosis. Similar events may also occur in macrophages and ECs, further implicating CREB in both normal and pathological vascular processes. CRE indicates cyclic AMP response element.

	So, Is CREB Good or Bad in the Vessel Wall?

Top Introduction CREB in Vascular Tissues So, Is CREB Good... Conclusions References

 
Transcriptional regulation has an impact on SMC phenotype, in terms of modulation between a synthetic (active) and a contractile (quiescent) phenotype. It is a very complex system with numerous checks and balances. Russell Ross¹² described cyclic AMP as the mitogenic gate, which was an inhibitor in most instances of smooth muscle proliferation, with the exception of a change in cellular context where agents such as endothelin and angiotensin 2 could drive SMC proliferation. Because of high cyclic nucleotide abundance in SMCs, most CREB that is present in SMCs is in the phosphorylated or active form. CREB content is very high in the vascular SMCs of young healthy animals, both rodents and monkeys (K.D. O’Brien and T.O. McDonald, unpublished data, 2002). Vascular CREB content is diminished in numerous rodent models of atherosclerosis disease–prone states, such as the insulin-resistant ob/ob mouse, Zucker rat, and most recently, the fat-fed LDL receptor–null mouse¹³ (J.E.B. Reusch, MD, and K.D. O’Brien, unpublished data, 2002). In addition, vascular CREB content is decreased with aging in rodent models).¹⁴ We have therefore postulated that CREB is a gatekeeper for protection from transition to the proliferative SMC phenotype. We have taken that hypothesis further to suggest that loss of CREB in the rodent models is a permissive step for SMC activation.

If CREB is an important vasculoprotective transcription factor, how would one explain CREB as a target of the vasculotoxic agents endothelin and angiotensin or as a prime regulator of the procoagulant factor thrombin? The simple answer is that we don’t yet know. The more speculative response (Figure 1) is that CREB is an immediate early gene, which responds in concert with AP1 factors and nuclear factor-B to numerous toxic stimuli, not only in vascular SMCs, but also in other cell types such as neurons, beta cells, fat cells, and various lymphocytes. In the context of metabolic and cytokine stress, we have observed in vitro and in vivo that there is an acute and robust activation of CREB and numerous other inflammatory cytokines and immediate early genes. With time, chronic metabolic stress leads to loss of both acute signaling to CREB through the AKT signaling pathway and decreased CREB expression in numerous target tissues, including the heart, the vasculature, and the nervous system. An important study by Mabuchi et al¹⁵ demonstrated that acute neuronal injury to the brain activated cyclic AMP response element (CRE)–responsive genes very robustly. This was interpreted to be a cytoprotective response. It is also likely that the acute activation and phosphorylation of CREB with balloon injury is a cytoprotective response and that CREB working in concert with other immediate early genes is an appropriate response to injury.

	Conclusions

Top Introduction CREB in Vascular Tissues So, Is CREB Good... Conclusions References

 
The article in the present issue of Circulation by Tokunou et al³ demonstrates that adenoviral delivery of DNA-binding dominant negative CREB M1 at the time of balloon injury leads to decreased neointimal lesion formation and slows down restenosis. Our interpretation of these data is that dominant negative CREB acts by increasing apoptosis of ECs and vascular SMCs and interfering with recruitment and activation of macrophages. Because this CREB M1 vector decreases intimal lesions, it could be a reasonable pharmacological target. In contrast, the interpretation that adenoviral delivery of that dominant negative CREB interferes with SMC proliferation is unlikely, according to their in vitro studies and the literature. Transgenic or SMC-specific adenoviral delivery of CREB would be necessary to clear up any remaining controversy. In order to leave the reader with a more general understanding of how CREB may be contributing to the vascular lesion, the model shown in Figure 2 is proposed.

View larger version (34K): [in this window] [in a new window]   Figure 2. Vascular response to dominant negative CREB. Introduction of dominant negative CREB M1 reduces CREB transcriptional activity. In vascular SMCs (top), reduced CREB activity promotes SMC apoptosis and increases proliferative potential. Recent evidence suggests that CREB may also play a role in the differentiation of monocytes to macrophages. Therefore, CREB M1 is expected to block the development of macrophages and transition to foam cells that contribute to atherosclerosis (middle). The role of CREB in ECs is largely unexplored, but it may participate in EC survival or in cytokine and growth factor production (bottom). Thus, dominant negative CREB M1 may beneficially decrease neointima formation by stimulating SMC apoptosis, blocking macrophage and foam cell development, and altering EC function.

	Acknowledgments

 
This work is supported by VA Merit review and Research Enhancement Awards Program (Drs Reusch and Klemm); the National Institutes of Health (RO1DK53969 to Dr Klemm and PO1 HL 14985 to Drs Reusch and Klemm); the American Heart Association; the American Diabetes Association; and the Juvenile Diabetes Research Foundation (Dr Reusch).

	Footnotes

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

	References

Top Introduction CREB in Vascular Tissues So, Is CREB Good... Conclusions References

 
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2. Bayes-Genis ACJ, Carlson PJ, Holmes DR, et al. Macrophages, myofibroblasts and neointimal hyperplasia after coronary artery injury and repair. Atherosclerosis. 2002; 1: 89–98.

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4. Klemm DJ, Watson PA, Frid MG, et al. cAMP response element-binding protein content is a molecular determinant of smooth muscle cell proliferation and migration. J Biol Chem. 2001; 276: 46132–46141.[Abstract/Free Full Text]

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6. Reusch JE, Klemm DJ. Inhibition of cAMP-response element-binding protein activity decreases protein kinase B/Akt expression in 3T3-L1 adipocytes and induces apoptosis. J Biol Chem. 2002; 277: 1426–1432.[Abstract/Free Full Text]

7. Pugazhenthi S, Nesterova A, Sable C, et al. Akt/protein kinase B up-regulates Bcl-2 expression through cAMP-response element-binding protein. J Biol Chem. 2000; 275: 10761–10766.[Abstract/Free Full Text]

8. Jambal P, Masterson S, Nesterova A, et al. Cytokine-mediated down-regulation of the transcription factor cAMP-response element-binding protein in pancreatic beta-cells. J Biol Chem. 2003; 278: 23055–23065.[Abstract/Free Full Text]

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11. Ishizuka TMK, Matsui T, Takase B, et al. Ramatroban, a thromboxane A2 receptor antagonist, prevents macrophage accumulation and neointimal formation after balloon arterial injury in cholesterol-fed rabbits. J Cardiovasc Pharmacol. 2003; 41: 571–578.[CrossRef][Medline] [Order article via Infotrieve]

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14. Watson PA, Nesterva A, Grippa J, et al. Aging mediated changes in vascular CREB can be reversed by caloric restriction. In: Endocrine Society Program and Abstracts Book, Annual 85th Meeting. Chevy Chase, Md: The Endocrine Society; 2003.

15. Mabuchi T, Kitagawa K, Kuwabara K, et al. Phosphorylation of cAMP response element-binding protein in hippocampal neurons as a protective response after exposure to glutamate in vitro and ischemia in vivo. J Neurosci. 2001; 21: 9204–9213.[Abstract/Free Full Text]

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