doi: 10.1161/01.RES.0000125646.08156.4D
© 2004 American Heart Association, Inc.
Editorials |
Beyond the Endothelium
NF-
B Regulation of Smooth Muscle Function
From the Department of Pathology, University of Washington School of Medicine, Seattle, Wash. Present address for N.F. is Department of Pharmacological Sciences, University of Milan, Milan, Italy.
Correspondence to Elaine W. Raines, Department of Pathology, Harborview Medical Center, 325 9th Ave, Box 359675, Seattle, WA 98104-2499. E-mail ewraines{at}u.washington.edu
Key Words: restenosis • inflammation • cell adhesion molecules • smooth muscle cells • chemokines
Vascular smooth muscle cells (SMCs) are important for structural integrity of the medial wall but are also central to vascular remodeling in response to injury. Although highly differentiated cells with the ability to regulate vascular tone and extracellular matrix synthesis, SMCs are also very plastic cells that can rapidly respond to local injury and dramatically modify their phenotype. A transcriptional factor implicated in vascular disease is nuclear factor-B (NF-B), a pleiotropic protein complex activated by proinflammatory signals and cellular stress that has been shown to regulate over a hundred cellular genes and different transcriptional programs (Figure). An intensive effort over the past 10 years has focused on the potential role of NF-B in the regulation of endothelial and inflammatory cell responses in vascular pathologies.1 However, recent in vitro and in vivo evidence highlights the importance of NF-B in regulating SMC gene expression and cellular functions after injury.
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In Vivo Evidence Supporting a Critical Role for NF-B in the Vascular Response to Injury
NF-B and its inhibitory proteins (IB, see Figure) form an autoregulatory system that has been linked to vascular disease. Activated NF-B is detected in human atherosclerosis within intimal lesions in SMCs, macrophages, and endothelial cells.2 In contrast, little activated NF-B is detected in normal healthy vessels. After balloon injury of the rat carotid artery, the levels of IB are rapidly reduced in medial SMCs and NF-B activation correlates with SMC proliferation and induced expression of NF-B-dependent genes.3 Despite the link between NF-B activation and vascular disease, it remains unclear to what extent it is a driving force or simply a readout of the inflammatory response.1
Very recently, several studies have tested the functional significance of NF-B activity in the formation of vascular lesions after acute injury of the artery wall.4–7 In the most comprehensive study, blockade of NF-B by adenoviral delivery of IB at the time of balloon injury of the rabbit iliac artery reduced by 30% to 50% the expression of two NF-B–dependent genes, intracellular adhesion molecule-1 and monocyte chemotactic protein-1, and decreased macrophage recruitment by 90% 5 days after injury.4 Further, blockade of NF-B was associated with a failure to upregulate expression of inhibitors of apoptosis (IAPs) and a 3-fold increase in the number of apoptotic cells, primarily in the media at 5 days. A corresponding decrease in lumen narrowing was also shown at 5 weeks that resulted in a lumen gain of 40% due to positive remodeling and an increase in vessel diameter. The neointimal area at 5 weeks was not different between control and IB-treated vessels, and no difference in the rate of SMC proliferation was seen at earlier time points.
Similar inhibition of adhesion molecule expression and macrophage accumulation was seen with administration of NF-B decoy oligodeoxynucleotides in the rat carotid injury model6 and in a porcine balloon injury model in the coronary artery,5 and with adenoviral overexpression of IB after balloon injury of the rat carotid artery.7 However, a difference in these later three studies is that a 30% to 50% reduction in neointimal area was observed at 2 weeks after sacrifice6,7 and at 4 weeks as assessed by intravascular ultrasound and histopathology.5 The demonstration that in vivo NF-B blockade can alter the cellular response to injury and lumen narrowing is exciting, but the critical genes targeted by the treatment and the precise mechanisms remain to be defined. Studies of altered SMC cell phenotype in culture after blockade of NF-B activation, such as the article appearing in this issue describing proinflammatory properties of neointimal SMCs,8 provide some clues.
NF-B Regulates a Proinflammatory Transcriptional Program in Smooth Muscle
In atherosclerosis and after acute injury of the vessel wall, circulating inflammatory cells are recruited to the site of injury.9 Although endothelial cell expression of adhesion molecules is critical for this recruitment, inflammatory infiltrate is still observed after balloon injury that removes the endothelium. The study by Weber and colleagues appearing in this issue of Circulation Research demonstrates that after balloon injury, neointimal SMCs in vivo and in vitro express increased levels of the adhesion molecule P-selectin that support a 2.5-fold enhanced arrest of leukocytes under flow compared with cultured medial SMCs.8 Increased P-selectin expression in neointimal SMCs is stable in culture without exogenous cytokine addition, is associated with enhanced NF-B activity, and blockade of NF-B by overexpression of IB significantly reduces leukocyte arrest. While increased expression of the adhesion molecule P-selectin is critical to enhanced rolling and arrest of leukocytes on neointimal SMCs, arrest is triggered by the chemokines fractalkine and GRO-
for monocytes and by stromal cell-derived factor-1 (SDF-1) for "memory" T cells. In vivo blockade of NF-B–dependent leukocyte arrest and its proinflammatory transcriptional program depicted in the Figure8 could be a significant contributor to the 90% reduction in macrophage recruitment, and possibly retention, after adenoviral delivery of IB to the rabbit iliac artery.4 However, a major limitation of the studies presented is that the functional significance of the SMC "inflammatory phenotype" is primarily drawn from in vitro studies using cultured neointimal SMCs. The in vitro phenotype has been correlated with NF-B activation and inflammatory gene expression in vivo, but it remains to be determined whether SMC-specific inhibition of this proinflammatory transcriptional program in vivo will have a meaningful effect on the vascular response to injury.
Smooth Muscle Remodeling of Extracellular Matrix Is Controlled by NF-B
SMCs are also central to the matrix remodeling associated with acute and chronic injury of the artery wall. We recently demonstrated that NF-B is activated in SMCs cultured on fibrillar collagen gels, and that blockade of NF-B prevents collagen gel contraction and reduces collagen degradation.10 As shown in the Figure, this effect was rescued by the overexpression of two genes regulated by NF-B, 2 integrin and the matrix metalloproteinase-1 (MMP-1). The rescue of collagen gel contraction by 2 integrin was further dependent on MMP activity,10 and MMP-1, MMP-3, and MMP-9 expression are NF-B–dependent in SMCs.11 The identification of at least two NF-B–specific gene products as key mediators of SMC collagen gel remodeling in vitro raises the possibility that these gene products may also contribute to observed changes in vessel remodeling in vivo as recently described in the rabbit iliac artery.4
NF-B and Smooth Muscle Survival and Proliferation
Increased SMC accumulation is thought to contribute to lesion progression in atherosclerosis and restenosis after angioplasty, but evidence also exists for SMC apoptosis, especially in advanced lesions of atherosclerosis. NF-B activation has been shown to be important for proliferation and the prevention of terminal differentiation of multiple cell types,12 including SMCs.13 In several cells, NF-B activation has been linked to cell cycle progression through its transcriptional activation of cyclin D1,12 but a direct connection has not been established for SMCs. Biomechanical strain promotes activation of NF-B and has also been shown to induce expression of iex-1, a gene that inhibits growth of SMCs in vitro and in vivo.14
Within the last decade, NF-B activation has also become synonymous with enhanced cell survival through its ability to regulate a large number of antiapoptotic proteins, although the functional consequences of NF-B activation from different receptors vary and not all signals that activate NF-B are antiapoptotic.15 Cultured SMCs with high NF-B activity have been shown to be less sensitive to apoptosis, express high levels of IAP-1 that is transcriptionally regulated by NF-B, and antisense inhibition of IAP-1 induces SMC death.16 Organ cultures of mouse carotid arteries also demonstrate a 3-fold increase in NF-B DNA binding activity in arteries exposed to high pressure compared with vessels at normal pressure, and SMC apoptosis increases 3-fold if NF-B activation is inhibited.17 While in vivo blockade of NF-B has had variable effects on SMC proliferation, the pronounced 3-fold induction of SMC apoptosis after IB overexpression in the rabbit iliac artery4 suggests a potentially critical role for the induction of antiapoptotic genes such as the IAPs.
Conclusions and Future Directions
A key question in understanding the powerful effects of IB overexpression on vascular injury in vivo is to determine which of the many genes that can be induced by NF-B are responsible for the phenotypic changes in SMCs. Interplay between the different transcriptional programs may also significantly impact the outcome in vivo. SMC-specific loss-of-function experiments will be particularly useful in understanding direct and indirect effects of the multipotent transcriptional program induced by NF-B activation in SMCs.
Acknowledgments
This work was supported by NIH grant HL18645 (E.W.R.).
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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