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Nuclear Factor-B Induction by Visfatin in Human Vascular Endothelial Cells

Its role in MMP-2/9 production and activation

From the Endocrinology & Metabolism Group, Clinical Sciences Research Institute, Warwick Medical School, University of Warwick, Coventry, U.K.

Address correspondence and reprint requests to Dr. Harpal S. Randeva, MBCHB, FRCP, MD, PhD, Endocrinology & Metabolism Group, Clinical Sciences Research Institute, Warwick Medical School, University of Warwick, Coventry CV4 7AL, U.K. E-mail: harpal.randeva{at}warwick.ac.uk

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESEARCH DESIGN AND METHODS-- RESULTS-- CONCLUSIONS-- References

 
OBJECTIVE—Visfatin is elevated in obesity and type 2 diabetes and is thought to be an inflammatory mediator within atherosclerotic lesions and to induce gelatinase activity. We investigated the activation of nuclear factor-B (NF-B), a well-known proinflammatory transcription factor, by visfatin in endothelial cells.

RESEARCH DESIGN AND METHODS—Human endothelial cells were transfected with pNF-B-Luc plasmid. Using quantitative PCR, Western blot analysis, and gelatin zymography, we studied NF-B signaling in gelatinase-mediated vascular inflammation by visfatin using the NF-B inhibitor BAY 11-7085.

RESULTS—Visfatin significantly increased NF-B transcriptional activity (P < 0.001). We also found a significant inhibition of tumor necrosis factor- (TNF-)-induced NF-B activity by visfatin (P < 0.001). Furthermore, the NF-B inhibitor significantly negated visfatin-induced matrix metalloproteinase (MMP)-2/9 mRNA expression, protein levels, and gelatinolytic activity (P < 0.001).

CONCLUSIONS—Visfatin-induced NF-B signaling in human endothelial cells affects the activation of gelatinases MMP-2 and -9, suggesting an important role of visfatin in the pathogenesis of vascular inflammation in obesity and type 2 diabetes.

Abbreviations: HUVEC, human umbilical vein endothelial cell • MMP, matrix metalloproteinase • NF-B, nuclear factor-B • TNF-, tumor necrosis factor-

	INTRODUCTION

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Cardiovascular disease is more common in individuals with diabetes and obesity (1). Adipocytes and stromal vascular cells within adipose tissue directly augment systemic inflammation. Circulating mediators of inflammation participate in the mechanisms of vascular insult and atheromatous change, and many of these inflammatory proteins are secreted directly from adipocytes and adipose tissue–derived macrophages (2).

Visfatin, an adipokine, has been shown to be elevated in obesity, insulin resistance states, and type 2 diabetes (3–5). More recently, it has been suggested that visfatin is an inflammatory mediator, based on its localization in macrophages within atherosclerotic lesions and its ability to induce matrix metalloproteinase (MMP)-9 in monocytes (6). Moreover, we have described the ability of visfatin to induce gelatinases (MMP-2 and -9) in human endothelial cells (7). Interestingly, systemic inflammation mediates multiple pathogenic mechanisms in the well-known associations between obesity and cardiovascular pathology and comorbidities such as type 2 diabetes and the metabolic syndrome (2); these associations, however, are poorly understood.

Nuclear transcription factor B (NF-B) is a major transcription factor in inflammatory responses that regulates a plethora of genes, playing a vital role in the initiation, progression, and rupture of atherosclerotic plaques (8). Crucial enzymes involved in this process are the gelatinases (MMP-2 and MMP-9), the transcription of which is regulated by NF-B (9).

With the aforementioned in mind, we sought to investigate whether visfatin activates NF-B, inducing inflammatory effects in the vascular endothelium.

	RESEARCH DESIGN AND METHODS—

TOP ABSTRACT INTRODUCTION RESEARCH DESIGN AND METHODS-- RESULTS-- CONCLUSIONS-- References

 
We studied NF-B activation by visfatin by stably transfecting a human endothelial cell line, EAHy926 (hybridoma of human umbilical vein endothelial cells [HUVECs] and epithelioma A549 cells), or by transient transfection of HUVECs with a cis-reporter plasmid containing luciferase reporter gene linked to five repeats of NF-B binding sites (pNF-B-Luc; Stratagene, La Jolla, CA). Multiple clones were selected for the analysis of NF-B activation. Furthermore, using quantitative PCR, Western blot analysis, and gelatin zymography, we investigated the involvement of NF-B signaling in gelatinase-mediated vascular inflammation by visfatin using the NF-B inhibitor BAY 11-7085 (see online appendix available at http://dx.doi.org/10.2337/dc07-1544).

	RESULTS—

TOP ABSTRACT INTRODUCTION RESEARCH DESIGN AND METHODS-- RESULTS-- CONCLUSIONS-- References

 
In pNF-B-Luc stably transfected endothelial cells, visfatin induced a significant dose-dependent increase in NF-B–mediated transcriptional activity (Fig. 1A) with a potency comparable with tumor necrosis factor- (TNF-) (10 ng/ml) (data not shown), a robust inducer of NF-B activity. Similar significant results were obtained with transiently transfected HUVECs (online appendix, Fig. A).

View larger version (30K): [in this window] [in a new window]   Figure 1— A: Serum-starved endothelial cells stably transfected with pNF-B-luciferase were treated with or without visfatin (0–1,600 ng/ml) for 2 h. Cells were lysed, and luciferase activities were measured. Visfatin induced a dose-dependent increase in luciferase activity at 2 h. *P < 0.05, **P < 0.01, ***P < 0. 001 vs. basal. B: Serum-starved endothelial cells stably transfected with pNF-B-luciferase were preincubated with or without visfatin (0–1,600 ng/ml) for 16 h, followed by TNF- (10 ng/ml) for 2 h. Similarly, cells were lysed, and luciferase activities were measured. Results showed significant inhibition of TNF-–induced NF-B–mediated transcriptional activity by visfatin, ***P < 0. 001 vs. basal, ##P < 0.01, ###P < 0.001 vs. TNF-–treated endothelial cells. C: Serum-starved endothelial cells treated with visfatin (0–1,600 ng/ml) for 4 h showed a significant dose-dependent increase in MMP-2/9 glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression, **P < 0.01, ***P < 0.001 vs. basal. Furthermore, serum-starved endothelial cells treated with visfatin (1,600 ng/ml) preincubated with BAY 11-7085 (10 µmol/l) for 1 h significantly decreased MMP-2/9 mRNA expression, ###P <0.001 vs. visfatin treated. D–E: Serum-starved endothelial cells treated with visfatin (0–1,600 ng/ml) for 24 h showed a significant dose-dependent increase in MMP-2/9 gelatinolytic activity, **P < 0.01, ***P < 0.001 vs. basal. Furthermore, serum-starved endothelial cells treated with visfatin (1,600 ng/ml) for 24 h and preincubated with BAY 11-7085 (10 µmol/l) for 1 h significantly decreased MMP-2/9 gelatinolytic activity, ###P < 0.001 vs. visfatin treated. Data are means ± SEM of three experiments. Each experiment was carried out in triplicate.

In light of our current observations that visfatin increases NF-B transcriptional activity, we used the NF-B inhibitor BAY 11-7085 to determine its role in visfatin-mediated MMP activation. Interestingly, we found that visfatin-induced MMP-2/9 mRNA expression and gelatinolytic activity were significantly negated with BAY 11-7085 (10 µmol/l) (Fig. 1C–E). Likewise, in visfatin/TNF-–treated endothelial cells, MMP-2/9 protein levels were significantly decreased by preincubation with BAY 11-7085 (10 µmol/l) (online appendix, Fig. B1 and 2).

	CONCLUSIONS—

TOP ABSTRACT INTRODUCTION RESEARCH DESIGN AND METHODS-- RESULTS-- CONCLUSIONS-- References

 
We present novel data showing that visfatin is a profound stimulator of NF-B transcriptional activity in human endothelial cells. Our results also demonstrate the crucial involvement of NF-B signaling in visfatin-induced activation of gelatinases, factors that are important in the pathogenesis of vascular inflammation.

Furthermore, we present novel data that visfatin induces hyporesponsiveness of NF-B–mediated transcriptional activity in human endothelial cells. These findings are of importance given the fact that obesity and type 2 diabetes are states of proinflammatory cytokine "overload" (10). It can be said, therefore, that this dysregulation of NF-B signaling induced by visfatin in endothelial cells may affect the fine balance of the varied inflammatory responses present in these dysmetabolic states.

In vascular inflammatory responses, NF-B signaling is an important regulator of endothelial adhesion molecules, chemokines, and MMPs (11), all key enzymes involved in disruption of atherosclerotic plaques and in vessel wall remodelling as part of an inflammatory response. Interestingly, Dahl et al. (6) have recently suggested that visfatin may play a role in plaque destabilization, given that macrophages are laden with visfatin, which induces MMP-9 in human THP-1 monocytes. More recently, we have demonstrated visfatin's angiogenic potential in endothelial cells (7) and that dysregulated angiogenesis, as seen in diabetes or chronic inflammation, involves the MMP system. Thus, activation of NF-B by visfatin may play an important role in vascular pathology associated with obesity and type 2 diabetes. Our data support this idea because visfatin-induced MMP-2/9 production and activities were profoundly negated by the NF-B inhibitor BAY 11-7085.

The physiological/pathophysiological significance of our findings may pertain to the observations that visfatin levels are raised in obesity and diabetes and that MMP-2 and -9 play a critical role in vascular pathology. Given visfatin's involvement in plaque destabilization (6), our novel findings of NF-B induction by visfatin in endothelial cells add a new perspective to visfatin's proinflammatory role. The limitation of our in vitro study needs to be further clarified in vivo. In summary, our findings introduce a novel insight into visfatin's diverse role in the development of the metabolic syndrome and reaffirm the emerging role of adipokines as mediators of inflammatory responses.

	Acknowledgments

 
The General Charities of the City of Coventry funded this study.

H.S.R. acknowledges S. Waheguru, University of Warwick, for his continual support. We also acknowledge Dr. C.J. Edgell for kindly gifting the EAHy926 cell line.

	Footnotes

 
Published ahead of print at http://care.diabetesjournals.org on 9 January 2008. DOI: 10.2337/dc07-1544.

Additional information for this article can be found in an online appendix at http://dx.doi.org/10.2337/dc07-1544.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C Section 1734 solely to indicate this fact.

Received for publication August 6, 2007. Accepted for publication December 25, 2007.

	References

TOP ABSTRACT INTRODUCTION RESEARCH DESIGN AND METHODS-- RESULTS-- CONCLUSIONS-- References

 

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This Article Abstract Full Text (PDF) Online-Only Appendix All Versions of this Article: dc07-1544v1 31/4/758    most recent Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Adya, R. Articles by Randeva, H. S. Search for Related Content PubMed PubMed Citation Articles by Adya, R. Articles by Randeva, H. S. Social Bookmarking                What's this?