Fibrinogen regulates the expression of inflammatory chemokines through NF-κB activation of endothelial cells

Min Guo; Sanjeev K. Sahni; Abha Sahni; Charles W. Francis

doi:10.1160/TH04-04-0261

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2004; 92(04): 858-866
DOI: 10.1160/TH04-04-0261

Endothelium and Vascular Development

Schattauer GmbH

Fibrinogen regulates the expression of inflammatory chemokines through NF-κB activation of endothelial cells

Min Guo

¹Hematology-Oncology Unit, Department of Medicine, University of Rochester School of Medicine and Dentistry, Rochester, New York, USA

,

Sanjeev K. Sahni

¹Hematology-Oncology Unit, Department of Medicine, University of Rochester School of Medicine and Dentistry, Rochester, New York, USA

,

Abha Sahni

¹Hematology-Oncology Unit, Department of Medicine, University of Rochester School of Medicine and Dentistry, Rochester, New York, USA

,

Charles W. Francis

¹Hematology-Oncology Unit, Department of Medicine, University of Rochester School of Medicine and Dentistry, Rochester, New York, USA

› Author Affiliations

Abstract

Summary

The objective of this study was to characterize the role of fibrinogen in stimulating expression of inflammatory chemokines in endothelial cells through NF-κB activation. Human umbilical vein endothelial cells (HUVEC) were exposed to fibrinogen up to 3,000 µg/ml, and NF-κB activation was assessed using electrophoretic mobility shift assay (EMSA). Fibrinogen exposure resulted in a concentration dependent increase in NF-κB activation that reached a maximum at 1,000 µg/ml after 4 hours and was sustained up to 24 hours. The effect was inhibited by antibodies to α_vβ₃ and α₅β₁ and by the GRGDS peptide, indicating integrin involvement. Preincubation with Mn²⁺ lowered the fibrinogen concentration-dependence, consistent with integrin activation. Supershift assays demonstrated involvement of the p50, p65 and c-Rel components of NF-κB. Fibrinogen exposure also resulted in up-regulation of expression of monocyte chemoattractant protein-1 (MCP-1) and of interleukin-8 as shown by RNase protection assays and by real-time RT-PCR. Increased secretion of MCP-1 was confirmed by ELISA. Parthenolide, an IκB kinase inhibitor, prevented up-regulation of MCP-1 by fibrinogen, linking this response to NF-κB activation. From our findings, we conclude that fibrinogen regulates NF-κB activation and expression of inflammatory chemokines in endothelial cells and may be involved in mediating inflammatory processes.

Keywords

Fibrinogen - NF-κB - chemokines - endothelial cells

Full Text

References

References
1 Conforti G, Dominguez-Jimenez C, Zanetti A. et al. Human endothelial cells express integrin receptors on the luminal aspect of their membrane. Blood 1992; 80: 437-46.
2 Bini A, Kudryk BJ. Fibrinogen in human atherosclerosis. Ann N Y Acad Sci 1995; 748: 461-71 discussion 71-3..
3 Danesh J, Collins R, Appleby P. et al. Association of fibrinogen, C-reactive protein, albumin, or leukocyte count with coronary heart disease: meta-analyses of prospective studies. JAMA 1998; 279: 1477-82.
4 Ross R. Atherosclerosis—an inflammatory disease. N Engl J Med 1999; 340: 115-26.
5 Shin WS, Szuba A, Rockson SG. The role of chemokines in human cardiovascular pathology: enhanced biological insights. Atherosclerosis 2002; 160: 91-102.
6 Gerszten RE, Garcia-Zepeda EA, Lim YC. et al. MCP-1 and IL-8 trigger firm adhesion of monocytes to vascular endothelium under flow conditions. Nature 1999; 398: 718-23.
7 Rollins BJ, Yoshimura T, Leonard EJ. et al. Cytokine-activated human endothelial cells synthesize and secrete a monocyte chemoattractant, MCP-1/JE. Am J Pathol 1990; 136: 1229-33.
8 Zeiher AM, Fisslthaler B, Schray-Utz B. et al. Nitric oxide modulates the expression of monocyte chemoattractant protein 1 in cultured human endothelial cells. Circ Res 1995; 76: 980-6.
9 Lind L. Circulating markers of inflammation and atherosclerosis. Atherosclerosis 2003; 169: 203-14.
10 Tak PP, Firestein GS. NF-κB: a key role in inflammatory diseases. J Clin Invest 2001; 107: 7-11.
11 De Martin R, Hoeth M, Hofer-Warbinek R. et al. The transcription factor NF-kappa B and the regulation of vascular cell function. Arterioscler Thromb Vasc Biol 2000; 20: E83-8.
12 Bunce LA, Sporn LA, Francis CW. Endothelial cell spreading on fibrin requires fibrinopeptide B cleavage and amino acid residues 15-42 of the beta chain. J Clin Invest 1992; 89: 842-50.
13 Gimbrone Jr MA, Cotran RS, Folkman J. Human vascular endothelial cells in culture. Growth and DNA synthesis. J Cell Biol 1974; 60: 673-84.
14 Sitrin RG, Pan PM, Srikanth S. et al. Fibrinogen activates NF-κ- B transcription factors in mononuclear phagocytes. J Immunol 1998; 161: 1462-70.
15 Mukaida N, Shiroo M, Matsushima K. Genomic structure of the human monocytederived neutrophil chemotactic factor IL-8. J Immunol 1989; 143: 1366-71.
16 Shyy YJ, Li YS, Kolattukudy PE. Structure of human monocyte chemotactic protein gene and its regulation by TPA. Biochem Biophys Res Commun 1990; 169: 346-51.
17 Brink N, Szamel M, Young AR. et al. Comparative quantification of IL-1β, IL-10, IL-10r, TNFalpha and IL-7 mRNA levels in UV-irradiated human skin in vivo. Inflamm Res 2000; 49: 290-6.
18 Hehner SP, Hofmann TG, Droge W. et al. The antiinflammatory sesquiterpene lactone parthenolide inhibits NF-κ B by targeting the I κ B kinase complex. J Immunol 1999; 163: 5617-23.
19 Byzova TV, Goldman CK, Pampori N. et al. A mechanism for modulation of cellular responses to VEGF: activation of the integrins. Mol Cell 2000; 06: 851-60.
20 Herrick S, Blanc-Brude O, Gray A. et al. Fibrinogen. Int J Biochem Cell Biol 1999; 31: 741-6.
21 Hoffmann A, Levchenko A, Scott ML. et al. The IκB-NF-κB signaling module: temporal control and selective gene activation. Science 2002; 298: 1241-5.
22 Gnant MF, Turner EM, Alexander Jr HR. Effects of hyperthermia and tumour necrosis factor on inflammatory cytokine secretion and procoagulant activity in endothelial cells. Cytokine 2000; 12: 339-47.
23 Bootle-Wilbraham CA, Tazzyman S, Marshall JM. et al. Fibrinogen E-fragment inhibits the migration and tubule formation of human dermal microvascular endothelial cells in vitro. Cancer Res 2000; 60: 4719-24.
24 Karin M, Lin A. NF-κB at the crossroads of life and death. Nat Immunol 2002; 03: 221-7.
25 Scatena M, Almeida M, Chaisson ML. et al. NF-κB mediates α_vβ₃ integrin-induced endothelial cell survival. J Cell Biol 1998; 141: 1083-93.
26 Kadish JL, Butterfield CE, Folkman J. The effect of fibrin on cultured vascular endothelial cells. Tissue Cell 1979; 11: 99-108.
27 Watanabe K, Tanaka K. Influence of fibrin, fibrinogen and fibrinogen degradation products on cultured endothelial cells. Atherosclerosis 1983; 48: 57-70.
28 Kaplan KL, Mather T, DeMarco L. et al. Effect of fibrin on endothelial cell production of prostacyclin and tissue plasminogen activator. Arteriosclerosis 1989; 09: 43-9.
29 Qi J, Kreutzer DL. Fibrin activation of vascular endothelial cells. Induction of IL-8 expression. J Immunol 1995; 155: 867-76.
30 Dejana E, Languino LR, Polentarutti N. et al. Interaction between fibrinogen and cultured endothelial cells. Induction of migration and specific binding. J Clin Invest 1985; 75: 11-8.
31 Suehiro K, Gailit J, Plow EF. Fibrinogen is a ligand for integrin α₅β₁ on endothelial cells. J Biol Chem 1997; 272: 5360-6.
32 Harley SL, Powell JT. Fibrinogen up-regulates the expression of monocyte chemoattractant protein 1 in human saphenous vein endothelial cells. Biochem J 1999; 341: 739-44.
33 Takagi J, Springer TA. Integrin activation and structural rearrangement. Immunol Rev 2002; 186: 141-63.
34 Takagi J, Petre BM, Walz T. et al. Global conformational rearrangements in integrin extracellular domains in outside-in and insideout signaling. Cell 2002; 110: 599-11.
35 Ueda A, Ishigatsubo Y, Okubo T. et al. Transcriptional regulation of the human monocyte chemoattractant protein-1 gene. Cooperation of two NF-κB sites and NF-κB/Rel subunit specificity. J Biol Chem 1997; 272: 31092-9.
36 Roebuck KA. Regulation of interleukin-8 gene expression. J Interferon Cytokine Res 1999; 19: 429-38.
37 Hipp MS, Urbich C, Mayer P. et al. Proteasome inhibition leads to NF-κB-independent IL-8 transactivation in human endothelial cells through induction of AP-1. Eur J Immunol 2002; 32: 2208-17.
38 Collins T, Cybulsky MI. NF-κB: pivotal mediator or innocent bystander in atherogenesis?. J Clin Invest 2001; 107: 255-64.
39 Aiello RJ, Bourassa PA, Lindsey S. et al. Monocyte chemoattractant protein-1 accelerates atherosclerosis in apolipoprotein E-deficient mice. Arterioscler Thromb Vasc Biol 1999; 19: 1518-25.