Presence of NGAL/MMP-9 complexes in human abdominal aortic aneurysms

Summary

It has been suggested that the intraluminal thrombus of abdominal aortic aneurysms (AAAs) predisposes for AAA enlargement and rupture.The growth of theAAA is dependent on proteolytic degradation of elastin. Here, we analysed whether the neutrophil gelatinase-associated lipocalin (NGAL) is expressed within the thrombus and the aneurysm wall. NGAL can bind to metalloproteinase- 9 (MMP-9) and inhibit its degradation,thereby preserving enzymatic activity. Biopsies were obtained from thrombus- free and thrombus-covered aneurysm wall and the intraluminal thrombus from patients undergoing elective surgery for AAA. Immunohistochemistry and real-time PCR were used to study NGAL and MMP-9 expression. Immunoprecipitation, gel zymography,Western blot and ELISA were used to detect and quantify NGAL/MMP-9 complexes. NGAL was detected in the thrombus, the interface between the thrombus and the underlying wall and in the wall itself.Double staining showed that neutrophils are the major source of NGAL expression. Immunoprecipitation of MMP-9 with antibody against NGAL showed that complexes of NGAL and active MMP-9 were present in thrombus, the interface fluid and the aneurysm wall.Western blot analyses using non-reducing conditions and gel zymography demonstrated that high-molecular-weight complexes of NGAL/MMP-9 were present within the different regions.The concentration of the NGAL/MMP-9 complex was highest in the luminal part of the thrombus. In conclusion, NGAL in complex with activated MMP-9 is present in AAA wall and thrombus. Neutrophil-derived NGAL could enhance the proteolytic activity associated with AAA, but the importance of this mechanism for aneurysm growth remains to be shown.

Keywords

Abdominal aortic aneurysm - NGAL - matrix metalloproteinases-9 - thrombus

^* These authors contributed equally to this work.

• References

• 1 Choke E, Cockerill G, Wilson R. et al. A review of biological factors implicated in abdominal aortic aneurysm rupture. Eur J Vasc Endovasc Surg 2005; 30: 227-244.
• 2 Kjeldsen L, Cowland JB, Borregaard N. Human neutrophil gelatinase-associated lipocalin and homologous proteins in rat and mouse. Biochim Biophys Acta 2000; 1482: 272-283.
• 3 Kjeldsen L, Johnsen AH, Sengelov H. et al. Isolation and primary structure of NGAL, anovel protein associated with human neutrophil gelatinase. J Biol Chem 1993; 268: 10425-10432.
• 4 Yan L, Borregaard N, Kjeldsen L. et al. The high molecular weight urinary matrix metalloproteinase (MMP) activity is a complex of gelatinase B/MMP-9 and neutrophil gelatinase-associated lipocalin (NGAL). Modulation of MMP-9 activity by NGAL. J Biol Chem 2001; 276: 37258-37265.
• 5 Hraba-Renevey S, Turler H, Kress M. et al. SV40-induced expression of mouse gene 24p3 involves a post-transcriptional mechanism. Oncogene 1989; 4: 601-608.
• 6 Triebel S, Blaser J, Reinke H. et al. A 25 kDa alpha 2-microglobulin-related proteinis a component of the 125 kDa form of human gelatinase. FEBS Lett 1992; 314: 386-388.
• 7 Hans SS, Jareunpoon O, Balasubramaniam M. et al. Size and location of thrombus in intact and ruptured abdominal aortic aneurysms. J Vasc Surg 2005; 41: 584-588.
• 8 Wolf YG, Thomas WS, Brennan FJ. et al. Computed tomography scanning findings associated with rapid expansion of abdominal aortic aneurysms. J Vasc Surg 1994; 20: 529-535 discussion535-538.
• 9 Satta J, Laara E, Juvonen T. Intraluminal thrombus predicts rupture of an abdominal aortic aneurysm. J Vasc Surg 1996; 23: 737-739.
• 10 Stenbaek J, Kalin B, Swedenborg J. Growth of thrombus is a better predictor for rupture than diameter in patients with abdominal aortic aneurysms. Eur J Vasc Endovasc Surg 2000; 20: 466-469.
• 11 Kazi M, Thyberg J, Religa P. et al. Influence of intraluminal thrombuson structural and cellular composition of abdominal aortic aneurysm wall. J Vasc Surg 2003; 38: 1283-1292.
• 12 Kazi M, Zhu C, Roy J. et al. Difference in matrix-degrading protease expression and activity between thrombus-free and thrombus-covered wall of abdominal aortic aneurysm. Arterioscler Thromb Vasc Biol 2005; 25: 1341-1346.
• 13 Fontaine V, Jacob MP, Houard X. et al. Involvement of the mural thrombus as a site of protease release and activation in human aortic aneurysms. Am J Pathol 2002; 161: 1701-1710.
• 14 Fontaine V, Touat Z, Mtairag el M. et al. Role of leukocyte elastase in preventing cellularre-colonization of the mural thrombus. Am J Pathol 2004; 164: 2077-2087.
• 15 Yoshimura K, Aoki H, Ikeda Y. et al. Regression of abdominal aortic aneurysm by inhibition of c-Jun N-terminal kinase. Nat Med 2005; 11: 1330-1338.
• 16 Morgan AR, Rerkasem K, Gallagher PJ. et al. Differences in matrix metalloproteinase-1 and matrix metalloproteinase-12 transcript levels among carotid atherosclerotic plaques with different histopathological characteristics. Stroke 2004; 35: 1310-1315.
• 17 Zerega B, Cermelli S, Michelis B. et al. Expression of NRL/NGAL (neu-related lipocalin/neutrophil gelatinase-associated lipocalin) during mammalian embryonic development and in inflammation. Eur J Cell Biol 2000; 79: 165-172.
• 18 Cowland JB, Sorensen OE, Sehested M. et al. Neutrophil gelatinase-associated lipocalin is up-regulated in human epithelial cells by IL-1 beta, butnot by TNF-alpha. J Immunol 2003; 171: 6630-6639.
• 19 Liu Q, Nilsen-Hamilton M. Identification of a new acute phase protein. J Biol Chem 1995; 270: 22565-22570.
• 20 Meheus LA, Fransen LM, Raymackers JG. et al. Identification by microsequencing of lipopolysaccharide-induced proteins secreted by mouse macrophages. J Immunol 1993; 151: 1535-1547.
• 21 Hemdahl A-L, Gabrielsen A, Zhu C. et al. Expression of neutrophil gelatinase-associated lipocalin in atherosclerosis and myocardial infarction. Arterioscler Thromb Vasc Biol 2006; 26: 136-142.
• 22 Bu D-X, Hemdahl A-L, Gabrielsen A. et al. Induction of neutrophilgelatinase-associated lipocalin in vascular injury via activation of NF-k B. Am J Pathol 2006; 169: 2245-2253.
• 23 Eliason JL, Hannawa KK, Ailawadi G. et al. Neutrophil depletion inhibits experimental abdominalaortic aneurysm formation. Circulation 2005; 112: 232-240.
• 24 Akerstrom B, Flower DR, Salier JP. Lipocalins: unity in diversity. Biochim Biophys Acta 2000; 1482: 1-8.
• 25 Bundgaard JR, Sengelov H, Borregaard N. et al. Molecular cloning and expression of a cDNA encoding NGAL: a lipocalin expressed in human neutrophils. Biochem Biophys Res Commun 1994; 202: 1468-1475.
• 26 Goetz DH, Holmes MA, Borregaard N. et al. The neutrophillipocalin NGAL is a bacteriostatic agent that interferes with siderophore-mediated iron acquisition. Mol Cell 2002; 10: 1033-1043.
• 27 Kol A, Sukhova GK, Lichtman AH. et al. Chlamy-dial heat shock protein 60 localizes in human atheroma and regulates macrophagetumor necrosis factor-alpha and matrix metalloproteinase expression. Circulation 1998; 98: 300-307.
• 28 Juvonen T, Biancari F, Juvonen J. Chlamydia pneumoniae and aortic aneurysms. Scand Cardiovasc J 2002; 36: 327-328.