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Enhanced matrix-degrading proteolytic activity within the thin thrombus-covered wall of human abdominal aortic aneurysms
Atherosclerosis 212 (2010) 161–165
Contents lists available at ScienceDirect
Atherosclerosis journal homepage: www.elsevier.com/locate/atherosclerosis
Enhanced matrix-degrading proteolytic activity within the thin thrombus-covered wall of human abdominal aortic aneurysms Ireneusz Wiernicki a,∗ , Ewa Stachowska b , Krzysztof Safranow b , Miloslaw Cnotliwy a , Marta Rybicka b , Mariusz Kaczmarczyk c , Piotr Gutowski a a b c
Department of Vascular Surgery and Angiology, Pomeranian Medical University, Powstancow Wielkopolskich 72, 70-111 Szczecin, Poland Department of Biochemistry and Medical Chemistry, Pomeranian Medical University, Szczecin, Poland Department of Clinical and Molecular Biochemistry, Pomeranian Medical University, Szczecin, Poland
a r t i c l e
i n f o
Article history: Received 25 November 2009 Received in revised form 23 April 2010 Accepted 26 April 2010 Available online 6 May 2010 Keywords: Abdominal aortic aneurysm Intraluminal thrombus Proteolytic activity
a b s t r a c t Objective: The maintenance of an arterial elastin’s integrity is essential in the prevention of abdominal aortic aneurysm (AAA) development. So far, the effect of intraluminal thrombus (ILT) thickness on the elastolytic activity within the AAA wall has not been studied. In the present study the hypothesis that thin thrombus is associated with enhanced proteolytic activity within human AAA wall was investigated. Methods: The specimens for analysis, from both thin (≤10 mm) thrombus-covered and thick (≥25 mm) thrombus-covered wall, had been taken from 40 patients undergoing elective repair of AAA. We evaluated neutrophil elastase activity with the enzymatic assay. Concentrations of active matrix metalloproteinase9 (MMP-9), total matrix metalloproteinase-8 (MMP-8), and tissue inhibitor of matrix metalloproteinases1 (TIMP-1) were measured by ELISA. Biochemical parameters were compared with the Wilcoxon signedrank test. Results: Statistical analysis showed that the activity of elastase (P < 0.0001) as well as concentrations of active MMP-9 (P = 0.001), total MMP-8 (P < 0.0001) and active MMP-9/total TIMP-1 ratio (P = 0.002) were significantly higher in the thin thrombus-covered wall. Furthermore the TIMP-1 was found to have a lower concentration in the thin thrombus-covered in comparison with the thick thrombus-covered wall (P = 0.003). There was a significant positive correlation between measurements in AAA wall sites with thin and thick thrombus for elastase, TIMP-1, MMP-9/TIMP-1 ratio, and a borderline correlation was observed for MMP-8. Active MMP-9 concentration did not correlate between sites. Conclusion: The current study demonstrates the differentiation of protease activity within the same AAA wall and its enhancement within the thin thrombus-covered aneurysm wall. © 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The degradation of the extracellular matrix by proteases causing aortic disease is a terminal event, modulated by genetic background, hemodynamic strain, cellular events and thrombus formation. In the etiology of an abdominal aortic aneurysm (AAA), the release of elastase by the neutrophils and macrophages has been implicated. Elastin-derived peptides stimulate the release of elastase in the aortic wall by circulating neutrophils, which for patients with AAA leads to a predetermined raise in the amount of elastase [1]. Therefore, circulating neutrophils are an important initial component of experimental AAA formation. Neutrophils may also be an important source of matrix metalloproteinase-9 (MMP-9), a matrix-degrading enzyme thought to be crucial in the
∗ Corresponding author. Tel.: +48 91 466 11 56; fax: +48 91 466 11 57. E-mail address: [email protected] (I. Wiernicki). 0021-9150/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2010.04.033
development of AAA [2]. Moreover, some studies demonstrated that human neutrophil elastase could not only activate pro-MMP9 but could also degrade tissue inhibitor of metalloproteinases-1 (TIMP-1) [3]. Elastase isolated from the AAA hydrolyses elastin with the optimum activity, i.e. is poorly inhibited, at pH 7.6 [4]. The alkaline intramural pH 7.64 ± 0.10, in the aneurysm wall adjacent to the thin (≤10 mm) part of intraluminal thrombus (ILT) may accelerate the local destruction of the elastin fiber [5]. Loss of elastin is the initiating event in AAA formation, whereas loss of collagen is required for continued expansion. Wilson et al. showed that a high concentration of MMP-8 in AAAs represents a potent pathway for collagen degradation, and hence aneurysm formation and expansion [6]. Therefore, the aim of this paper was to estimate activity of elastase, MMP-9, MMP-9/TIMP-1 ratio (a relative index of proteolytic state), and MMP-8 concentration in the aneurysm wall tissue. Having taken into consideration all of the above, we hypothesize that the thin thrombus-covered wall of
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ing surgery. Samples were not obtained from the posterior wall due to potential risk addition to the surgery. Two 1 cm × 2 cm tissue sections were cut transversely from the thin thrombus and thick thrombus-covered aneurysm wall. The adventitia was dissected free of excess perivascular fat and soft tissues enveloping the abdominal aorta. Therefore, we analyzed the wall, including media and adventitia, while the intraluminal thrombus itself was excluded. Preparation of samples was performed on the ice pad. They were homogenized to powder consistency in a steel homogenizer cooled with liquid nitrogen. One gram of the powdered sample was weighed and frozen at −80 ◦ C till the final quantification. The activity of elastase and the concentrations of active MMP-9, total MMP-8 and tissue inhibitor of matrix metalloproteinases (TIMP-1), as well as total protein content were measured in homogenates prepared from the powder in phosphate-buffered saline (PBS).
2.3. Measurements
Fig. 1. Representative picture of computed tomography (CT) with the eccentric, perimural blood flow channel in the ILT demonstrating the sites of aneurysm wall sampling. Arrows indicate: A – site with the thinner of ILT, and B – site with the thicker of ILT. Arrowheads indicate areas of biopsy.
AAA is contributing to the enhanced matrix-degrading proteolytic activity. 2. Methods 2.1. Subjects and study design Material was prospectively sampled from 40 consecutively recruited patients (37 males and 3 females) during the process of repairing abdominal aortic aneurysms at the Department of Vascular Surgery and Angiology of Pomeranian Medical University in Szczecin. The mean age of the subjects was 66.8 ± 8.5 years (range 49–83); coronary artery disease was found in 14 patients, arterial hypertension in 19, chronic obstructive pulmonary disease in 21, diabetes mellitus in 3. Thirty-four patients were active smokers (up to 10 cigarettes/day). The mean value of the maximum AAA diameters appeared to be 62.7 ± 10.2 mm (range 50–90 mm). The nature of the study was explained to the patients and their written consent was obtained. The study protocol was accepted by the Bioethical Committee of the Pomeranian Medical University.
The kits used in the estimation of active MMP-9 (Fluorokine E – Human active MMP-9 Fluorescent Assay), concentration of MMP8 (Quantikine – Human total MMP-8 Immunoassay) and TIMP-1 (Quantikine – Human TIMP-1 Immunoassay) by immunoenzymatic methods (ELISA) were purchased from R&D Systems Europe GmbH (Germany). The elastase activity quantifying kit (Enzymatic Assay of ELASTASE, Leukocyte) was obtained from Sigma–Aldrich (Poland). The study protocol was performed strictly in conformity with the information booklets attached to the kits by the producers. In these assays no significant cross-reactivity or interference was observed. The active MMP-9 concentration was measured with DELFIA VICTOR2 fluorometer (PerkinElmer), MMP-8 and TIMP-1 with Titertek Multiskan plate reader (EFLAB, Finland) and the elastase activity with UV/vis Lambda 40 P spectrophotometer (Perkin Elmer). The protein concentration was estimated using the Bradford reagent (Sigma–Aldrich, Poland).
2.4. Statistical methods Biochemical parameters were compared between A and B sites in each patient by the Wilcoxon signed-rank test. Spearman’s rank correlation coefficient (Rs) was used to measure correlations between the examined parameters. A P-value of <0.05 was considered statistically significant.
2.2. Tissue sampling
3. Results
In each case two sections of the aneurysm wall were examined: one covered with a thin thrombus (≤10 mm – site A) and one with a thick thrombus (≥25 mm – site B) as shown in Fig. 1. We used such inclusion criteria to clearly differentiate a thin mural thrombus (up to 10 mm), which permits penetration of blood components to the wall, from a thicker thrombus [7]. All the measurements of ILT were performed in the maximum diameter (≥50 mm) of the aneurysm. In all cases the AAA imaging included computed tomography. All samples were obtained from elective cases. Tissue samples were gathered from the middle 1/3 of the longitudinal dimension, close to the point of maximum dilatation of the AAA sac at the same level (A and B sites). Biopsy samples were retrieved, after the clamping of the aorta, from the anterior or posterior-lateral position at the aneurysm wall. The samples taken from the visually thick thrombus-covered and thin thrombus-covered vessel wall (the thrombus thickness was measured for each site by using a laser micrometer before excision of specimen) were obtained dur-
The increased proteolytic activity was found in site A covered with thin thrombus (increased elastase, active MMP-9, total MMP8 and MMP-9/TIMP-1 ratio, decreased TIMP-1). The results of these biochemical parameters are summarized in Table 1 and graphically shown in Fig. 2a–d. Total protein content was similar in sites with thin and thick thrombus (82.6 ± 11.1 vs 84.3 ± 9.9 mg protein/g tissue wet weight, respectively, P = 0.44). Correlations for each pair of measured parameters (active MMP9, total MMP-8, TIMP-1, elastase) in sites A and B separately are shown in Table 2. The only significant association was positive correlation between MMP-8 and active MMP-9 in site B. The correlations between values measured in sites A and B for each parameter are presented in Table 3. There was a significant positive correlation between measurements in AAA wall sites with thin and thick thrombus for elastase, TIMP-1, MMP-9/TIMP-1 ratio, and a borderline correlation was observed for MMP-8. Active MMP-9 concentration did not correlate between sites.
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Table 1 Comparison of the biochemical parameters between A and B sites of the AAA wall. Biochemical parametersa
Site A (thrombus ≤10 mm)
Elastase activity Active MMP-9 Total MMP-8 TIMP-1 MMP-9/TIMP-1 ratio
7.60 16.33 120.26 133.61 0.183
a b
± ± ± ± ±
Site B (thrombus ≥25 mm)
4.72 2.63 106.42 71.81 0.151
4.29 14.62 47.17 175.42 0.131
± ± ± ± ±
Pb
2.97 1.98 41.87 84.87 0.163
<0.0001 0.0010 <0.0001 0.0032 0.0018
Elastase activity is expressed as U/mg protein; the unit for active MMP-9, total MMP-8 and TIMP-1 concentration is ng/mg protein. Wilcoxon signed-rank test.
Fig. 2. (a) Difference in the activity of elastase (U/mg protein) in the same patient, in the regions of the AAA wall with the thinner (≤10 mm – site A – open triangles) and thicker (≥25 mm – site B – filled squares) parts of ILT. (b) Difference in active forms of MMP-9 (ng/mg protein) in the same patient, in the regions of the AAA wall with the thinner (≤10 mm – site A – open triangles) and thicker (≥25 mm – site B – filled squares) parts of ILT. (c) Difference in concentration of total MMP-8 (ng/mg protein) in the same patient, in the regions of the AAA wall with the thinner (≤10 mm – site A – open triangles) and thicker (≥25 mm – site B – filled squares) parts of ILT. (d) Difference in concentration of TIMP-1 (ng/mg protein) in the same patient, in the regions of the AAA wall with the thinner (≤10 mm – site A – open triangles) and thicker (≥25 mm – site B – filled squares) parts of ILT.
4. Discussion Aortic diseases are diverse and involve a multiplicity of biological systems in the vascular wall and at the interface with blood. Recent studies show a preferential accumulation of neutrophils and release of major neutrophil chemotactic factors at the luminal layer
of the mural thrombus [8,9]. ILT contains a lot of neutrophils, which release elastase, pro-MMP-9 and -8, and are mainly found in the luminal part of the thrombus [10]. Inflammatory cells infiltrates are relevant sources of MMP-9 and -8, among others, in the AAA wall and may substantially contribute to aneurysm wall instability. Furthermore, significant inverse correlations were found between
Table 2 Correlations between biochemical parameters within A and B sites of the AAA wall. Correlated parameters
Active MMP-9
Total MMP-8
TIMP-1
Rs
P
Rs
P
Rs
P
Site A (thrombus ≤10 mm) Elastase activity Active MMP-9 Total MMP-8
+0.01 – –
0.96 – –
+0.27 +0.07 –
0.10 0.65 –
+0.13 −0.10 −0.03
0.42 0.52 0.85
Site B (thrombus ≥25 mm) Elastase activity Active MMP-9 Total MMP-8
+0.13 – –
0.41 – –
+0.17 +0.39 –
0.30 0.012 –
−0.08 +0.02 −0.05
0.63 0.88 0.75
Rs – Spearman rank correlation coefficient.
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Table 3 Correlations between biochemical parameters measured in sites A and B of the AAA wall. Biochemical parameters
Elastase activity Active MMP-9 Total MMP-8 TIMP-1 MMP-9/TIMP-1 ratio
Correlation between sites A and B Rs
P
+0.42 +0.18 +0.28 +0.43 +0.46
0.0072 0.27 0.078 0.0052 0.0027
Rs – Spearman rank correlation coefficient.
the amounts of inflammatory cells and elastin/collagen content of the aortic vessel wall [11]. MMP-9 activity was also distributed in macrophages by invading neovessels at the outer media and adventitia of the AAA [12,13]. Cellular penetration from the AAA lumen through ILT may occur up to 10 mm [7]. Therefore, thin ILT seems to be more permeable than thick one. The intima and inner media of the aortic wall are supplied by blood from the lumen [14]. Aneurysmal tissue elastase activity is situated in the intima and inner media of the wall [15]. Hemodynamic forces may modulate AAA inflammation and diameter enlargement via direct regulation of intimal macrophage adhesion and transmural migration [16]. The abovementioned observations are in agreement with the current results of our study. We found that part of the aneurysmal wall covered by thin ILT showed higher elastase, active MMP-9 and total MMP-8 levels. MMP-9 appears to be the predominant metalloproteinase in the AAA. This is due to the fact that its mRNA levels are more than 20 times higher than those of MMP-1 and 2 times higher than those of MMP-2 [17]. Elmore et al. [18] claim it is the growth of MMP-9 activity and not MMP-2 that is an important factor in the etiology of AAAs. The presence of MMP-9 is important not only because it appears to influence all stages of AAA pathogenesis but also because its substrate specificity toward partially degraded fibrillar collagen fragments complements the action of MMP-8, a potent type I collagenase [19]. Leukocyte-derived MMP-9 is associated with the aortic wall degeneration and the aneurysm formation [20]. It has been hypothesized that an imbalance between proteinases and their naturally occurring inhibitors is the cause of observed histologic abnormalities. An unbalanced relation in the MMPs/TIMPs activity ratio can underlie the pathogenesis of the abdominal aortic aneurysm. Decreased TIMP-1 and TIMP-2 expression in the extracellular matrix of the walls of AAAs has been demonstrated in several studies. In comparison with the control group, the MMP9 to TIMP-1 ratio (a relative index of proteolytic state) was raised in the aortic aneurysms tissue [21]. Interaction of TIMP-1 (a natural inhibitor of MMP-9) with MMP-9 plays an important role [22]. In our study, the active MMP-9/total TIMP-1 ratio was greater in the thin thrombus-covered wall compared with a thicker thrombuscovered wall. The assessment of the posttranslational regulation of protease activity showed a threefold increase in MMP-8 activation in growing as well as ruptured AAAs [23]. Enhanced expression of MMPs mRNA was found in the aneurysm wall without thrombus in contrast to the thrombus-covered wall. Furthermore, MMP-9 activity was also discovered in the interface between the thrombus and the underlying wall [24]. Our results for the first time showed quantitatively that active forms of elastase, MMP-9, active MMP9/total TIMP-1 ratio, and MMP-8 concentration were strongly raised within the thin thrombus-covered wall compared to samples from thick thrombus-covered AAA wall. The reports proving that mural thrombus was significantly thinner in ruptured than in non-ruptured AAA (9 vs. 19 mm), as well as those describing that the aneurysm biopsies from the rupture site show higher MMP-8 and -9 levels compared to samples taken from the paired
anterior wall [19,25] are in accordance with our study. Some studies demonstrated that human neutrophil elastase could activate pro-MMP-9 and also degrade TIMP-1 [3]. In accordance with the aforementioned facts, in the present study, the aneurysmal wall covered by a thin ILT shows a higher elastase and MMP-9 activity and a lower TIMP-1 concentration. On the basis of the above data, we suggest that increased elastase activity may influence the activity of MMP-9, which is thought to play an important role in aneurysm development. We also found that all biochemical parameters, except active MMP-9, correlated positively between thin thrombus-covered and thick thrombus-covered AAA wall. It suggests that MMP-9 penetration to AAA wall with thin and thick thrombus may be regulated by different mechanisms. The uptake of (18)F-fluorodeoxyglucose (FDG) in the aneurysm wall may be connected to the accumulation of inflammatory cells responsible for the production and the activation of degrading enzymes [26]. Truijers et al. [27] reported that FDG uptake is located within the non-calcified aneurysm wall. This indirectly confirms that transfer of inflammatory cells and subsequently elastase, MMP-9 and MMP-8 occurs from the lumen through thin ILT to the underlying wall to a greater extent than from the vasa vasorum of the human AAA. The mean annual growth rate was significantly lower in men with an AAA wall calcification above than below 50% [28]. AAA wall thickness varied regionally – from low at a rupture site to high at a calcified site [29]. It has been suggested that calcified plaques may be a significant barrier for the transport of inflammatory cells to the wall of the AAA. The development of heterogeneous multilayered intraluminal thrombi and calcified plaques likely has an effect on the thickness variability of the vascular wall. The thin thrombus-covered wall associated with proteolytic elastase and MMP-9 hyperactivity could lead to focal weakening of the aneurysm wall. These observations may explain why some small aneurysms rupture and larger ones do not, and why rupture is rarely located in the largest diameter. We suggest that the thickness of ILT may be a surrogate marker for the other changes that are associated directly with the AAA growth rate and a potentially earlier rupture in thin thrombus-covered wall of aneurysms. Furthermore, a halo-type, uniform thin intraluminal thrombus in small AAAs should lead to more frequent evaluations of the aneurysm size or suggest early surgery. Therefore the distribution of proteolytic activity within the AAA wall might be a good indicator of its susceptibility to rupture. These observations are in agreement with our pathologic concepts describing the role of local factors in the pathogenesis of AAA rupture [Fig. 3 online Supplementary material]. Our study points to thin ILT as a significant factor responsible for high protease activity in AAA wall, but it is not conclusive as regards the mechanism of this phenomenon: is it just the lack of protection associated with thick thrombus, or active participation of thin thrombus in the elevated activity of proteases? Measurement of protease activity in thrombus-free regions of AAA could explain this issue. Unfortunately, we found that AAAs with even a small surface of truly thrombus-free wall are very rare in our patients. Even when CT imaging suggested presence of thrombus-free wall, visual examination usually showed thin layer of thrombus, so we could not classify such samples as thrombus-free. The lack of proteolytic activity measurements within thrombus-free AAA wall is a limitation of our study which does not allow us to make firm conclusions about the role of the thrombus. However, Kazi et al. demonstrated that MMP-1, -7, -9 and -12 expressions were upregulated in the wall segments clearly without thrombus compared with the thrombus-covered wall [24]. It seems that elevated protease activity might be a common feature of thin thrombus-covered wall and thrombus-free wall, but further studies are needed to confirm this hypothesis. Another limitation of our study is the lack of measurements of protease activity in thrombus. The results of pre-
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vious studies, referenced above, showed that neutrophils releasing proteases (including MMP-9) are located in the luminal sublayer of the thrombus. This was confirmed by a recent paper using in vivo MR imaging and histologic analysis in high-risk AAAs [30]. Therefore, our results could be explained by the fact that thin thrombi convey more protease activity to the AAA wall than thick thrombi. In conclusion, our study demonstrates for the first time that the part of abdominal aortic aneurysm wall covered with thin thrombus is exposed to higher proteolytic activity than the part protected by thick thrombus. This phenomenon can be considered as one of the mechanisms responsible for the association of thrombus size, shape and location with the risk of aneurysm rupture. Acknowledgement We thank Mr. Gary Stewart, a native English speaking translator, for his assistance in preparation of the manuscript. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.atherosclerosis.2010.04.033. References [1] Cohen JR, Parikh S, Grella L, et al. Role of the neutrophil in abdominal aortic aneurysm development. Cardiovasc Surg 1993;1:373–6. [2] Eliason JL, Hannawa KK, Ailawadi G, et al. Neutrophil depletion inhibits experimental abdominal aortic aneurysm formation. Circulation 2005;112:232–40. [3] Itoh Y, Nagase H. Preferential inactivation of tissue inhibitor of metalloproteinases-1 that is bound to the precursor of matrix metalloproteinase 9 (progelatinase B) by human neutrophil elastase. J Biol Chem 1995;270:16518–21. [4] Powell J, Greenhalgh RMJ. Cellular, enzymatic, and genetic factors in the pathogenesis of abdominal aortic aneurysms. J Vasc Surg 1989;9:297–304. [5] Wiernicki I, Cnotliwy M, Baranowska-Bosiacka I, et al. Elastin degradation within the abdominal aortic aneurysm wall – relationship between intramural pH and adjacent thrombus formation. Eur J Clin Invest 2008;38:883–7. [6] Wilson WR, Schwalbe EC, Jones JL, Bell PR, Thompson MM. Matrix metalloproteinase 8 (neutrophil collagenase) in the pathogenesis of abdominal aortic aneurysm. Br J Surg 2005;92:828–33. [7] Adolph R, Vorp DA, Steed DL, Webster MW, Kameneva MV, Watkins SC. Cellular content and permeability of intraluminal thrombus in abdominal aortic aneurysm. J Vasc Surg 1997;25:916–26. [8] Houard X, Touat Z, Ollivier V, et al. Mediators of neutrophil recruitment in human abdominal aortic aneurysms. Cardiovasc Res 2009;82:532–41. [9] Houard X, Ollivier V, Louedec L, Michel JB, Bäck M. Differential inflammatory activity across human abdominal aortic aneurysms reveals neutrophil-derived leukotriene B4 as a major chemotactic factor released from the intraluminal thrombus. FASEB J 2009;23:1376–83. [10] Fontaine V, Touat Z, Mtairag el M, et al. Role of leukocyte elastase in preventing cellular re-colonization of the mural thrombus. Am J Pathol 2004;164:2077–87.
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[11] Reeps C, Pelisek J, Seidl S, et al. Inflammatory infiltrates and neovessels are relevant sources of MMPs in abdominal aortic aneurysm wall. Pathobiology 2009;76:243–52. [12] Thompson RW, Holmes DR, Mertens RA, et al. Production and localization of 92-kilodalton gelatinase in abdominal aortic aneurysms. An elastolytic metalloproteinase expressed by aneurysm-infiltrating macrophages. J Clin Invest 1995;96:318–26. [13] Tsuruda T, Kato J, Hatakeyama K, et al. Adventitial mast cells contribute to pathogenesis in the progression of abdominal aortic aneurysm. Circ Res 2008;102:1368–77. [14] Dobrin PB, Baker WH, Gley WC. Elastolytic and collagenolytic studies of arteries. Arch Surg 1984;119:405–8. [15] Busuttil RW, Rinderbriecht H, Flesher A, Carmack C. Elastase activity: the role of elastase in aortic aneurysm formation. J Surg Res 1982;32:214–7. [16] Sho E, Sho M, Hoshina K, Kimura H, Nakahashi TK, Dalman RL. Hemodynamic forces regulate mural macrophage infiltration in experimental aortic aneurysms. Exp Mol Pathol 2004;76:108–16. [17] Tamarina NA, McMillan WD, Shively VP, Pearce WH. Expression of matrix metalloproteinases and their inhibitors in aneurysms and normal aorta. Surgery 1997;122:264–71. [18] Elmore JR, Keister BF, Franklin DP, Youkey JR, Carey DJ. Expression of matrix metalloproteinases and TIMPs in human abdominal aortic aneurysms. Ann Vasc Surg 1998;12:221–8. [19] Wilson WR, Anderton M, Schwalbe EC, et al. Matrix metalloproteinase-8 and -9 are increased at the site of abdominal aortic aneurysm rupture. Circulation 2006;113:438–45. [20] Saito S, Zempo N, Yamashita A, Takenaka H, Fujioka K, Esato K. Matrix metalloproteinase expressions in arteriosclerotic aneurysmal disease. Vasc Endovasc Surg 2002;36:1–7. [21] Koullias GJ, Ravichandran P, Korkolis DP, Rimm DL, Elefteriades JA. Increased tissue microarray matrix metalloproteinase expression favors proteolysis in thoracic aortic aneurysms and dissections. Ann Thorac Surg 2004;78: 2106–10. [22] Nishimura K, Ikebuchi M, Kanaoka Y, et al. Relationships between matrix metalloproteinases and tissue inhibitor of metalloproteinases in the wall of abdominal aortic aneurysms. Int Angiol 2003;22:229–38. [23] Abdul-Hussien H, Soekhoe RG, Weber E, et al. Collagen degradation in the abdominal aneurysm: a conspiracy of matrix metalloproteinase and cysteine collagenases. Am J Pathol 2007;170:809–17. [24] 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;25:1341–6. [25] Kushihashi T, Munechika H, Matsui S, Moritani T, Horichi Y, Hishida T. CT of abdominal aortic aneurysms: aneurysmal size and thickness of intraaneurysmal thrombus as risk factors of rupture. Nippon Igaku Hoshasen Gakkai Zasshi 1991;51:219–27. [26] Sakalihasan N, Hustinx R, Limet R. Contribution of PET scanning to the evaluation of abdominal aortic aneurysm. Semin Vasc Surg 2004;17:144–53. [27] Truijers M, Kurvers HA, Bredie SJ, Oyen WJ, Blankensteijn JD. In vivo imaging of abdominal aortic aneurysms: increased FDG uptake suggests inflammation in the aneurysm wall. J Endovasc Ther 2008;15:462–7. [28] Lindholt JS. Aneurysmal wall calcification predicts natural history of small abdominal aortic aneurysms. Atherosclerosis 2008;197:673–8. [29] Raghavan ML, Kratzberg J, Castro de Tolosa EM, Hanaoka MM, Walker P, da Silva ES. Regional distribution of wall thickness and failure properties of human abdominal aortic aneurysm. J Biomech 2006;39:3010–6. [30] Nchimi A, Defawe O, Brisbois D, et al. MR imaging of iron phagocytosis in intraluminal thrombi of abdominal aortic aneurysms in humans. Radiology 2010;254:973–81.
Contents lists available at ScienceDirect
Atherosclerosis journal homepage: www.elsevier.com/locate/atherosclerosis
Enhanced matrix-degrading proteolytic activity within the thin thrombus-covered wall of human abdominal aortic aneurysms Ireneusz Wiernicki a,∗ , Ewa Stachowska b , Krzysztof Safranow b , Miloslaw Cnotliwy a , Marta Rybicka b , Mariusz Kaczmarczyk c , Piotr Gutowski a a b c
Department of Vascular Surgery and Angiology, Pomeranian Medical University, Powstancow Wielkopolskich 72, 70-111 Szczecin, Poland Department of Biochemistry and Medical Chemistry, Pomeranian Medical University, Szczecin, Poland Department of Clinical and Molecular Biochemistry, Pomeranian Medical University, Szczecin, Poland
a r t i c l e
i n f o
Article history: Received 25 November 2009 Received in revised form 23 April 2010 Accepted 26 April 2010 Available online 6 May 2010 Keywords: Abdominal aortic aneurysm Intraluminal thrombus Proteolytic activity
a b s t r a c t Objective: The maintenance of an arterial elastin’s integrity is essential in the prevention of abdominal aortic aneurysm (AAA) development. So far, the effect of intraluminal thrombus (ILT) thickness on the elastolytic activity within the AAA wall has not been studied. In the present study the hypothesis that thin thrombus is associated with enhanced proteolytic activity within human AAA wall was investigated. Methods: The specimens for analysis, from both thin (≤10 mm) thrombus-covered and thick (≥25 mm) thrombus-covered wall, had been taken from 40 patients undergoing elective repair of AAA. We evaluated neutrophil elastase activity with the enzymatic assay. Concentrations of active matrix metalloproteinase9 (MMP-9), total matrix metalloproteinase-8 (MMP-8), and tissue inhibitor of matrix metalloproteinases1 (TIMP-1) were measured by ELISA. Biochemical parameters were compared with the Wilcoxon signedrank test. Results: Statistical analysis showed that the activity of elastase (P < 0.0001) as well as concentrations of active MMP-9 (P = 0.001), total MMP-8 (P < 0.0001) and active MMP-9/total TIMP-1 ratio (P = 0.002) were significantly higher in the thin thrombus-covered wall. Furthermore the TIMP-1 was found to have a lower concentration in the thin thrombus-covered in comparison with the thick thrombus-covered wall (P = 0.003). There was a significant positive correlation between measurements in AAA wall sites with thin and thick thrombus for elastase, TIMP-1, MMP-9/TIMP-1 ratio, and a borderline correlation was observed for MMP-8. Active MMP-9 concentration did not correlate between sites. Conclusion: The current study demonstrates the differentiation of protease activity within the same AAA wall and its enhancement within the thin thrombus-covered aneurysm wall. © 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The degradation of the extracellular matrix by proteases causing aortic disease is a terminal event, modulated by genetic background, hemodynamic strain, cellular events and thrombus formation. In the etiology of an abdominal aortic aneurysm (AAA), the release of elastase by the neutrophils and macrophages has been implicated. Elastin-derived peptides stimulate the release of elastase in the aortic wall by circulating neutrophils, which for patients with AAA leads to a predetermined raise in the amount of elastase [1]. Therefore, circulating neutrophils are an important initial component of experimental AAA formation. Neutrophils may also be an important source of matrix metalloproteinase-9 (MMP-9), a matrix-degrading enzyme thought to be crucial in the
∗ Corresponding author. Tel.: +48 91 466 11 56; fax: +48 91 466 11 57. E-mail address: [email protected] (I. Wiernicki). 0021-9150/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2010.04.033
development of AAA [2]. Moreover, some studies demonstrated that human neutrophil elastase could not only activate pro-MMP9 but could also degrade tissue inhibitor of metalloproteinases-1 (TIMP-1) [3]. Elastase isolated from the AAA hydrolyses elastin with the optimum activity, i.e. is poorly inhibited, at pH 7.6 [4]. The alkaline intramural pH 7.64 ± 0.10, in the aneurysm wall adjacent to the thin (≤10 mm) part of intraluminal thrombus (ILT) may accelerate the local destruction of the elastin fiber [5]. Loss of elastin is the initiating event in AAA formation, whereas loss of collagen is required for continued expansion. Wilson et al. showed that a high concentration of MMP-8 in AAAs represents a potent pathway for collagen degradation, and hence aneurysm formation and expansion [6]. Therefore, the aim of this paper was to estimate activity of elastase, MMP-9, MMP-9/TIMP-1 ratio (a relative index of proteolytic state), and MMP-8 concentration in the aneurysm wall tissue. Having taken into consideration all of the above, we hypothesize that the thin thrombus-covered wall of
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ing surgery. Samples were not obtained from the posterior wall due to potential risk addition to the surgery. Two 1 cm × 2 cm tissue sections were cut transversely from the thin thrombus and thick thrombus-covered aneurysm wall. The adventitia was dissected free of excess perivascular fat and soft tissues enveloping the abdominal aorta. Therefore, we analyzed the wall, including media and adventitia, while the intraluminal thrombus itself was excluded. Preparation of samples was performed on the ice pad. They were homogenized to powder consistency in a steel homogenizer cooled with liquid nitrogen. One gram of the powdered sample was weighed and frozen at −80 ◦ C till the final quantification. The activity of elastase and the concentrations of active MMP-9, total MMP-8 and tissue inhibitor of matrix metalloproteinases (TIMP-1), as well as total protein content were measured in homogenates prepared from the powder in phosphate-buffered saline (PBS).
2.3. Measurements
Fig. 1. Representative picture of computed tomography (CT) with the eccentric, perimural blood flow channel in the ILT demonstrating the sites of aneurysm wall sampling. Arrows indicate: A – site with the thinner of ILT, and B – site with the thicker of ILT. Arrowheads indicate areas of biopsy.
AAA is contributing to the enhanced matrix-degrading proteolytic activity. 2. Methods 2.1. Subjects and study design Material was prospectively sampled from 40 consecutively recruited patients (37 males and 3 females) during the process of repairing abdominal aortic aneurysms at the Department of Vascular Surgery and Angiology of Pomeranian Medical University in Szczecin. The mean age of the subjects was 66.8 ± 8.5 years (range 49–83); coronary artery disease was found in 14 patients, arterial hypertension in 19, chronic obstructive pulmonary disease in 21, diabetes mellitus in 3. Thirty-four patients were active smokers (up to 10 cigarettes/day). The mean value of the maximum AAA diameters appeared to be 62.7 ± 10.2 mm (range 50–90 mm). The nature of the study was explained to the patients and their written consent was obtained. The study protocol was accepted by the Bioethical Committee of the Pomeranian Medical University.
The kits used in the estimation of active MMP-9 (Fluorokine E – Human active MMP-9 Fluorescent Assay), concentration of MMP8 (Quantikine – Human total MMP-8 Immunoassay) and TIMP-1 (Quantikine – Human TIMP-1 Immunoassay) by immunoenzymatic methods (ELISA) were purchased from R&D Systems Europe GmbH (Germany). The elastase activity quantifying kit (Enzymatic Assay of ELASTASE, Leukocyte) was obtained from Sigma–Aldrich (Poland). The study protocol was performed strictly in conformity with the information booklets attached to the kits by the producers. In these assays no significant cross-reactivity or interference was observed. The active MMP-9 concentration was measured with DELFIA VICTOR2 fluorometer (PerkinElmer), MMP-8 and TIMP-1 with Titertek Multiskan plate reader (EFLAB, Finland) and the elastase activity with UV/vis Lambda 40 P spectrophotometer (Perkin Elmer). The protein concentration was estimated using the Bradford reagent (Sigma–Aldrich, Poland).
2.4. Statistical methods Biochemical parameters were compared between A and B sites in each patient by the Wilcoxon signed-rank test. Spearman’s rank correlation coefficient (Rs) was used to measure correlations between the examined parameters. A P-value of <0.05 was considered statistically significant.
2.2. Tissue sampling
3. Results
In each case two sections of the aneurysm wall were examined: one covered with a thin thrombus (≤10 mm – site A) and one with a thick thrombus (≥25 mm – site B) as shown in Fig. 1. We used such inclusion criteria to clearly differentiate a thin mural thrombus (up to 10 mm), which permits penetration of blood components to the wall, from a thicker thrombus [7]. All the measurements of ILT were performed in the maximum diameter (≥50 mm) of the aneurysm. In all cases the AAA imaging included computed tomography. All samples were obtained from elective cases. Tissue samples were gathered from the middle 1/3 of the longitudinal dimension, close to the point of maximum dilatation of the AAA sac at the same level (A and B sites). Biopsy samples were retrieved, after the clamping of the aorta, from the anterior or posterior-lateral position at the aneurysm wall. The samples taken from the visually thick thrombus-covered and thin thrombus-covered vessel wall (the thrombus thickness was measured for each site by using a laser micrometer before excision of specimen) were obtained dur-
The increased proteolytic activity was found in site A covered with thin thrombus (increased elastase, active MMP-9, total MMP8 and MMP-9/TIMP-1 ratio, decreased TIMP-1). The results of these biochemical parameters are summarized in Table 1 and graphically shown in Fig. 2a–d. Total protein content was similar in sites with thin and thick thrombus (82.6 ± 11.1 vs 84.3 ± 9.9 mg protein/g tissue wet weight, respectively, P = 0.44). Correlations for each pair of measured parameters (active MMP9, total MMP-8, TIMP-1, elastase) in sites A and B separately are shown in Table 2. The only significant association was positive correlation between MMP-8 and active MMP-9 in site B. The correlations between values measured in sites A and B for each parameter are presented in Table 3. There was a significant positive correlation between measurements in AAA wall sites with thin and thick thrombus for elastase, TIMP-1, MMP-9/TIMP-1 ratio, and a borderline correlation was observed for MMP-8. Active MMP-9 concentration did not correlate between sites.
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Table 1 Comparison of the biochemical parameters between A and B sites of the AAA wall. Biochemical parametersa
Site A (thrombus ≤10 mm)
Elastase activity Active MMP-9 Total MMP-8 TIMP-1 MMP-9/TIMP-1 ratio
7.60 16.33 120.26 133.61 0.183
a b
± ± ± ± ±
Site B (thrombus ≥25 mm)
4.72 2.63 106.42 71.81 0.151
4.29 14.62 47.17 175.42 0.131
± ± ± ± ±
Pb
2.97 1.98 41.87 84.87 0.163
<0.0001 0.0010 <0.0001 0.0032 0.0018
Elastase activity is expressed as U/mg protein; the unit for active MMP-9, total MMP-8 and TIMP-1 concentration is ng/mg protein. Wilcoxon signed-rank test.
Fig. 2. (a) Difference in the activity of elastase (U/mg protein) in the same patient, in the regions of the AAA wall with the thinner (≤10 mm – site A – open triangles) and thicker (≥25 mm – site B – filled squares) parts of ILT. (b) Difference in active forms of MMP-9 (ng/mg protein) in the same patient, in the regions of the AAA wall with the thinner (≤10 mm – site A – open triangles) and thicker (≥25 mm – site B – filled squares) parts of ILT. (c) Difference in concentration of total MMP-8 (ng/mg protein) in the same patient, in the regions of the AAA wall with the thinner (≤10 mm – site A – open triangles) and thicker (≥25 mm – site B – filled squares) parts of ILT. (d) Difference in concentration of TIMP-1 (ng/mg protein) in the same patient, in the regions of the AAA wall with the thinner (≤10 mm – site A – open triangles) and thicker (≥25 mm – site B – filled squares) parts of ILT.
4. Discussion Aortic diseases are diverse and involve a multiplicity of biological systems in the vascular wall and at the interface with blood. Recent studies show a preferential accumulation of neutrophils and release of major neutrophil chemotactic factors at the luminal layer
of the mural thrombus [8,9]. ILT contains a lot of neutrophils, which release elastase, pro-MMP-9 and -8, and are mainly found in the luminal part of the thrombus [10]. Inflammatory cells infiltrates are relevant sources of MMP-9 and -8, among others, in the AAA wall and may substantially contribute to aneurysm wall instability. Furthermore, significant inverse correlations were found between
Table 2 Correlations between biochemical parameters within A and B sites of the AAA wall. Correlated parameters
Active MMP-9
Total MMP-8
TIMP-1
Rs
P
Rs
P
Rs
P
Site A (thrombus ≤10 mm) Elastase activity Active MMP-9 Total MMP-8
+0.01 – –
0.96 – –
+0.27 +0.07 –
0.10 0.65 –
+0.13 −0.10 −0.03
0.42 0.52 0.85
Site B (thrombus ≥25 mm) Elastase activity Active MMP-9 Total MMP-8
+0.13 – –
0.41 – –
+0.17 +0.39 –
0.30 0.012 –
−0.08 +0.02 −0.05
0.63 0.88 0.75
Rs – Spearman rank correlation coefficient.
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Table 3 Correlations between biochemical parameters measured in sites A and B of the AAA wall. Biochemical parameters
Elastase activity Active MMP-9 Total MMP-8 TIMP-1 MMP-9/TIMP-1 ratio
Correlation between sites A and B Rs
P
+0.42 +0.18 +0.28 +0.43 +0.46
0.0072 0.27 0.078 0.0052 0.0027
Rs – Spearman rank correlation coefficient.
the amounts of inflammatory cells and elastin/collagen content of the aortic vessel wall [11]. MMP-9 activity was also distributed in macrophages by invading neovessels at the outer media and adventitia of the AAA [12,13]. Cellular penetration from the AAA lumen through ILT may occur up to 10 mm [7]. Therefore, thin ILT seems to be more permeable than thick one. The intima and inner media of the aortic wall are supplied by blood from the lumen [14]. Aneurysmal tissue elastase activity is situated in the intima and inner media of the wall [15]. Hemodynamic forces may modulate AAA inflammation and diameter enlargement via direct regulation of intimal macrophage adhesion and transmural migration [16]. The abovementioned observations are in agreement with the current results of our study. We found that part of the aneurysmal wall covered by thin ILT showed higher elastase, active MMP-9 and total MMP-8 levels. MMP-9 appears to be the predominant metalloproteinase in the AAA. This is due to the fact that its mRNA levels are more than 20 times higher than those of MMP-1 and 2 times higher than those of MMP-2 [17]. Elmore et al. [18] claim it is the growth of MMP-9 activity and not MMP-2 that is an important factor in the etiology of AAAs. The presence of MMP-9 is important not only because it appears to influence all stages of AAA pathogenesis but also because its substrate specificity toward partially degraded fibrillar collagen fragments complements the action of MMP-8, a potent type I collagenase [19]. Leukocyte-derived MMP-9 is associated with the aortic wall degeneration and the aneurysm formation [20]. It has been hypothesized that an imbalance between proteinases and their naturally occurring inhibitors is the cause of observed histologic abnormalities. An unbalanced relation in the MMPs/TIMPs activity ratio can underlie the pathogenesis of the abdominal aortic aneurysm. Decreased TIMP-1 and TIMP-2 expression in the extracellular matrix of the walls of AAAs has been demonstrated in several studies. In comparison with the control group, the MMP9 to TIMP-1 ratio (a relative index of proteolytic state) was raised in the aortic aneurysms tissue [21]. Interaction of TIMP-1 (a natural inhibitor of MMP-9) with MMP-9 plays an important role [22]. In our study, the active MMP-9/total TIMP-1 ratio was greater in the thin thrombus-covered wall compared with a thicker thrombuscovered wall. The assessment of the posttranslational regulation of protease activity showed a threefold increase in MMP-8 activation in growing as well as ruptured AAAs [23]. Enhanced expression of MMPs mRNA was found in the aneurysm wall without thrombus in contrast to the thrombus-covered wall. Furthermore, MMP-9 activity was also discovered in the interface between the thrombus and the underlying wall [24]. Our results for the first time showed quantitatively that active forms of elastase, MMP-9, active MMP9/total TIMP-1 ratio, and MMP-8 concentration were strongly raised within the thin thrombus-covered wall compared to samples from thick thrombus-covered AAA wall. The reports proving that mural thrombus was significantly thinner in ruptured than in non-ruptured AAA (9 vs. 19 mm), as well as those describing that the aneurysm biopsies from the rupture site show higher MMP-8 and -9 levels compared to samples taken from the paired
anterior wall [19,25] are in accordance with our study. Some studies demonstrated that human neutrophil elastase could activate pro-MMP-9 and also degrade TIMP-1 [3]. In accordance with the aforementioned facts, in the present study, the aneurysmal wall covered by a thin ILT shows a higher elastase and MMP-9 activity and a lower TIMP-1 concentration. On the basis of the above data, we suggest that increased elastase activity may influence the activity of MMP-9, which is thought to play an important role in aneurysm development. We also found that all biochemical parameters, except active MMP-9, correlated positively between thin thrombus-covered and thick thrombus-covered AAA wall. It suggests that MMP-9 penetration to AAA wall with thin and thick thrombus may be regulated by different mechanisms. The uptake of (18)F-fluorodeoxyglucose (FDG) in the aneurysm wall may be connected to the accumulation of inflammatory cells responsible for the production and the activation of degrading enzymes [26]. Truijers et al. [27] reported that FDG uptake is located within the non-calcified aneurysm wall. This indirectly confirms that transfer of inflammatory cells and subsequently elastase, MMP-9 and MMP-8 occurs from the lumen through thin ILT to the underlying wall to a greater extent than from the vasa vasorum of the human AAA. The mean annual growth rate was significantly lower in men with an AAA wall calcification above than below 50% [28]. AAA wall thickness varied regionally – from low at a rupture site to high at a calcified site [29]. It has been suggested that calcified plaques may be a significant barrier for the transport of inflammatory cells to the wall of the AAA. The development of heterogeneous multilayered intraluminal thrombi and calcified plaques likely has an effect on the thickness variability of the vascular wall. The thin thrombus-covered wall associated with proteolytic elastase and MMP-9 hyperactivity could lead to focal weakening of the aneurysm wall. These observations may explain why some small aneurysms rupture and larger ones do not, and why rupture is rarely located in the largest diameter. We suggest that the thickness of ILT may be a surrogate marker for the other changes that are associated directly with the AAA growth rate and a potentially earlier rupture in thin thrombus-covered wall of aneurysms. Furthermore, a halo-type, uniform thin intraluminal thrombus in small AAAs should lead to more frequent evaluations of the aneurysm size or suggest early surgery. Therefore the distribution of proteolytic activity within the AAA wall might be a good indicator of its susceptibility to rupture. These observations are in agreement with our pathologic concepts describing the role of local factors in the pathogenesis of AAA rupture [Fig. 3 online Supplementary material]. Our study points to thin ILT as a significant factor responsible for high protease activity in AAA wall, but it is not conclusive as regards the mechanism of this phenomenon: is it just the lack of protection associated with thick thrombus, or active participation of thin thrombus in the elevated activity of proteases? Measurement of protease activity in thrombus-free regions of AAA could explain this issue. Unfortunately, we found that AAAs with even a small surface of truly thrombus-free wall are very rare in our patients. Even when CT imaging suggested presence of thrombus-free wall, visual examination usually showed thin layer of thrombus, so we could not classify such samples as thrombus-free. The lack of proteolytic activity measurements within thrombus-free AAA wall is a limitation of our study which does not allow us to make firm conclusions about the role of the thrombus. However, Kazi et al. demonstrated that MMP-1, -7, -9 and -12 expressions were upregulated in the wall segments clearly without thrombus compared with the thrombus-covered wall [24]. It seems that elevated protease activity might be a common feature of thin thrombus-covered wall and thrombus-free wall, but further studies are needed to confirm this hypothesis. Another limitation of our study is the lack of measurements of protease activity in thrombus. The results of pre-
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vious studies, referenced above, showed that neutrophils releasing proteases (including MMP-9) are located in the luminal sublayer of the thrombus. This was confirmed by a recent paper using in vivo MR imaging and histologic analysis in high-risk AAAs [30]. Therefore, our results could be explained by the fact that thin thrombi convey more protease activity to the AAA wall than thick thrombi. In conclusion, our study demonstrates for the first time that the part of abdominal aortic aneurysm wall covered with thin thrombus is exposed to higher proteolytic activity than the part protected by thick thrombus. This phenomenon can be considered as one of the mechanisms responsible for the association of thrombus size, shape and location with the risk of aneurysm rupture. Acknowledgement We thank Mr. Gary Stewart, a native English speaking translator, for his assistance in preparation of the manuscript. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.atherosclerosis.2010.04.033. References [1] Cohen JR, Parikh S, Grella L, et al. Role of the neutrophil in abdominal aortic aneurysm development. Cardiovasc Surg 1993;1:373–6. [2] Eliason JL, Hannawa KK, Ailawadi G, et al. Neutrophil depletion inhibits experimental abdominal aortic aneurysm formation. Circulation 2005;112:232–40. [3] Itoh Y, Nagase H. Preferential inactivation of tissue inhibitor of metalloproteinases-1 that is bound to the precursor of matrix metalloproteinase 9 (progelatinase B) by human neutrophil elastase. J Biol Chem 1995;270:16518–21. [4] Powell J, Greenhalgh RMJ. Cellular, enzymatic, and genetic factors in the pathogenesis of abdominal aortic aneurysms. J Vasc Surg 1989;9:297–304. [5] Wiernicki I, Cnotliwy M, Baranowska-Bosiacka I, et al. Elastin degradation within the abdominal aortic aneurysm wall – relationship between intramural pH and adjacent thrombus formation. Eur J Clin Invest 2008;38:883–7. [6] Wilson WR, Schwalbe EC, Jones JL, Bell PR, Thompson MM. Matrix metalloproteinase 8 (neutrophil collagenase) in the pathogenesis of abdominal aortic aneurysm. Br J Surg 2005;92:828–33. [7] Adolph R, Vorp DA, Steed DL, Webster MW, Kameneva MV, Watkins SC. Cellular content and permeability of intraluminal thrombus in abdominal aortic aneurysm. J Vasc Surg 1997;25:916–26. [8] Houard X, Touat Z, Ollivier V, et al. Mediators of neutrophil recruitment in human abdominal aortic aneurysms. Cardiovasc Res 2009;82:532–41. [9] Houard X, Ollivier V, Louedec L, Michel JB, Bäck M. Differential inflammatory activity across human abdominal aortic aneurysms reveals neutrophil-derived leukotriene B4 as a major chemotactic factor released from the intraluminal thrombus. FASEB J 2009;23:1376–83. [10] Fontaine V, Touat Z, Mtairag el M, et al. Role of leukocyte elastase in preventing cellular re-colonization of the mural thrombus. Am J Pathol 2004;164:2077–87.
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