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From the Atherosclerosis Research Unit (M.K., C.Z., A.H., P.E.), King Gustav V Research Institute, Department of Medicine, Karolinska Institute; the Department of Vascular Surgery (M.K., J.S., U.H.), Karolinska Hospital, Karolinska Institute; St G?rans Hospital (J.R.); and Cardiovascular Research, Center for Molecular Medicine (G.P.-B.), Karolinska Institute, Stockholm, Sweden.
Correspondence to Per Eriksson, King Gustav V Research Institute, Karolinska Institutet, SE-171 76 Stockholm, Sweden. E-mail per.eriksson@medks.ki.se
Abstract
Objective— It has been suggested that the intraluminal thrombus of abdominal aortic aneurysms (AAAs) predisposes for AAA rupture. Here, we examined the possibility that the intraluminal thrombus influences expression and activity of matrix-degrading proteases in the AAA wall.
Methods and Results— Twenty patients undergoing elective repair of AAAs were included. From each patient, specimens from both thrombus-covered and thrombus-free wall were taken for analysis. Gene arrays and quantitative real-time polymerase chain reaction showed that matrix metalloproteinase (MMP)-1, -7, -9, and -12 expressions were upregulated in the thrombus-free wall compared with the thrombus-covered wall. Immunohistochemistry confirmed the differential expression of MMP-9 but also localized MMP-9 to the interface between the thrombus and the underlying vessel wall. MMP-9 expression was colocalized with the presence of macrophages. Similar expression patterns were observed for urokinase plasminogen activator (uPA), uPA receptor, and plasminogen activator inhibitor-1. Gelatinase activity was detected in the same regions as MMP-9 protein expression, ie, within the thrombus-free wall and in the interface between the thrombus and the underlying wall.
Conclusion— The present work demonstrates that protease expression and activity differs within the aneurysm wall. The source and activity of the proteases responsible for the degradation of the thrombus-covered wall need to be further determined.
It has been suggested that the intraluminal thrombus of abdominal aortic aneurysms (AAAs) predisposes for AAA rupture. Differences in gene expression pattern revealed that many MMPs were upregulated in the thrombus-free wall. The source of the proteases responsible for the degradation of the thrombus-covered wall needs to be determined.
Key Words: abdominal aortic aneurysm ? thrombus ? gene expression ? matrix metalloproteinases
Introduction
The risk of rupture of an abdominal aortic aneurysm (AAA) is determined by its size, whereas growth of the AAA is dependent on proteolytic degradation of collagen and elastin. The degradation of extracellular matrix has been associated with increased activities of matrix-degrading proteases, especially matrix metalloproteinases (MMPs). Recently, the increased expression of several MMPs and tissue inhibitors of metalloproteinases (TIMPs) were described in the aortic wall from patients with AAAs.1,2 In addition, the elastolytic proteases cathepsin K and S have been shown to be upregulated in aneurysm tissue whereas the cathepsin inhibitor cystatin C is downregulated.3
However, several questions regarding the pathogenesis of rupture remain unresolved. It is known that AAAs are filled to a varying extent with a laminated thrombus.4–7 The presence of thrombus has been suggested to be involved in the weakening of the underlying vessel wall leading to aneurysm rupture, but the exact mechanisms are not known. Previous studies analyzing the expression and activity of matrix degrading proteases have usually not considered the presence of thrombus,1,8–14 although both aneurysm growth and rupture have been associated with growth of the thrombus.15,16 Furthermore, it has been suggested that rupture may be attributed to hemorrhage into the mural thrombus. Arita et al17 performed computed tomographic analysis on both ruptured and nonruptured aneurysms and found that clefts with seeping blood in the mural thrombus were associated with rupture. Increased gelatinolytic activity has been demonstrated in the thrombus,18 and MMP-9 activity has been colocalized with polymorphonuclear leukocytes trapped by the thrombus.19 The wall covered by thrombus is also thinner, and contains less elastin and fewer smooth muscle cells (SMCs) but more B- and T-cells than the thrombus free wall.20,21 Together, this implies that the thrombus affects the underlying vessel wall and predisposes for rupture.
In the present study we analyzed gene expression in wall segments with and without thrombus from patients undergoing elective AAA repair. The aim was to establish whether differences in gene expression pattern could be linked to an increased risk for AAA rupture. Expression and activity of matrix-degrading proteases were analyzed with gene arrays, quantitative real-time polymerase chain reaction (PCR), immunohistochemistry, and in situ zymography.
Materials and Methods
Biochemical Reagents
PBS was purchased from Life Technologies, hydrogen peroxide (H2O2), methanol, and xylene were from Merck, mounting media (Mountex) was from Histolab, and bovine serum albumin was from Sigma Diagnostics. Vectastain ABC kit, 3,3-diaminobenzidine (DAB-) peroxidase substrate kit, antigen unmasking solution, normal goat serum, biotinylated anti-mouse, secondary antibodies, and normal mouse immunoglobulins were purchased from Vector Laboratories, Inc (Burlingame, Calif). The primary antibodies used for the immunostainings are presented in Table I (available online at http://atvb.ahajournals.org). For in situ zymography, gelatinase films (Wake Chemicals Ltd.) were used.
Sample Collection
Patients undergoing elective surgery for infrarenal AAAs with preoperative computed tomography (CT) scan demonstrating eccentric intraluminal thrombus, and with a thrombus-free aneurysm wall segment, were selected for the study. The method for obtaining the biopsies has been described in detail previously.21 As shown in Figure 1, the location of the thrombus varied among the patients and also within the same patient. Biopsies of thrombus-free wall were taken both from the anterior and the lateral wall. Added risk to the surgery prevented biopsies from the posterior wall. Two 2x4-cm tissue sections were cut transversely from thrombus-free and thrombus-covered aneurysm wall, respectively. The adventitia was dissected free of excess perivascular fat, and the sections were fixed in 4% paraformaldehyde for light microscopy or immediately snap frozen in liquid nitrogen (LN2) for RNA isolation and in situ zymography. Selection criteria restricted the number of patients. Altogether, biopsies from 20 patients were used in this study, of which biopsies from 12 patients were used for RNA preparations and the rest for immunostaining and zymography. All patients approved the intraoperative retrieval of tissue from the aneurysm wall according to informed consent procedures and approval by the local ethic committee.
Figure 1. Representative pictures of computed tomography (CT) demonstrating that the location of the thrombus varied among the patients and also within the same patient. a and b, Images from a single patient demonstrating thrombus-free and thrombus-covered area as a site of biopsy from different levels of the aneurysm. c and d, Two different patients where biopsies were taken at same level in each patient. Arrowheads indicate areas of biopsy.
Microarray Analysis and Quantitative Real-Time PCR
Frozen samples were homogenized in a dismembranator (B. Braun Melsungen AG, Germany). Lysis buffer was added to the homogenate, and RNA was isolated using RNeasy mini kit including a DNase I step (Qiagen). Quality and quantity of RNA were analyzed by an Agilent 2100 bioanalyzer (Agilent Technologies). RNA originating from either of the two wall segments were pooled, and 10 μg was used for microarray chip analysis (Affymetrix, U95A array). cDNA synthesis, hybridization, and analyses of results were conducted as recommended by the manufactures. Three separate experiments were performed for both locations.
For quantification using real-time PCR, 0.5 μg of RNA from each individual patient sample was reverse transcribed using superscript II according to the manufacturer’s manual (Invitrogen). After dilution of the cDNA to 100 μL, 3 μL of cDNA were amplified by real-time PCR with 1x TaqMan universal PCR master mix (Applied Biosystem). Assay On-Demand Kits containing corresponding primers and probes from Applied Biosystem were used. ?-Actin and acidic ribosomal phosphoprotein P0 (RPLP0) were used as housekeeping genes to normalize for RNA loading. For ?-actin and RPLP0, 1 μmol/L of each primer and 0.25 μmol/L of probe were used. ?-Actin primers were designed using the Primer Express software (Applied Biosystem). The primers for ?-actin were: ?-actin-FW, 5-CTGGCTGCTGACCGAGG-3 and ?-actin-RW, 5-GAAGGTC-TCAAACATGATCTGGGT-3; the probe was: ?-actin-TM, 6FAM5'-CCCTGAACCCCAAGGCCAACCG-3'TAMRA. The primers for RPLP0 were as described22: RPLP0-FW, 5-CCATTCT-ATCATCAACGGGTACAA-3 and RPLP0-RW, 5-AGCAAGTGG-GAAGGTGTAATCC-3; the probe was: RPLP0-TM, 6FAM5'-TCTCCACAGACAAGGCCAGGACTCGT-3'TAMRA. Each sample was analyzed in duplicate using ABI prism 7000 (Applied Biosystem). The PCR amplification was related to a standard curve.
Immunohistochemistry
Paraffin sections were cleared in xylene, rehydrated in graded ethanol (100% to 70%), immersed in water for 5 to 10 minutes, and incubated in 0.3% hydrogen peroxide (H2O2) in 70% methanol for 20 minutes to inhibit endogenous peroxidase activity. The specimens were then rinsed 3x5 minutes in PBS. Epitopes were unmasked by boiling in citrate buffer (pH 6.0) for 10 to 15 minutes when necessary. After rinsing in PBS, the sections were blocked for 30 to 60 minutes in 3% bovine serum albumin (BSA) in PBS or 5% goat or rabbit serum in PBS and then incubated with and without primary antibodies (Table I) in 0.1% BSA in PBS overnight at 4°C in a humidified chamber. The samples were then rinsed in PBS and incubated with 7.5 μg/mL biotinylated secondary antibody in 0.1% BSA in PBS for one hour at room temperature followed by avidin-biotin amplification (ABC Elite) for 30 minutes, and developed with DAB substrate. Sections were counterstained with Mayer hematoxylin for 2 to 5 minutes and mounted. Negative controls were obtained by substituting the primary antibody with PBS.
In Situ Zymography
Gelatinolytic activity was analyzed using a MMP in situ Zymo-Film (Wake Chemicals Ltd.). MMP-PT in situ Zymo film that contains 1,10-Phenanthroline as a MMP inhibitor and 0.01% EDTA to block protease activity was used as control. Frozen sections (5 to 6 μm) were placed on the film and incubated in a moist chamber at 37°C for 6 to 18 hours according to the manufacturer’s instructions. The specimens were then stained with Amido black 10B (Sigma) for 15 to 20 minutes and destained for 10 minutes. The areas of gelatinolytic activity were visualized under light microscope.
Statistical Analysis
Quantitative RT-PCR data were evaluated with a paired t test. Probability values below 0.05 were considered statistically significant. Spearman Rank correlations were computed to analyze relationships between cell-specific mRNA markers and investigated genes.
Results
Differential Gene Expression of Matrix-Degrading Proteases in the Aneurysm Wall
Gene array analysis on pooled RNA from 8 patients was performed to screen for MMP expression. MMP-1, MMP-7, MMP-9, and MMP-12 showed significant differential expression when comparing the thrombus-covered wall and the thrombus-free wall; all were overexpressed in the wall without thrombus. The expression of MMP-1, MMP-7, MMP-9, and MMP-12 was 3.1-, 7.5-, 3.0-, and 3.0-fold higher, respectively, in the thrombus-free wall (Table II, available online at http://atvb.ahajournals.org).
Because the array analyses were performed on pooled samples, the findings were verified with quantitative real-time PCR on individual samples from 5 patients using ?-actin and RPLP0 as house-keeping genes. Normalization to either house-keeping genes gave the same results, and the presented results were normalized to ?-actin. Quantitative real-time PCR analyses of 13 genes present on the array confirmed the results of the gene-array analysis (Table III, available online at http://atvb.ahajournals.org). Expression of MMP-1 (P<0.05), MMP-7 (P=0.06), and MMP-9 (P<0.01) was increased in thrombus-free wall (Figure 2a through 2c), whereas increased expression of MMP-12 in the thrombus-free wall was only seen in one patient (Figure 2d). Furthermore, TIMP-1 expression was enhanced in the thrombus-free wall as compared with the thrombus-covered wall (P<0.05; Figure 2e). MMP-1, MMP-7, and MMP-12 mRNA were exclusively expressed in the thrombus-free wall whereas MMP-9 and TIMP-1 showed a low expression also in the thrombus-covered wall in some patients (Figure 2a through 2e).
Figure 2. mRNA expression of genes potentially involved in matrix degrading processes in the AAA wall measured by quantitative real-time PCR in the aneurysm wall segments exposed to flowing blood (NT) and segments covered by thrombus (T). Values are arbitrary units after correction for ?-actin expression. The highest signal is referred as 100%. Each symbol corresponds to samples from the two wall regions of the same patient.
Several other genes implicated in matrix remodeling were also differentially expressed. The elastolytic protease cathepsin S (P<0.05), urokinase-type plasminogen activator (uPA; P<0.05), uPA receptor (uPAR; P<0.05), and plasminogen activator inhibitor-1 (PAI-1; P=0.05) were all upregulated in the thrombus-free wall, but there was no significant difference in tPA mRNA expression between the two wall segments (P=0.51; Figure 2f through 2j).
Quantitative real-time PCR analysis of mRNA concentrations of cell-specific markers in the different wall segments demonstrated CD68 mRNA (macrophages) mainly in the wall without thrombus (P<0.05; Figure 3b), whereas CD20 mRNA expression (B-cells) was more abundant in the thrombus-covered wall (P=0.07; Figure 3d). SM22 mRNA, a smooth muscle cell-specific transcript, was present in both wall segments (Figure 3a). CD3 mRNA expression (T cells; Figure 3c) appeared to be more abundant in the thrombus-covered wall although the differences did not reach statistical significance (P=0.19). There was a strong correlation between CD68 mRNA concentrations and mRNA concentrations of MMP-1 (R=0.87; P<0.01), MMP-12 (R=0.67; P<0.05), uPAR (R=0.86; P<0.05), uPA (R=0.82; P<0.05), PAI-1 (R=0.81; P<0.05), and cathepsin S (R=0.98; P<0.01). None of the protease genes correlated with SM22, CD3, or CD20 except for uPA mRNA, which correlated positively with SM22 mRNA (R=0.70, P<0.05) and negatively with CD20 mRNA concentrations (R=–0.70, P<0.05).
Figure 3. mRNA expression of cell-specific markers measured by quantitative real-time PCR in AAA wall segments exposed to flowing blood (NT) and AAA wall covered by thrombus (T). Values are arbitrary units after correction for ?-actin expression. The highest signal is referred as 100%. Each symbol corresponds to samples from the two wall regions of the same patient.
Localization of MMP-9 Protein in the Aneurysm Wall
Protein expression in the two wall segments was analyzed by immunohistochemistry. In accordance with the mRNA analysis, MMP-9 protein was detected mainly in the wall without thrombus (Figure 4a and 4c), but MMP-9 protein was also detected in the luminal interface between the thrombus and the underlying wall (Figure 4e and 4g). The expression of MMP-9 was colocalized with the presence of macrophages (CD68 positive cells) both in the thrombus-free wall (Figure 4b and 4d) and in the interphase between the thrombus and the underlying wall (Figure 4f and 4h). uPA and uPAR protein were mainly detected in the thrombus-free wall (Figure 5a and 5c), whereas PAI-1 protein expression differed among the patients (Figure 5d). In common with MMP-9, uPAR and PAI-1 were also detected in the region between the thrombus and the underlying wall (Figure 5d and 5f). Staining for MMP-7 and MMP-12 was too weak to allow firm conclusions (data not shown).
Figure 4. Immunohistochemical demonstration of MMP-9 protein expression in thrombus-free AAA wall (NT; a and c), AAA wall covered with thrombus (T; e and g). CD68-positive cells demonstrating macrophages in AAAs exposed to flowing blood (b and d) and in AAA wall covered with thrombus (f and h). Bars represent 100 μm. A indicates adventitia; M, media; I, intima. Dotted line outlines the thrombus.
Figure 5. Immunohistochemical demonstration of uPA (a and b), uPAR (c and d), and PAI-1 (e and f) protein expression in AAA wall exposed to flowing blood (NT) and in AAA wall covered with thrombus (T). Arrows indicate positive staining. Bars represent 100 μm. A indicates adventitia; M, media; I, intima. Dotted line outlines the thrombus.
In Situ Zymography
Because MMPs are synthesized as inactive proenzymes, protease activity was analyzed using in situ zymography. Gelatinase activity was localized to the intima and media of the thrombus-free wall and to the region between the thrombus and the underlying wall (Figure 6a, 6c, and 6e). MMP inhibitor 10-phenanthroline (Figure 6b) and protease inhibitor EDTA (Figure 6d and 6f) were used as controls.
Figure 6. In situ zymography demonstrating gelatinase activity in AAA wall exposed to flowing blood (NT; a and c) and in AAA wall covered by thrombus (T; e). Control sections were treated with the MMP inhibitor 10-phenanthroline (PT; b) or EDTA (d and f). Blue color represents gelatinase activity. Bars represent 500 μm. M indicates media; I, intima. Dotted line outlines the thrombus.
Discussion
The intraluminal thrombus has been associated with growth and rupture of AAAs.15,16 Clinical data support the concept that rupture occurs through the thrombus-covered wall after bleeding into the thrombus.17 Structurally, the thrombus-covered aneurysm wall is thinner than the thrombus-free wall and contains fewer and fragmented elastin fibers as well as a reduced number of SMCs.20,21 Elastin degradation has been suggested to be important for aneurysm growth, and elevated plasma levels of elastin-derived peptides are predictive of rupture in humans.23,24 Taken together, these observations suggest that the thrombus is associated with an increased risk of aneurysm rupture, possibly by influencing the degradation of vessel wall extracellular matrix. To further explore the causes for increased elastin degradation in the thrombus-covered wall, mRNA and protein expression as well as activity of matrix-degrading proteases were characterized in AAA wall segments and related to the presence of the luminal thrombus.
In contrast to our initial hypothesis, gene array analysis on pooled RNA samples as well as quantitative real-time PCR on individual specimens demonstrated upregulation of matrix-degrading proteases and CD68 in the thrombus-free wall. Accumulation of macrophages in the thrombus-free wall was also supported by the upregulation of CD11b and MARCO mRNA within this wall segment. Immunohistochemistry and in situ zymography localized MMP-9 protein expression and gelatinase activity to the media of the thrombus-free wall but also to the interface between the thrombus and the underlying wall. Immunohistochemical evaluation showed colocalization of staining for MMP-9 and CD68-positive cells at both these sites. Furthermore, there was a strong correlation between the expression of mRNA for CD68 and MMPs. Together this suggests that macrophages may be the main source of protease expression, which is an integrated part of an ongoing inflammatory process in the vessel wall. We have recently shown that macrophage differentiation in vitro is associated with a significant increase in their proteolytic capacity.25
Similar to the MMPs, cathepsin S mRNA expression was detected in the thrombus-free wall. Increased expression of this cysteine protease, which belongs to a family of proteases with potent elastolytic activity,26,27 has recently been demonstrated in AAAs.3 Because monocyte-derived macrophages have the capacity to synthesize cathepsin S,28 the accumulation of CD68-positive cells within the media of the thrombus-free wall may explain the increased expression of this protease. However, the apparent lack of elastin degradation within the thrombus-free wall indicates that there is not a high degree of proteolytic activity, although gelatinase activity could be detected by using in situ zymography. In this context it should be noted that TIMP-1 was upregulated in the thrombus free wall.
Gene array analyses showed that other genes previously linked to coronary atherosclerosis such as interleukin-1 receptor antagonist,29 lectin-type oxidized LDL receptor (LOX-1),30 and lipoprotein lipase31 were upregulated in the thrombus-free wall. The main difference compared with atherosclerosis appears to be a localization of macrophages in the media of the AAA wall rather than the intima as seen in atherosclerosis. The difference between medial versus intimal inflammation is at present not understood.
Protease expression and activity localized to the border region between the thrombus and the underlying vessel wall may explain the previously demonstrated lack of elastin fibers in the thrombus-covered wall.21 However, protease activity derived from the thrombus may also contribute, because the thrombus itself is a source of proteolytic activity.18,19 Leukocytes trapped within the luminal part of the thrombus have been shown to secrete MMP-9 and elastase.32 Leukocyte elastase is mainly found in the luminal part of the thrombus, whereas MMP-9 activity is also found in the abluminal part.32 The thrombus-covered aneurysm wall showed an increased expression of CD3 and CD20 mRNA, confirming our previous observations of an aggregation of T-cells (CD3-positive cells) and B-cells (CD20-positive cells) in this wall segment.21 These observations indicate the presence of intense inflammation in the thrombus-covered aneurysm wall. Whether leukocytes in association with the thrombus are also the source of the uPAR and PAI-1 detected remains to be shown. uPAR is a glycosyl-phosphatidylinositolanchored glycoprotein on the surface of various cell types that serves to localize the activation reactions in the proteolytic cascade system of plasminogen activation.33 Circulating neutrophils have been demonstrated to express uPAR on their cell surface,34 and the thrombus has been shown to be able to trap plasma PAI-1.35 Furthermore, platelets store significant amounts of PAI-1 in their -granules, which are released on platelet activation.36 The significance of this is, however, questionable because platelet PAI-1 is less active than plasma PAI-1.37
The degradation of elastin probably precedes the destruction of collagen during AAA development,23 and local treatment with elastase causes aneurysms in experimental animals.38,39 Thus, it is reasonable to assume that elastin degradation leading to elongation and dilatation of the aorta precedes thrombus formation in the aneurysm. Macrophage accumulation and MMP-9 activity in the media of the aneurysm wall without thrombus could possibly represent this early phase of wall degradation and aneurysm formation.
In conclusion, the present work demonstrates that protease expression differs within the aneurysm wall. Upregulation of MMPs was found in the aneurysm wall without thrombus, but MMP-9 activity was also found in the interface between the thrombus and the underlying wall. The differential expression of proteolytic enzymes should be remembered when evaluating reports of proteolytic activity in specimens obtained from AAAs. The importance of proteases derived from the thrombus related to aneurysm growth and rupture needs to be further evaluated to fully explain elastin degradation seen in the thrombus-covered wall. This could possibly be performed in an appropriate AAA animal model containing an intraluminal thrombus.
Acknowledgments
This project was supported by grants from the Swedish Research Council (12660 and 6623), the Swedish Heart-Lung foundation, the Torsten and Ragnar S?derberg foundation, the King Gustaf V 80th Birthday Foundation, the King Gustaf V and Queen Victoria Foundation, and AFA Health Fund. We are grateful to Siw Frebelius, Ann-Britt Wikstr?m, and Mariette Lengquist for excellent technical assistance.
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