Asian Cardiovasc Thorac Ann 2007;15:185-190
© 2007 Asia Publishing EXchange Ltd
Proteomics of Ascending Aortic Aneurysm with Bicuspid or Tricuspid Aortic Valve
Peter Matt, MD,
Anne von Orelli, MD1,
Franziska Bernet, MD,
Thomas Grussenmeyer, PhD1,
Ivan Lefkovits, PhD1,
Hans-Reinhard Zerkowski, MD
Division of Cardiothoracic Surgery
1 Cardiovascular Proteomics Research Laboratory, University Hospital, Basel, Switzerland
For reprint information contact: Peter Matt, MD Tel: 41 61 328 6102 Fax: 41 61 265 8854 Email: pmatt{at}uhbs.ch, Division of Cardiothoracic Surgery, University Hospital, Spitalstrasse 21, CH-4031 Basel, Switzerland.
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ABSTRACT
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Bicuspid aortic valve is often associated with lesions of the ascending aorta, which differ histologically from those in tricuspid valve patients. We undertook proteomic analyses to assess differences at the proteome level. Aortic samples were collected from 20 patients undergoing aortic valve and/or ascending aortic replacement; 9 had a bicuspid valve: 5 with aortic aneurysm (diameter > 50 mm) and 4 without dilation; 11 had a tricuspid valve: 6 with aortic aneurysm and 5 without dilation. Patients with histologically proven connective tissue disorders were excluded. Samples were dissected, solubilized, and subjected to 2-dimensional gel electrophoresis. Gel patterns showed an average of 580 protein spots in samples from bicuspid valve patients, and 564 spots in those with tricuspid valves. Comparative analysis revealed a correlation coefficient of 0.93 for protein expression in the bicuspid valve group compared to the tricuspid group. Three protein spots were significantly over-expressed and 4 were significantly down-regulated in the bicuspid group compared to the tricuspid group. The lowest correlation in protein expression was between non-dilated aortic tissues. These differences between aortic tissues of bicuspid and tricuspid valve patients suggest that mechanisms of aortic dilation might differ, at least in part, between such patients.
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INTRODUCTION
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Bicuspid aortic valve (BAV) is a common congenital valve malformation with a prevalence of 1%–2% in the general population, based on autopsy studies.1,2 Bicuspid aortic valve has a hereditary component and affects males four times more often than females. Bicuspid aortic valve may lead to aortic valve insufficiency and/or stenosis, and is often associated with acquired lesions of the aorta, such as aortic aneurysm formation, tubular ring dilation, and aortic dissection. Aortic dilation is also found in patients with a normally functioning valve and is typically located on the convexity of the ascending aorta.3 Flow turbulence due to a BAV may be one of the causes of aortic complications.4 Aortic valve cusps and the ascending aortic media are known to be derived from cells of the same neural crest.5 Thus a growing body of evidence suggests a common pathogenetic mechanism underlying both BAV formation and aortic lesions. Genomic investigations showed no difference between the aortic tissues of people with BAV and those with a tricuspid aortic valve (TAV).1,6 Histological studies revealed less cystic medial necrosis, reduced elastic lamellae, and increased smooth muscle cell apoptosis in BAV-associated aorta.7–9 Some differences in matrix metalloproteinases (MMP-2, MMP-9, and MMP-1) with loss of fibrillin-1 have been described in aortic tissue associated with BAV compared with that of TAV.10–12 Additional matrix changes might be present at the transcriptional/proteome level; therefore, we compared the proteome analyses of ascending aortic wall segments of patients with BAV and those with TAV, with and without aortic dilation.
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PATIENTS AND METHODS
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Ascending aortic wall segments were obtained from 20 patients undergoing elective aortic valve and/or ascending aorta replacement; 9 had a BAV and 11 had a TAV. Five patients with BAV had an ascending aortic aneurysm > 50 mm in diameter (group 1) and 4 had no aortic dilation (diameter < 30 mm; group 2). Six patients with TAV had an ascending aortic diameter > 50 mm (group 3) and 5 had no aortic dilation (group 4). Group 4 represents the control group. Only patients with a true congenital BAV or TAV, assessed by patient history and intraoperative anatomical examination, were included. Patients whose aortic valve became functionally bicuspid during their lifetime were excluded. All samples were examined histologically to exclude patients with arteriosclerosis, connective tissue disorders, and inflammatory diseases. Patients with coexisting coronary artery disease and/or peripheral arterial disease were also excluded. Hospital records of all patients were reviewed, focusing on aortic valve history, demographics, and operative procedures.
Aortic wall segments were excised from the anterior ascending aorta 2 to 4 cm above the aortic annulus. All specimens were immediately placed in a physiological solution (Krebs-Henseleit) to remove blood components, frozen in liquid nitrogen, and stored at –80°C until the time of preparation and solubilization. Aortic samples were solubilized using a buffer containing 4% 3[(3-cholamidopropyl) dimethylammonio]-propanesulfonic acid, 2 M thiourea, 7 M urea, 20 mM dithiothreitol, and 2% Ampholine (pH 9-11).13 The equivalent of 2 mg of tissue was used for each sample. The 1st and 2nd dimensions of the two-dimensional gel electrophoresis (2-DE) were carried out in Anderson’s ISODALT system for the simultaneous analysis of 20 samples (instruments produced in the workshop of the former Basel Institute for Immunology, Switzerland).14 The 1st dimension was based on the separation of polypeptides according to their molecular charge (isoelectric point). The charge separation matrix was based on the use of carrier ampholytes (BioRad, Richmond) with a high buffering capacity near their isoelectric point. This was a broad range of ampholytes covering isoelectric points between 3 and 10. The gel rod of the 1st dimension with the separated polypeptides was applied to the 2nd dimension of an 11%–19% acrylamide gradient, and electric current applied for overnight size separation (constant 140 V). Polypeptide spots were visualized on 2-DE gels by silver staining.13 Four runs of 2-DE were performed to reproduce proteomic patterns and to confirm proteome differences between samples. The 2-DE gels were scanned in an ImageScanner (Amersham, Zurich). Image analysis was carried out using PDQuest (BioRad). Image files were processed for noise, streak removal, and background correction, and converted into spot files by spot modelling and fitting. In the final spot list, each spot was defined by x- and y-coordinates and by the spot volume. Upon spot detection, the entire pattern was inspected for artefacts that were not removed by the software algorithms. Such spots were eliminated or merged with other spots if appropriate. At the end of the matching process, a master pattern containing all the spots was obtained in each of the images. Quantitative evaluation and comparison of gel patterns was carried out upon normalization. The normalization procedure corrects for differences in the overall intensity of silver staining in 2-DE gel images. For comparative analysis of all 20 samples, interest was focused on one region that was comparable among all samples (Figure 1
). The aim was to identify a set of spots that reflect modulation (over-expression or down-regulation) of the final polypeptide products in the BAV and TAV samples.
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Figure 1. Representative 2-D gel patterns in the 4 groups. The red frames mark the areas of interest that were compared. Gel size, 17 x 17 cm. Group 1 = bicuspid aortic valve + ascending aneurysm; group 2 = bicuspid aortic valve + no aneurysm; group 3 = tricuspid aortic valve + ascending aneurysm; group 4 = tricuspid aortic valve + no aneurysm. pI = isoelectric point, Mr = molecular weight.
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Quantitative parameters of all spots were stored in a database and analyzed. For comparative pattern analysis, histograms, scatterplots, coefficients of correlation, linear regression, and the Mann-Whitney U test were used. A value of p < 0.05 was considered significant.
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RESULTS
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The patient’s baseline characteristics are presented in Table 1. At the time of surgery, patients with BAV were significantly younger than those with TAV ( p < 0.02). No other baseline characteristics differed significantly between patients with BAV and those with TAV.
Figure 1
shows representative 2-DE gel patterns for all 4 groups. The 2-DE image areas that were compared and analyzed in the further investigation are marked by red frames. Comparative pattern analysis revealed quantitative differences between protein spot expression in BAV and TAV samples. The correlation of protein expression in BAV and TAV samples is presented in 3 scatterplots in Figure 2. The BAV and TAV specimens showed a strong correlation (correlation coefficient 0.93, slope of regression line 0.97), but some spots were significantly different: Figure 3A presents 3 protein spots that were significantly increased in BAV compared with TAV samples ( p < 0.05), while Figure 3B shows 4 protein spots that were significantly increased in TAV compared to BAV specimens ( p < 0.05). The non-dilated aortic samples had a much weaker correlation (correlation coefficient 0.83, slope of the regression line 0.84). Quantitative pattern analysis revealed 2 spots with at least a 10-fold increase in protein expression in non-dilated BAV vs non-dilated TAV samples, but none with a 10-fold decrease. In contrast, comparison of dilated aortic specimens showed a good correlation of protein expression (correlation coefficient 0.93, slope of the regression line 0.98). Three spots showed at least a 10-fold increase in protein expression in BAV compared with TAV dilated aortic samples, but no spots showed a 10-fold decrease. Specific protein differences were found in each of the 4 subgroups; however, not all of these protein spots were shown in each member of the subgroup. Protein identification of spots of interest using mass spectrometry is under scrutiny.
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Figure 2. Three scatterplots showing correlation of protein expression between BAV- and TAV-associated aortic samples. BAV = bicuspid aortic valve, Corr coeff = correlation coefficient, slope = slope of the green regression line, TAV = tricuspid aortic valve.
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Figure 3. Protein spots expressed differently among aortic samples. (A) Area of interest in 2-D gel of a patient with bicuspid aortic valve and aortic aneurysm. Blue arrows mark the spots with significant over-expression compared to tricuspid valve patients. (B) Area of interest in 2-D gel of a patient with tricuspid aortic valve and aortic aneurysm. Red arrows mark the spots with significant over-expression compared to bicuspid valve patients. Histograms show spot number (SSP) and spot quantity in bicuspid (blue bar) and tricuspid valve (red bar) samples.
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DISCUSSION
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Bicuspid aortic valve is a congenital malformation affecting over 4 million US citizens.1 Many patients with a BAV develop aortic complications during their lifetime, requiring invasive or noninvasive treatment, even with a normally functioning aortic valve.15 The inheritance of BAV is multifactorial, and occasionally autosomally dominant. Interestingly, Lee and colleagues16 showed a correlation between endothelium-derived nitric oxide synthetase knockout mice and the occurrence of BAV. Why patients with BAV develop aortic complications more often and earlier than those with TAV is uncertain. Consequently, it remains unclear whether patients with BAV should be monitored and treated differently to those with TAV; should they receive prophylactic treatment vs observation, valve repair vs replacement, and ascending aortic replacement with smaller diameters vs observation?3,17,18 Basic questions about a probable intrinsic defect of the aortic wall, the influence of flow turbulence on the aortic matrix, and aneurysm formation are unsolved. Genomic-based studies found no differences between BAV and TAV aortic wall specimens.1,6 Histopathological investigations of ascending aortic aneurysmal tissue revealed that BAV samples had less fibrosis, less arteriosclerosis, less cystic media necrosis, reduced elastic lamellae, and increased distance between elastic lamellae with decreased thickness of the elastic lamellae, compared to TAV samples. In addition, these samples demonstrated less smooth muscle cell disorientation, an increased proportion of apoptotic smooth muscle cells, increased mediators of cell death, but similar infiltrates consisting of macrophages, T- and B-lymphocytes, and natural killer cells.7–9,19 Biomolecular studies of BAV and TAV aortic specimens have shown several, although somewhat inconsistent, differences. Fedak and colleagues10 found increased MMP-2 activity and decreased fibrillin-1 levels in BAV samples, but no differences in elastin, collagen and MMP-9. Boyum and colleagues11 found increased activity of MMP-2 and MMP-9 in BAV compared to TAV samples. LeMaire and colleagues12 showed higher MMP-2 levels and lower MMP-9 levels in BAV compared to TAV samples. Tissue inhibitor metalloproteinases did not differ significantly between the groups.
We performed 2-DE of ascending aortic tissue to reveal quantitative and qualitative differences in protein expression between BAV and TAV samples. We included non-dilated aortic samples to obtain information on the course of aneurysm formation. Aortic specimens were taken from the anterior ascending aorta 2 to 4 cm superior to the aortic annulus to standardize the anatomical areas compared. Cotrufo and colleagues20 recently described matrix variations between the convexity and concavity of the ascending aorta. Comparison of baseline characteristics showed that patients with BAV were operated on at a younger age than those with TAV, but other characteristics did not differ significantly, as found in previous studies.8,12 Although our 2-DE of aortic samples showed an average of 580 protein spots in the BAV group and 564 spots in patients with TAV, which was not significantly different, comparative pattern analysis revealed quantitative and qualitative differences in spot expression between BAV and TAV samples. Specific spots were found in each of the 4 subgroups; however, not all spots were shown in each member of the subgroup. Scatterplot analysis revealed a good correlation in protein expression between all BAV and all TAV samples (correlation coefficient 0.93; Figure 2). The lowest correlation was between non-dilated BAV and non-dilated TAV aortic tissue. With progressive dilation of the ascending aorta, the correlation in spot expression increased (correlation coefficient in dilated aortic samples 0.94, slope of the regression line 0.98). The marked protein spots demonstrated in Figure 3 could not be identified because the amount of protein in the 2-DE gels was too low for mass spectrometry. Concentration of these proteins using different biochemical techniques is under investigation.
These preliminary results suggest that there are relevant differences at the proteome level between BAV-associated and TAV-associated ascending aortic tissue, although the variations are limited. The fact that we found the lowest correlation in non-dilated tissue supports the hypothesis of a pre-existing intrinsic aortic wall defect before aortic dilation. This could predispose patients with BAV to earlier aortic aneurysm formation. The influence of differences in flow turbulence on aortic matrix changes remains unclear, but our data show a probable adaptation process in protein expression with progressive dilation of the ascending aorta in both groups. Therefore, differences in hemodynamics may have less influence on aortic matrix variations between BAV and TAV. These results provide some evidence that molecular processes in the course of ascending aortic dilation may differ between patients with BAV and those with TAV.
There were some limitations to this study, including the small sample size, heterogenous patient population (male/female ratio, aortic valve stenosis/insufficiency ratio), and the difference in patient age at surgery. However, patients with a BAV are often operated on at a younger age due to the increased risks of valve failure and aortic complications. We tried to minimize this bias by examining all aortic samples histologically and excluding any patients showing arteriosclerosis or a connective tissue disorder. In addition, there were some difficulties related to the 2-DE technique. Performing 2-DE with ascending aortic tissue is difficult because of the fibrous nature of the tissue, and it has been seldom described in the literature. Quantitative analysis of silver-stained gels is difficult. Therefore, all 2-DE gels underwent a normalization process before pattern analysis, and protein quantities were considered to be significantly different if the p value was less than 0.05. Nevertheless, proteome analysis revealed differences between tissues of the ascending aortic wall associated with BAV and TAV. The lowest correlation at the proteome level was shown in non-dilated aortic samples. This suggests that molecular processes in the course of ascending aortic dilation might differ, at least in part, between patients with BAV and those with TAV.
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ACKNOWLEDGMENTS
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We thank Dr. Stephan Dirnhofer for analysis of histological samples, and Emmanuel Traunecker and Regina Decker for their excellent technical help.
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ABSTRACT
INTRODUCTION
