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Combined plasma and tissue proteomic study of atherogenic model mouse: approach to elucidate molecular determinants in atherosclerosis development Hiroko Hanzawa, Takeshi Sakamoto, Akihito Kaneko, Naomi Manri, Yan Zhao, Songji Zhao, Nagara Tamaki, and Yuji Kuge J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.5b00405 • Publication Date (Web): 01 Sep 2015 Downloaded from http://pubs.acs.org on September 4, 2015

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Journal of Proteome Research

Combined plasma and tissue proteomic study of atherogenic model mouse: approach to elucidate molecular determinants in atherosclerosis development AUTHOR NAMES Hiroko Hanzawa‡1,3, Takeshi Sakamoto*‡2,4, Akihito Kaneko†2, Naomi Manri2,3, Yan Zhao†4, Songji Zhao5, Nagara Tamaki4, and Yuji Kuge3,6

AUTHOR ADDRESS 1

Center for Exploratory Research, Research & Development Group, Hitachi, Ltd., 350-0395,

Hatoyama, Saitama, Japan 2

Center for Technology Innovation - Healthcare, Research & Development Group, Hitachi, Ltd.,

185-8601, Kokubunji, Japan 3

Central Institute of Isotope Science, Hokkaido University, 060-0814, Sapporo, Japan

ACS Paragon Plus Environment

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Department of Nuclear Medicine, Graduate School of Medicine, Hokkaido University, 060-

8638, Sapporo, Japan 5

Department of Tracer Kinetics & Bio-analysis, Graduate School of Medicine, Hokkaido

University, 060-8638, Sapporo, Japan 6

Department of Integrated Molecular Imaging, Graduate School of Medicine, Hokkaido

University, 060-8638, Sapporo, Japan

KEYWORDS proteomic study, apoE deficient mouse, atherosclerosis, inflammation, complement, coagulation, fibrinolysis, extracellular matrix, macrophage

ABSTRACT Atherogenic cardiovascular diseases are the major cause of mortality. Prevention and prediction of incidents is important; however, biomarkers that directly reflect the disease progression remain poorly investigated. To elucidate molecular determinants of atherogenesis, proteomic approaches are advantageous by using model animals for comparing changes occurring systematically (blood stream) and locally (lesion) in accordance with the disease progression stages. We conducted differential mass spectrometric analysis between apolipoprotein E deficient (apoED) and wild-type (wt) mice using the plasma and arterial tissue of both types of mice obtained at four pathognomonic time points of the disease. One hundred proteins in the


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plasma and 390 in the arterial tissues were continuously detected throughout the four time points; 29 were identified in common. Thirteen proteins in the plasma and 36 in the arterial tissues showed significant difference in abundance between the apoED and wt mice at certain time points. Importantly, we found that quantitative variation patterns regarding the pathognomonic time points did not always correspond between the plasma and arterial tissues, resulting in gaining insight into atherosclerotic plaque progression. These characteristic proteins were found to be components of inflammation, thrombus formation, and vascular remodeling, suggesting drastic and integrative alteration in accordance with atherosclerosis development.

TEXT Introduction Atherosclerosis is an inflammatory disease mainly accounting for acute coronary syndrome, myocardial infarction, and stroke, which are the most common causes of mortality worldwide1. Accurate evaluation of the developmental stages of atherosclerosis, vulnerability, and rupture risk of atherosclerotic plaques is important for risk prediction and guiding treatment decisions of patients with atherosclerosis-related diseases. Therefore, a variety of approaches to establish novel biomarkers have been conducted to date2. Due to the complexity of the disease, however, there is currently a lack of appropriate evaluation indexes that directly reflect atherosclerosis development. Furthermore, identification of the main contributors to each pathological event arising with atherosclerosis progression provides a fundamental understanding of pathogenesis. It is likely that they are strictly controlled by a set of genes and proteins expressed by a variety of cell types present in the vessel wall and/or provided from circulating blood3-5, but it is still not


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fully known how they are controlled. In other words, despite strenuous attempts by research groups worldwide and accumulation of pathophysiological knowledge, substantial gaps in understanding may still exist. Disease proteomics is a promising, proteomic profiling technology for filling in such gaps, which makes it possible to exhaustively identify disease-related alteration of protein expression at the whole-cell or tissue levels in biological specimens. Numerous studies have been conducted to develop diagnostic biomarkers, enabling early detection of disease and identification of new targets for therapeutics6. Recent efforts using mass spectrometry to determine changes in disease-related protein expression have involved quantitative proteomics by using differential stable isotopic labeling, such as cleavable isotope-coded affinity tags (cICAT)7 and isobaric tags for relative and absolute quantitation (iTRAQ)8. Either light or heavy cICATs are labeled at the free thiol groups of isolated intact proteins from two sample states (such as disease and healthy states) and the ratio of a protein amount between these states is estimated by comparing MS signal intensity of the corresponding proteolytic peptide fragments7. This enables highthroughput quantitative proteome profiling and applicability to be applied to a broad range of samples, including body fluids (e.g., serum or plasma) or extracts from tissues of interests. In fact, to determine the major proteinaceous factors contributing to the pathogenesis of atherosclerosis and related diseases, there have been several studies involving body fluid or tissue samples obtained from atherosclerotic model animals9 and symptomatic patients with atherosclerosis-related coronary artery diseases10-11. Since mutual interaction among factors in circulating blood and tissues contributes to atherosclerosis progression, a simultaneously integrated proteomic analysis of serum/plasma and arterial tissues12-13 is preferable, but has been rarely done.


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To analyze complex interaction among molecules in depth, the experimental conditions should be simplified by eliminating various influential factors. In this regard, atherogenic animal models, in which the genetic background and growth conditions are aligned, are the most useful. Using an apolipoprotein-E deficient (apoED) mouse model is particularly popular because the spontaneously hyperlipidemic and atherogenic model is known for developing all phases of atherosclerotic plaques in an age-dependent manner with similar features to human lesions14-16. Therefore, apoED mice have been used for nutritional intervention studies, pharmacological studies, drug development, and pathophysiological studies of atherosclerotic plaque development by investigative groups worldwide17. It is known that genetic and environmental factors, such as diet, are likely to affect lesion formation. On a high-fat diet, in fact, the rate and extent of lesion progression are accelerated in the arteries of apoED mice5, 18-19. As we previously demonstrated on a high-fat diet, early lesions were observed at 18 weeks of age, and advanced lesions were prominent in the proximal brachiocephalic arteries and the lesser curvature of the aortic arches at 25 and 35 weeks of age, but no atherosclerotic lesions were found in wild-type (wt) mice20. In addition, atheromatous lesions at 25 weeks were more abundant, but fibroatheromatous lesions at 25 weeks were less abundant than those at 35 weeks. In this study, we conducted combined differential proteomic studies of plasma and aortas as arterial tissue obtained from both wt and apoED mice at four pathognomonic time points (12, 18, 25 and 35 weeks), using cICATs as the stable isotopic labeling tags for relative quantitation.

Materials and Methods


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Animals: Animal care and animal experiments were conducted at Japan SLC, Inc., (Hamamatsu, Japan) under the approval of the company’s animal care committee. The apoED (C57BL/6.KOR/StmSlc-Apoeshl) and wt (C57BL/6) mice were fed a high-fat diet containing 0.15% by weight cholesterol (Nosan Corporation, Yokohama, Japan) from eight weeks of age. Each group of nine male or female mice at ages of 12, 18, 25 and 35 weeks was euthanized. Then plasma (EDTA-2K) was collected individually and pooled. Whole artery tissues from carotid to femoral obtained from six mice of each group were perfused with ice-cold phosphatebuffered saline (PBS) and frozen in liquid nitrogen for protein extractions, and the other three were rinsed carefully for fixation in 10% neutral buffered formalin (pH 7.2) for immunohistochemical analyses. Depletion of high-abundant proteins in pooled plasma: The pooled plasma of each group was applied to the multiple affinity removal column (MARC, Ms-3, 4.6 x 100 mm, Agilent Technologies, Inc., Santa Clara, USA), which removed a total of three high-abundant proteins (albumin, immunoglobulin G and transferrin) following the vendor’s instructions. Plasma proteins eluted from the MARC were further concentrated and used in subsequent procedures. Protein extraction from arterial tissues: Before extraction, the frozen aortas were partially thawed and weighed on ice. The aortic samples were then homogenized into powder form in liquid nitrogen. A protein extraction buffer (9.5 M Urea, 2 % CHAPS, 65 mM DTT, 40 mM Tris, pH 8.3) was added to the frozen powder, and supernatant was obtained with centrifugation (12,000 x rpm, for 20 minutes at 4°C). Ice-cold acetone was mixed with the supernatant to precipitate and concentrate extracted proteins with centrifugation (12,000 x rpm, for 20 minutes at 4°C). The pellets were dried and resolved in a re-solubilization buffer (0.2 % SDS, 50 mM Tris, pH 8.5). The concentration of protein extracted from the arteries was determined from a


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BCA protein assay reagent (Thermo Scientific Pierce, Rockford, USA) according to the manufacturer’s instructions. Protein labeling with isotope-coded affinity tags: Proteins prepared from plasma and arterial tissue as described above were labeled with isotope-coded affinity tags using the Cleavable ICAT® Reagent Kit (Applied Biosystems, Foster City, USA) according to the manufacturer’s instructions with the following minor modifications. One milligram of each extracted protein from age-matched apoED and wt mice was denatured by the addition of a denaturing buffer (6 M Urea, 0.05 % SDS, 50 mM Tris, 5 mM EDTA, 10 mM TBP, pH8.5) up to 800 µL for 30 minutes at 37°C. Next, 200 µL of “Cleavable ICAT Reagent Light” dissolved in acetone was mixed with the denatured proteins from the wt, and 200 µL of “Cleavable ICAT Reagent Heavy” was mixed with the denatured proteins from the apoED. Each portion was incubated for 2 hours at 37°C. Then, 800 µL of 10 mM Tris-HCl (pH 8.5) and 160 µL of TPCK-treated trypsin (Applied Biosystems) at a concentration of 125 µg/mL were added to each labeled protein described above. Subsequently, equal amounts of “Light” and “Heavy” labeled proteins were mixed and incubated for 16 hours at 37°C for complete trypsin digestion. Prefractionation of trypsin-digested peptides: Tryptic peptides were applied to the strong cation exchange (SCX) column (Poly Sulfoethyl A; 4.6 φ x 100 mm, PolyLC Inc., Columbia, USA) and the eluted solution was fractionated into 25 fractions. The separation conditions were as follows. Elution buffers A [10 mM KH2PO4 (pH 2.8), 25% ACN] and B [10 mM KH2PO4 (pH 2.8), 25% ACN, 0.5 M KCl] were used for the linear gradient; 0% at 10 minutes, 20% at 70 minutes, 50% at 85 minutes, 60% at 90 minutes, 60% at 95 minutes, and 100% at 100 minutes for buffer B. Each eluted fraction was concentrated into a quarter of the volume by using a


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vacuum concentrator (SpeedVac SPD1010, Thermo Fisher Scientific, Waltham, USA) and added to the desalting column (CAPCELL PAK C18 MG; 2.0 φ x 10 mm, Shiseido, Co. Ltd., Tokyo, Japan). Desalting buffers A (0.05% TFA in 2% ACN, 98% MS-grade water) and B (0.05% TFA in 80% ACN, 20% MS-grade water) were used, and the desalted fractions were dried for mass spectrometry analysis. Nanoflow LC-MS/MS: The SCX-separated fractions were analyzed using a liquid chromatography mass spectrometry (LC-MS) system (NanoFrontier LD, Hitachi HighTechnologies Corporation, Tokyo, Japan). The dried samples were dissolved in 4 to 10 µL of solvent A (0.1% formic acid in 2% ACN, 98% MS-grade water or equivalent) and 1 µL of the dissolved sample was used for LC-MS analysis connected to a separation column (MonoCap for Fast Flow C18, 50 µm φ x 150 mm, GL Sciences Inc., Torrance, USA). The separation conditions were as follows. Solvents A and B (0.1 % formic acid in 98% ACN, 2% MS-grade water or equivalent) were used with a linear gradient for 120 minutes (2 to 30% B) at a 200 nL/min flow rate. A trap column (Monolith Trap, 50 µm φ x 150 mm, Hitachi HighTechnologies Corporation) and a silica spray tip (Pico Tips™, Tubing OD/ID, 360/50 µm, Tip ID 10 µm, New Objective Inc., Woburn, USA) were coupled to the system. Spectra were collected from an ion trap mass spectrometer in a negative ion electro-spray mode. The MS and MS/MS spectra of each SCX pre-separated 25-peptide fraction were collected twice in each MS scan with the information-based-acquisition (IBA) technique21. The other analytical conditions were as follows: curtain gas flow, 0.7 L/min; spray potential, 1700 V; detector potential, 2200 V; isolation time, 5 ms; isolation width, 10 Da; and CID time, 10 ms.


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Data analysis: Original MS/MS peak lists (PLs) were generated using built-in PL generation software in the mass spectrometer and further protein/peptide identification and cICAT quantitation analyses were conducted using a custom developed software platform with the relational database NanoFProt (Oracle, 10.2.000). For each MS precursor ion, the charge state, monoisotopic mass, and area of isotope peaks were calculated and stored in the NanoFProt database. Total signal intensity of the precursor ion were calculated by accumulating the area of corresponding isotopic peaks in the mass chromatogram and stored in the NanoFProt. The majority of analyzed peptide ions contained cysteine residue(s) fully labeled with either cICAT. Therefore, each MS precursor ion was classified into one of three categories: 1) fully lightlabeled peptides, 2) fully heavy-labeled peptides, or 3) other peptides, by searching the counterpart peptide (ICAT pair) in the corresponding mass spectra. The quantitative data for ICAT pair candidates, such as monoisotopic mass and total signal intensity, were also stored in the NanoFProt database. Three kinds of MS/MS PLs were created in Mascot generic format: an original PL containing all PLs generated, an ICAT-Light PL containing the PLs for 1) fully lightlabeled peptides and 3) other peptides, and an ICAT-Heavy PL containing the PLs for 2) fully heavy-labeled peptides and 3) other peptides. These PLs were then searched using Mascot (Ver. 2.1, Matrix Science Ltd.) against the SwissProt database (2011_10v2). One trypsin-missed cleavage was allowed, and the peptide mass tolerances for both MS/MS and MS were set to ± 0.2 Da. Variable modification of ICAT-C at cysteine (+227.13 Da) and ICAT-C:13C(9) at cysteine (+236.16 Da) were applied to the original PL. Fixed modification of ICAT-C at cysteine and ICAT-C:13C(9) at cysteine were applied for the ICAT-Light PL and ICAT-Heavy PL, respectively. Other options used for Mascot search were variable modifications of oxidation at methionine/ tryptophane/ histidine (+ 15.99 Da) and carbamylation of lysine or the N-terminus


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(+ 43.01 Da). The Mascot results were stored in the NanoFProt database and the results of peptide identification and ICAT quantitation were linked to each other. Basically, the proteins containing peptides with ion scores having identical or extensive homology (p 1.5) did not have such age-related tendency (Figure 1A). In the arterial tissues, the percentage of both ‘up-regulated’ and ‘down-regulated’ proteins increased on and after 25 weeks of age (Figure 1B). The total number of ‘overlapped’ proteins, which were identified both in the plasma and arterial tissues, was 51 to 61 throughout all four time points (Figure 2). The percentage of the proteins that showed no obvious change in abundance between the apoED and wt mice in both plasma and arterial tissues was over 55% at the ages of 12 to 18 weeks and decreased to 19% afterward; hence, the percentage of ‘not-correlated’ proteins, which showed a significant difference in the expression ratio (apoED/wt) between plasma and arterial tissues, was over 65% on and after 25 weeks of age (Figure 2). Proteins identified in plasma: Thirteen of the 100 identified proteins throughout all four time points were determined as up- or down-regulated, which showed more than a 1.5 times increase or decrease in abundance in the apoED plasma compared with that in the wt plasma, or showed 1.5 times or more of the maximum/minimum expression ratios (apoED/wt) throughout the four characteristic time points (Table 1). In Table 1, the variation patterns of the expression ratios were classified into six types: monotonic increase (pattern 1) or decrease (pattern 2) of the expression ratios throughout the four characteristic time points, showing the maximum (pattern 3) or minimum (pattern 4) of the expression ratios at 18 weeks of age and showing the maximum (pattern 5) or minimum (pattern 6) of the expression ratios at 25 weeks of age. CD5L (male,


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pattern 3; female, pattern 5), properdin (pattern 2), and thrombospondin-4 (TSP4) (female, pattern 6) showed characteristic changes in the expression ratios in the plasma throughout the four time points, whereas they showed an increasing trend in the expression ratios in the apoED arterial tissues, more than double that in the wt arterial tissues (Tables 1-2). The changing trends in the expression ratios of the von Willebrand factor (vWF) were also not correlated between the plasma (pattern 3) and arterial tissues (no significant change) as shown in Table 1 and Supplemental Table 3. The changes in the expression ratios of fetuin-B, properdin, and vWF showed similar trends between male and female mice (Supplemental Figure 1), and we also found proteins such as CD5L showing different changing trends between male and female mice (Table 1 and Supplemental Figure 2). It is known that CD5L exists in complex form with immunoglobulin M in plasma22. The changes in the expression ratios of CD5L were similar to those in the Ig mu chain C region (IGHM), though not equal to the trends between male and female mice (Supplemental Figure 2A and B). Complement component C8 is composed from three different subunits: alpha, beta, and gamma23. The variation in the expression ratios among the three subunits, complement component C8 alpha chain (CO8A), beta chain (CO8B), and gamma chain (CO8G), were essentially consistent, though overall changing trends were different between male (pattern 1) and female (pattern 5) (Table 1 and Supplemental Figures 2C and D). On the other hand, the variation patterns of the expression ratios between anti-coagulant proteins, vitamin K-dependent protein C (PROC) and vitamin K-dependent protein Z (PROZ), did not correspond with each other (Supplemental Figure 3). Proteins identified in arterial tissues: We found that thirty-three proteins showed an increasing tendency and three showed a decreasing tendency in the expression ratios of the 390 identified proteins in the arterial tissues throughout all four time points (Table 2). Unlike in the plasma


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analyses, proteins showing other changing patterns in the expression ratios were rarely observed in the arterial tissues (data not shown). At the age of 12 weeks, the abundance of two proteins, the Ig mu chain C region (IGHM) and properdin in the apoED arterial tissues, reached more than twice that of the wt arterial tissues (Table 2). At the age of 18 weeks, the abundance of 12 proteins including CD5L, plastin-2 (PLSL), thrombospondin-1 (TSP1), and vascular cell adhesion protein 1 (VCAM1) in the apoED arterial tissues reached more than double that of the wt arterial tissues and continued to increase (Table 2). At the ages of 25 to 35 weeks, the abundance of 12 proteins including collagen alpha-1(XIV) chain (COEA1), cartilage oligomeric matrix protein (COMP), fatty acid-binding protein, epidermal (FABP5), and TSP4, and seven proteins, including myosin-9 (MYH9) and vitamin K-dependent protein S (PROS) identified in the apoED arterial tissues, were up to more than double that of the wt arterial tissues (Table 2). By the age of 25 weeks, the abundance of cytosol aminopeptidase (AMPL) in the apoED arterial tissues reached one half of that in the wt arterial tissues (Table 2). At the age of 35 weeks, the abundance of two proteins, including calponin 1 (CNN1), in the apoED arterial tissues reached one half of that in the wt arterial tissues (Table 2). Most of these proteins in Table 2, showing significant and monotonic increase or decrease in the expression ratios in the arterial tissues, exhibited no difference or only subtle changes in expression ratios, or they were not identified at any of the four time points, in the plasma (Supplemental Table 4). Several exceptions, immunoglobulin-related proteins; IGHM, Immunoglobulin J chain (IGJ), and Ig kappa chain C region (IGKC), showed high levels of expressions in the plasma as well as arterial tissues obtained from the apoED mice compared with the wt mice (Table 1, Supplemental Table 4). In addition, the abundance of CD5L markedly elevated in the plasma as well as arterial tissues obtained from the apoED mice compared with that from the wt mice (Tables 1-2). In contrast,


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the changing patterns in the expression ratios of TSP1 showed significant and monotonic increases in the arterial tissues throughout the four time points; however, the expression ratio of TSP1 in the plasma was at a minimum at 25 weeks (Table 2, Supplemental Table 4). Proteins identified as down-regulated in the arterial tissues, although there were a few exceptions, were not detected in the plasma (Table 2, Supplemental Table 4). Changing patterns in expression ratios among family proteins: The changing patterns in the expression ratios of identified proteins and their families throughout the four time points are shown in Supplemental Table 5. Calponin-1 (CNN1) and families (calponin-2, CNN2 and calponin-3, CNN3), which are thin filament-associated proteins implicated in the regulation and modulation of smooth muscle cells, showed a decreasing tendency in the expression ratios in the arterial tissues. Two identified fatty-acid binding proteins, (adipocyte-type, FABP4 and epidermal-type, FABP5) showed different tendencies. While FABP5 showed a monotonic increase in expression ratios in the arterial tissues, FABP4 did not show a significant difference in expression levels between the apoED and wt arterial tissues. Among the plastin family of actin-binding proteins, only plastin-2 (L-plastin, PLSL) showed an increase in the expression ratios in the apoED arterial tissues. Members of the thrombospondin family, which are adhesive glycoproteins presumed to mediate cell-to-cell interactions, showed a basically increasing tendency in the arterial tissues, but not in the plasma. Validation of increase in expression ratios of CD5L both in plasma and arterial tissues of apoED mice: We conducted several experiments to validate the results including the expression ratios determined from comparative mass spectrometry. For a typical example of validation experiments we selected CD5L, which is a member of the scavenger receptor cysteine-rich super family and plays a role in the regulation of the immune system through the apoptosis inhibition


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of macrophages24. We conducted sandwich ELISA to determine concentrations of CD5L in pooled plasma (Table 3). The concentration of CD5L in pooled plasma of the apoED mice was approximately a 2-fold increase compared to that in the wt mice at all four time points. In addition, the expression ratios determined in the male mice decreased after 18 weeks of age, showing a similar tendency to that determined from differential mass spectrometry. We then conducted immunohistochemical analyses to detect CD5L using a specific antibody in atherosclerotic lesions in arterial tissues isolated from male mice. Figure 3 shows typical images of Mac-2 and CD5L immunohistochemical staining of the aortic arches obtained from male wt or apoED mice at the four time points. Unlike in the aortic arches of the wt mice, in the apoED mice atherosclerotic lesions developed depending on age. The CD5L was detected in the early to advanced lesions and CD5L expression in the lesions reached a maximal peak at 18 weeks and decreased subsequently (Supplemental Figure 4), showing a similar tendency to that obtained with the expression ratios in plasma determined from mass spectrometry (Table 1).

Discussion Atherosclerosis is an inflammatory disease, and atherosclerotic lesions are progressively formed in blood vessels through complicated molecular processes. To identify proteins showing changes in expression levels associated with atherosclerotic plaque progression, we conducted mass spectrometry-based proteomic analyses using both plasma and arterial tissues obtained from high-fat diet-administrated apoED and wt mice at four pathognomonic time points. Several similar proteomic studies using apoE knockout mice as an atherosclerotic model have been reported25-27. However, our combined plasma and tissue proteomic study involved a different approach from previous studies. This provided us a comprehensive understanding of the changes


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resulting from circulating blood stream and occurring in local lesions in association with atherosclerotic plaque progression. Among various quantitative proteomic methods, we selected cICAT because the relative quantitative analysis using two types of tags, ICAT-light and ICATheavy, matched our objectives to obtain comprehensive protein level difference between apoED and wt mice in a variety of experimental conditions such as age (12, 18, 25, and 35 weeks), gender (male and female), and specimen (plasma and artery tissue). In the first stage of this research we also tried to use the iTRAQ reagent but could not achieve satisfactory reproducibility due to possible incompatibility with the instrument. By using the cICAT reagent, we successfully achieved precise and reproducible results capable of comparing a 1.2 times difference in protein levels. We identified 100 and 390 proteins and determined their age-related (disease stage-related) expression profiling in the plasma and arterial tissues, respectively. It is interesting to note that the changing patterns in the expression ratios of identified proteins between the apoED and wt mice throughout the four time points were not always consistent in the plasma and arterial tissues. These results were not beyond our expectations. If we had used only plasma or arterial tissue, it would have been difficult to determine a small but significant change in the abundance of proteins in circulatory blood flow that may reflect atherosclerotic lesion progression. We also detected a small subset of proteins, including those previously reported as markers of plaque instability, at the various stages of atherosclerotic lesion progression28. Furthermore, our combined plasma and tissue proteomic study revealed that components of the complement pathway, which plays a central role in inflammatory response, the coagulation-fibrinolysis pathway, associated with thrombus formation, and the extracellular matrix (ECM), presumed to contribute directly to neovasculature and remodeling, showed changes in expression level associated with atherosclerosis development29.


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The percentage of up- or down-regulated proteins tended to increase after 25 weeks of age for both plasma and arterial tissues (Figure 1). More importantly, the number of proteins of which changing patterns in expression ratios were not correlated between the plasma and arterial tissues significantly increased after 25 weeks of age (Figure 2). After 25 weeks, the arterial tissue weight of the apoED mice significantly increased (Supplemental Table 2) and advanced atherosclerotic lesions formed at a high rate, as previously reported19, suggesting that a dramatic alteration at the molecular level in association with atherosclerosis plaque formation occurred at 25 weeks or later. Therefore, our results of quantitative analysis of identified proteins shown in Figures 1-2 may be consistent with the qualitative changes in lesion formation. Also, some of the proteins we detected as up- or down- regulated in the apoED arterial tissues have previously been reported as atherosclerosis- or vascular dysfunction-related proteins, indicating the robustness of the study designs and methods we used. Contrary to the monotonic trends of changing patterns in expression ratios in arterial tissues, one of the characteristic features of the changing patterns in the plasma is the presence of proteins showing the maximum or minimum expression ratios (apoED/wt) at specific time points such as 18 and 25 weeks of age (Table 1, patterns 3-6). This kind of characteristic time course might be applicable to an effective diagnosis of atherosclerosis development such as atherosclerotic plaque formation and progression. Components of complement pathway, coagulation-fibrinolysis cascades, and extracellular matrix (ECM) proteins: Table 4 shows the molecular functional classification of identified proteins together with the data from the Universal Protein Resource (UniProt). Many of the proteins listed in the table were classified as components or related to the complement pathway, the coagulation-fibrinolysis pathway, and the ECM. The complement system plays a central role


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in innate immunity and is closely related to a wide variety of inflammatory responses. In atherosclerotic lesion formation, the complement system is becoming increasingly implicated. In fact, complement activation can precede the development of lesions in experimental atheroma30. It was also reported that the deficiency in the complement components caused a reduction in lesion size; hence, the deficiency of regulatory/inhibitory components led to an increase in the size of atherosclerotic lesions determined using atherosclerosis rodent models31. On the other hand, Yasojima et al. demonstrated that complement components, but not inhibitory components, were up-regulated in human atherosclerotic plaque obtained from postmortem cases32. In contrast to these results, we found that the inhibitory complement components, C4b-binding protein (C4BP), complement factor H (CFAH), properdin, clusterin, and vitronectin, showed a monotonic increase in the apoED arterial tissues compared with that in the wt arterial tissues (Table 2). In addition, immunoglobulin M (IgM), which is a natural antibody, has been identified as being important for protection against atherosclerosis. Lewis et al. observed high serum IgM (sIgM) content and its deposition in atherosclerotic lesions in LDL-receptor (LDLR)-deficient mice. On the other hand, no sIgM deposition, but accelerated atherosclerotic lesion formation and increased lesion complexity, was observed in sIgM-LDLR double knockout mice33. We found that the levels of IGHM content in both plasma and arterial tissues of the apoED mice monotonically increased in accordance with the atherosclerotic plaque progression throughout the four time points from 12 to 35 weeks (Tables 1-2). These results suggest that innate and adaptive immune responses related to plasma and arterial tissue IgM might be a first clue to fight against progression of atherosclerotic lesions. As mentioned above, the role of complement components in atherosclerosis is still controversial. However, we identified complement components, inhibitory components, and immunoglobulins as proteins showing characteristic


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changing patterns of abundance with atherosclerosis progression, which is basically consistent with past reports. Acute thrombus formation on ruptured atherosclerotic plaques plays a key role during the onset of acute coronary syndrome, and the blood coagulation-fibrinolysis pathway is the molecular basis of obstructive clotting and thrombosis formation. It is known that the interaction between circulating blood flow and prothrombotic substances, such as the tissue factors existing in atherosclerotic lesions, leads to lesion disruption34. In this study, we identified a group of components related to the coagulation-fibrinolysis pathway such as coagulation components, anticoagulants, and thrombin activable fibrinolysis inhibitors, which showed significant changes in the expression ratios between the apoED and wt plasma and/or arterial tissues. In Supplemental Figure 5, we showed immunohistochemical staining results of the coagulationfibrinolysis components accumulated in arterial tissues (fibrinogen, prothrombin, alpha-2macroglobulin, vitamin K-dependent protein S, and carboxypeptidase B2), supporting the proteomic results shown in Tables 2 and 4. There are two major types of thrombus formation known as fibrin-rich or platelet-rich35,36. The above five proteins are associated with fibrin-rich thrombus formation. On the other hand, vWF was the only protein detected in arterial tissues among proteins playing main roles in platelet-rich thrombus formation, and the amount of vWF did not significantly change in arterial tissues throughout the four time points (Table 4). These results suggest that the fibrin-rich thrombus formation dominates in advanced atherosclerotic lesions in mouse models. It is also known that the ECM plays an important role in atherosclerotic plaque formation and progression. The vascular ECM is essential for the structural basis of the arterial vessel wall and functions as a frontier between signaling receptors on various vascular cells and secreted


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molecules coming from circulation. However, at present, it is not clear which molecules related to the ECM play pivotal roles at distinct stages of pathogenesis. In this study, we detected a considerable number of ECM proteins in arterial tissues that were up-regulated during plaque progression processes (Table 4, Supplemental Figure 6). Didangelos et al.37 conducted proteomic studies specifically targeting the vascular ECM and its associated proteins using human aortic specimens obtained upon aortotomy performed during routine aortic valve replacement. Among the 103 ECMs and their associated proteins they identified, we also identified ten ECM proteins (properdin, matrix metalloproteinase 2, tenascin, thrombospondin-1, clusterin, collagen alpha1(XIV), cartilage oligomeric matrix protein, fibronectin, fibromodulin and vitronectin) in mice arterial tissues identified as monotonically up-regulated along with atherosclerotic plaque progression (Table 2). Clutterbuck et al.38 detected five proteins (clusterin, cartilage oligomeric matrix protein, fibronectin, fibromodulin, and thrombospondin-1), as the major ECM proteins secreted in culture medium supernatants of articular cartilage determined in a targeted proteomic study. In our study, we found that the expression ratios (apoED/wt) of these five proteins showed a marked increase from 25 weeks. Further investigations are needed to find applicable explanations for the coincidence of expressed ECM proteins between atherosclerotic plaque and articular cartilage. It was also reported that fibromodulin activated the classical pathway of complement component(s) by physically binding to the globular heads of C1q, which leads to activation of complement component(s), suggesting that ECM proteins are directly implicated in the inflammatory process of atherosclerosis progression39. Other proteins: The CD5L, also known as an apoptosis inhibitor expressed by macrophages, AIM or SP alpha, is a soluble member of the scavenger receptor cysteine-rich domain superfamily of proteins. Arai et al.40 demonstrated that atherosclerotic lesions of CD5L and


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LDLR double knockout dramatically decreased. The inhibitory effect of CD5L for macrophage apoptosis in atherosclerotic lesions promotes atherosclerotic plaque development, suggesting that it is a survival strategy of macrophages in a lesion. We obtained the proteomics result showing a monotonic increase of CD5L in arterial tissues in accordance with atherosclerosis progression (Table 2). On the other hand, we also found that CD5L accumulates not only in atherosclerotic lesions with macrophage infiltration in a coordinated manner but also in smooth muscle areas adjacent to advanced lesions by immunohistochemical detection (Figure 3). Although the increasing tendency of the CD5L expression in arterial tissues was shown in the proteomics and the immunohistochemical results, the significance of the difference in the changing patterns between plasma (Table 1) and arterial tissues (Table 2) and the mechanism of CD5L accumulation in smooth muscle areas in arterial media remain to be solved in future. In addition, Tissot et al.22 indicated that CD5L is associated with sIgM purified from serum/plasma. In the present study, the expression ratios (apoED/wt) of IgM and CD5L were high throughout the atherosclerotic plaque progression stages determined in artery and plasma, and their increasing tendencies modestly paralleled each other (Tables 1-2, Supplemental Figure 2). Quantitative antagonistic activity between IgM and CD5L molecules might accommodate progression of atherosclerotic lesions. As shown in Supplemental Figure 2, we found proteins such as CD5L showing different change tendencies in the expression ratio in plasma between male and female mice. The presence of sex difference on atherosclerosis progression in the mouse model is still unclear, so further studies on immunohistochemical staining comparison of these proteins between male and female arterial tissues are needed for the validation. Limitations: In the present study, we failed to detect several known atherosclerotic plaquerelated proteins such as cytokines, monocyte chemoattractant protein-1 (MCP-1), intercellular


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adhesion molecule-1(ICAM-1), selectins, myeloperoxidase, and matrix metalloproteinase-926. Furthermore, the total number of identified proteins was less than that previously reported. We assumed that there were several technical reasons for not detecting these proteins. For example, the practical detection limit in our mass spectrometry system was assumed to be 1-10 ng/mL, which is much higher than normal concentrations of cytokines, which are estimated to be 1-10 pg/mL41. For protein extraction, we used only a single extraction buffer, which included urea, a detergent, and a reducing agent. Several trials using various buffers with different compositions might be required to increase protein extraction efficiency from fiber-rich arterial tissues. In addition, we used cICAT labeling reagents, which covalently bind to cysteine residue of targeted proteins through thiol linkage, for comparative quantitative mass spectrometry. Therefore, it becomes difficult to detect proteins with trypsin-digested peptides with few or no cysteine residues. Clinical perspectives: The proteins we identified in this study, which showed quantitative alteration in plasma and/or arterial tissues associated with atherosclerosis progression, may be potential biomarkers for diagnosis, risk assessment to prevent incidence, and therapy evaluation of atherosclerosis-related diseases. In recent years, imaging modalities such as positron emission tomography, single photon emission computed tomography, and magnetic resonance imaging have been applied to detect atherosclerotic plaques and their vulnerability2. Up-regulated proteins identified in apoED arterial tissues might be useful targets of multiple tracers for molecular imaging modalities to detect high-risk/vulnerable plaques and/or to accurately evaluate the progression stage of atherosclerosis-related diseases. Further studies with in vitro diagnostic and in vivo molecular imaging techniques are required to validate our findings and their clinical usefulness.


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Conclusion Most previous proteomic attempts to identify atherosclerosis-related proteins were done using only one series of samples, for example, serum/plasma or tissues. To identify proteins representing the pathophysiological changes in association with atherosclerotic plaque progression, we conducted combined plasma and arterial tissue proteomic analyses with samples obtained from wt and apoED mice at four pathognomonic time points. We found that the expression profile of proteins, many of which did not correspond between the plasma and arterial tissues, suggests that quantitative variation of a protein in the plasma was not predominantly caused by the progression of atherosclerosis in many cases. Our combined proteomic analyses demonstrated that the expression of components related to inflammation, coagulation-fibrinolysis, neovasculature, and remodeling, drastically altered in an integrative manner with atherosclerotic plaque progression, and we believe that they must be potent navigators for predicting the disease.

FIGURES Figure 1. Percentage of up- or down- regulated proteins detected in plasma (A) and arterial tissues (B) at each of four time points. We defined expression ratios (apoED/wt) of up- or downregulated proteins as more than 1.5 or less than 1/1.5. Numerical values in yellow rectangles indicate number of identified proteins at each of four time points.


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Figure 2. Correlation analyses of up- or down-regulated proteins between plasma and arterial tissues at each of four time points. Numerical values in yellow rectangles indicate number of proteins identified in both plasma and arterial tissues at each of four time points. Figure 3. Immunohistochemical detection of CD5L in wt and apoED arterial tissues. Paraffinembedded sections of aortic arches were stained using hematoxylin & eosin (H&E) for morphology, anti-machrophage (Mac2), and anti CD5L antibodies. Typical images are shown in figures. Bars are 100 µm.


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TABLES Table 1. Expression patterns of up- or down- regulated proteins detected in plasma Protein Description Complement C1q subcomponent subunit B

Swiss Prot Entry Name C1QB_MOUSE

CD5 antigen-like

CD5L_MOUSE

Complement factor D

CFAD_MOUSE

Complement component C8 alpha chain

CO8A_MOUSE

Complement component C8 beta chain

CO8B_MOUSE

Complement component C8 gamma chain

CO8G_MOUSE

Fetuin-B

FETUB_MOUSE

Ig mu chain C region

IGHM_MOUSE

Vitamin K-dependent protein C

PROC_MOUSE

Properdin

PROP_MOUSE

Vitamin K-dependent protein Z

PROZ_MOUSE

Thrombospondin-4

TSP4_MOUSE

von Willebrand factor

VWF_MOUSE

Sex Male Female Male Female Male Female Male Female Male Female Male Female Male Female Male Female Male Female Male Female Male Female Male Female Male Female

Expression Ratio in Plasma (apoED/wt) 12 weeks 18 weeks 25 weeks 35 weeks Max/Min 0.84‡ ND 4.07 3.60 0.95 1.00 1.13 1.23 1.13 1.23 1.14 1.31 0.98 0.91 4.85 1.81 1.11 1.02 2.15 2.07 0.91 1.07 0.98 1.22 0.95 0.94

1.10‡ 1.61‡ 6.73 3.59 ND 0.70 1.22 1.57 1.24 1.52 1.19 1.78 0.96 0.75 6.63 2.71 1.05 1.25 1.76 1.81 0.92 0.87 0.93 1.11 1.34 1.40

1.41‡ 1.67‡ 5.03 4.80 ND 1.19 1.45 2.65‡ 1.49 2.36 1.54‡ 2.14 1.07 1.00 4.47 4.67 0.99 0.90 1.56 1.50 1.53 0.86 1.01 0.88 0.97 0.75‡

1.22 1.52‡ 3.29 4.76 ND 1.24 1.83 2.11 1.89 2.08 1.78 1.26 1.29 1.19 3.71 4.98 0.55 0.83 1.45 1.42 0.56 0.88 1.10 1.36 1.02 1.28

1.68 ND 2.05 1.34 ND 1.77 1.63 2.15 1.67 1.92 1.56 1.70 1.35 1.59 1.79 2.75 2.01 1.51 1.48 1.46 2.73 1.21 1.19 1.54 1.40 1.87

Expression Pattern† 5‡ ND 3 5 ND 4 1 5‡ 1 5 1‡ 5 1 4 3 1 2 3 2 2 5 6 3 3‡

†Expression patterns were defined as follows: 1, monotonically increase; 2, monotonically decrease; 3, maximum at 18 weeks of age; 4, minimum at 18 weeks of age; 5, maximum at 25 weeks of age; 6, minimum at 25 weeks of age. Bold type indicates significant difference in expression ratio ( > 1.5 or < 1/1.5). ND, no unique peptide pairs derived from protein were detected. ‡ Ratio was determined once between two isolated trials.


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Table 2. Expression ratios determined by arterial analyses of up- or down- regulated proteins Protein Description

Swiss Prot Entry Name

Expression Ratio in Arterial Tissues (apoED/wt) 12 weeks 18 weeks 25 weeks 35 weeks

Up-regulated proteins Ig mu chain C region Properdin C4b-binding protein CD5 antigen-like Fibrinogen beta chain Fibrinogen gamma chain Immunoglobulin J chain Ig kappa chain C region 72 kDa type IV collagenase Mannan-binding lectin serine protease 2 Plastin-2 Tenascin Thrombospondin-1 Vascular cell adhesion protein 1 Alpha-2-macroglobulin ADAMTS-like protein 4 Cathepsin B Carboxypeptidase B2 Complement factor H Collagen alpha-1(XIV) chain Cartilage oligomeric matrix protein Fatty acid-binding protein, epidermal Fibronectin Fibromodulin Prothrombin Thrombospondin-4 Clusterin Complement factor B Inter-alpha-trypsin inhibitor heavy chain H1 Myosin-9 Vitamin K-dependent protein S

IGHM_MOUSE PROP_MOUSE C4BPA_MOUSE CD5L_MOUSE FIBB_MOUSE FIBG_MOUSE IGJ_MOUSE IGKC_MOUSE MMP2_MOUSE MASP2_MOUSE PLSL_MOUSE TENA_MOUSE TSP1_MOUSE VCAM1_MOUSE A2M_MOUSE ATL4_MOUSE CATB_MOUSE CBPB2_MOUSE CFAH_MOUSE COEA1_MOUSE COMP_MOUSE FABP5_MOUSE FINC_MOUSE FMOD_MOUSE THRB_MOUSE TSP4_MOUSE CLUS_MOUSE CFAB_MOUSE ITIH1_MOUSE MYH9_MOUSE PROS_MOUSE

2.51 2.06† ND ND 0.68† 1.12 ND 1.57† 1.03† ND ND ND 1.01† 1.98† 1.03† 1.43† 1.13† 0.76† 1.23 1.09† ND 1.28 1.47 ND 1.02† ND ND 1.06† ND 0.88 ND

3.72 4.12 3.43† 4.51† 2.38 2.50 2.52 2.80 (2.98)‡ 2.00† 3.67 3.75 2.68 2.97† 1.70 1.28 1.68 1.41† 1.80 1.47 0.85† 1.84 1.63 ND 1.57† (1.17) ‡ 1.50† 1.57 1.54† 1.44 1.42

4.86 9.76 6.88 7.57 6.42 8.09 3.86 3.25 ND 2.85 7.45 5.06 4.53 3.33 3.15 2.63 2.36 2.10† 2.71 2.50 4.92 3.55 4.36 4.67† 2.85 3.91 1.76† 1.61† 1.86† 1.57 1.94

6.50 6.62 6.48 8.89 6.83 6.75 6.18 5.91 12.2 4.53 7.83 7.44 11.4 4.95 3.63 2.85 2.85 3.16 4.96 3.06 13.7 3.47 4.47 18.7† 5.90† 6.77† 3.74 2.48 3.35 2.37 2.38


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Tropomyosin alpha-4 chain Vitronectin Down-regulated proteins Cytosol aminopeptidase Calponin-1 Guanylate cyclase soluble subunit alpha-3

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TPM4_MOUSE VTNC_MOUSE

ND 1.79†

ND 1.70

1.41† 1.79

2.78† 5.03

AMPL_MOUSE CNN1_MOUSE GCYA3_MOUSE

ND 1.01 ND

0.73 1.02 0.83

0.46† 0.58 0.68

0.63† 0.48 0.45

Bold type indicates significant difference in expression ratios ( >2 or < 1/2). †Ratio was determined once between two isolated trials. ND, no unique peptide pairs derived from protein were detected. ‡ Ratio was determined from a single peptide result.


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Table 3. Plasma CD5L level in wt and apoED determined using sandwich ELISA Sex Male

Female

Age wt apoED Ratio (apoED/wt) wt apoED Ratio (apoED/wt)

12 weeks 2.97 ± 0.20 6.36 ± 0.59 2.14 1.27 ± 0.23 3.55 ± 1.54 2.79

mCD5L (µg/mL) 18 weeks 25 weeks 3.57 ± 0.25 10.48 ± 1.91 2.93 2.24 ± 0.28 5.29 ± 0.96 2.36

8.68 ± 0.58 15.32 ± 2.48 1.77 3.18 ± 0.57 8.57 ± 2.88 2.7

35 weeks 9.40 ± 0.86 16.23 ± 2.15 1.73 3.96 ± 0.91 8.71 ± 2.99 2.2


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Table 4. Overview of identified proteins showing significant change in expression ratios between apoED and wt mice from plasma and/or arterial analyses Molecular functions Complement components and Immunoglobulins

Protein description Complement Component

Complement Regulator

Immunoglobulin

Coagulation and Fibrinolysis

Coagulation Component

Anticoagulant

Extracellular Matrix Components

Thrombin Activable Fibrinolysis Inhibitor ECM Protein

Mannan-binding lectin serine protease 2 Complement factor B Complement factor D Complement component C8 alpha chain Complement component C8 beta chain Complement component C8 gamma chain Complement C1q subcomponent subunit B C4b-binding protein Complement factor H Properdin Clusterin Vitronectin Ig kappa chain C region Ig mu chain C region Immunoglobulin J chain Fibrinogen beta chain Fibrinogen gamma chain Prothrombin von Willebrand factor Alpha-2-macroglobulin Vitamin K-dependent protein C Vitamin K-dependent protein S Vitamin K-dependent protein Z Carboxypeptidase B2 ADAMTS-like protein 4 Collagen alpha-1(XIV) chain Cartilage oligomeric matrix protein Fibronectin

Swiss Prot Entry Name MASP2_MOUSE

Plasma ↓

Arterial Tissue ↑

CFAB_MOUSE CFAD_MOUSE CO8A_MOUSE

↓ ↓↑ ↑

↑ ND ND

CO8B_MOUSE

↑

ND

CO8G_MOUSE

↑

ND

C1QB_MOUSE

↑↓

↑

C4BPA_MOUSE CFAH_MOUSE PROP_MOUSE CLUS_MOUSE VTNC_MOUSE IGKC_MOUSE IGHM_MOUSE IGJ_MOUSE FIBB_MOUSE FIBG_MOUSE THRB_MOUSE VWF_MOUSE A2M_MOUSE PROC_MOUSE PROS_MOUSE PROZ_MOUSE CBPB2_MOUSE

↑ → ↓ → ↑ ↑ ↑ ↑ → → → ↑↓ → ↓ → ↑↓ →

↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ → ↑ ND ↑ ND ↑

ATL4_MOUSE COEA1_MOUSE COMP_MOUSE FINC_MOUSE

ND ND → →

↑ ↑ ↑ ↑


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Others

Cell Adhesion Actin Binding

Lipid Binding Protease and Inhibitor

Apoptosis Blood Pressure Regulator

Fibromodulin 72 kDa type IV collagenase Tenascin Thrombospondin-4 Thrombospondin-1 Vascular cell adhesion protein 1 Calponin-1 Myosin-9 Plastin-2 Tropomyosin alpha-4 chain Fatty acid-binding protein, epidermal Cytosol aminopeptidase Cathepsin B Fetuin-B Inter-alpha-trypsin inhibitor heavy chain H1 CD5 antigen-like Guanylate cyclase soluble subunit alpha-3

FMOD_MOUSE MMP2_MOUSE TENA_MOUSE TSP4_MOUSE TSP1_MOUSE VCAM1_MOUSE CNN1_MOUSE MYH9_MOUSE PLSL_MOUSE TPM4_MOUSE FABP5_MOUSE

ND ND ND ↓↑ ↓↑ ND ND ND ND ND ND

↑ ↑ ↑ ↑ ↑ ↑ ↓ ↑ ↑ ↑ ↑

AMPL_MOUSE CATB_MOUSE FETUB_MOUSE ITIH1_MOUSE

ND ↑↓ ↓↑ →

↓ ↑ ND ↑

CD5L_MOUSE GCYA3_MOUSE

↑ ND

↑ ↓

↑, up-regulated in apoED mice; ↓, down-regulated in apoED mice; ↑↓, up-regulated followed by down-regulated in apoED mice during four time points; ↓↑, down-regulated followed by up-regulated in apoED mice during four time points; →, no significant change between apoED and wt mice; ND, not detected.


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SUPPORTING INFORMATION This material is available free of charge via http://pubs.acs.org/. Supplemental Figure 1. Changing patterns in expression ratio of (A) Fetuin-B (FETUB), (B) properdin (PROP), and (C) von Willebrand factor (vWF) determined through plasma analysis. Supplemental Figure 2. Changing patterns in expression ratio of (A) CD5 antigen-like (CD5L) and immunoglobulin M (IGHM) in male mouse, (B) CD5L and IGHM in female mouse, (C) complement component C8 alpha (CO8A), beta (CO8B), and gamma (CO8G) in male mouse, and (D) CO8A, CO8B, and CO8G in female mouse determined through plasma analysis. Supplemental Figure 3. Changing patterns in expression ratio of (A) vitamin K-dependent protein C (PROC) and vitamin K-dependent protein Z (PROZ) in male mouse, and (B) PROC and PROZ in female mouse determined through plasma analysis. Supplemental Figure 4. Immunohistochemical analyses of Mac-2 and CD5L positive areas. (A) shows lesion size determined by hematoxiline-eoxin staining of arterial tissue and (B) shows ratio of Mac-2 or CD5L positively staining areas. Supplemental Figure 5. Immunohistochemical detection of coagulation-fibrinolysis-related proteins: fibrinogen, prothrombin, alpha-2-macroglobulin, vitamin K-dependent protein S, and carboxypeptidase B2. Supplemental Figure 6. Immunohistochemical detection of MMP2, fibronectin, fibromodulin, and tenascin. Supplemental Table 1. Triglyceride, total cholesterol, HDL, and LDL levels of pooled plasma collected from apoED and wt mice. Supplemental Table 2. Body and arterial tissue weight change in apoED and wt mice according to age. Supplemental Table 3. Changes in expression ratios of proteins in arterial tissues identified as up- and/or down-regulated in apoED mouse plasma. Supplemental Table 4. Changes in expression ratios of proteins in plasma identified as up- or down-regulated in apoED arterial tissues. Supplemental Table 5. Changes in expression


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ratios in plasma and arterial tissues of protein families identified as up- and/or down-regulated. Supplemental Table 6. Identified proteins throughout the four time points.

AUTHOR INFORMATION Corresponding Author Takeshi Sakamoto Center for Technology Innovation - Healthcare, Research & Development Group, Hitachi, Ltd. 1-280, Higashi-koigakubo, Kokubunji-shi, Tokyo, 185-8601 Japan TEL: +81-50-3135-1024 EMAIL: [email protected]

Present Addresses The current address of Zhao, Y. is Kobe Medical Technology Office, ATOX Co., Ltd., Kobe, Japan. The current address of Kaneko, A. is Biosystems Design Department, Hitachi HighTechnologies Corporation, 312-8504, Hitachinaka-shi, Japan.

Author Contributions The manuscript was written with contributions of all authors. All authors have given approval to the final version of the manuscript. ‡These authors contributed equally to this work.


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Funding Sources This study was supported in part by the grant “The matching program for innovations in future drug discovery and medical care” from the Ministry of Education, Culture, Sports, Science, and Technology, Japan (To Tamaki, N.).

ACKNOWLEDGMENTS The authors thank Drs. Koichi Kokame, Hiroyasu Inoue, Chiaki Yokota, and Yoichi Shimizu for their valuable discussions, Dr. Hirokazu Nishida for his critical reading of the manuscript, and Yuko Komori, Yumi Yanagiya, and Megumi Hikichi for their technical assistance.

ABBREVIATIONS ACN, acetonitrile; A2M, α-2 macroglobulin; ApoED, Apolipoprotein E deficient mouse; CHAPS, 3-[(3-Cholamidopropyl) dimethylammonio] propanesulfonate; cICAT, cleavable isotope-coded affinity tag; DTT, dithiothreitol; HDL, high-density lipoprotein; IL-6, interleulin6; Immunoglobulin M, IgM; LC-MS, liquid chromatography MS; LDL, low-density lipoprotein; MS, mass spectrometry; PBS, phosphate-buffered saline; PPARγ, peroxisome proliferatoractivated receptor-γ; SCX column, strong cation exchange column; SDS, sodium dodecyl sulfate; TFA, trifluoroacetic acid; Tris, 2-amino-2-(hydroxymethyl)propane-1,3-diol; wt, wild-type

REFERENCES (1) Ross, R. Atherosclerosis--an inflammatory disease. N. Engl. J. Med. 1999, 340, 115-126.


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(2) Wang, X.; Connolly, T.M., Biomarkers of vulnerable atheromatous plaques: translational medicine perspectives. Adv. Clin. Chem. 2010, 50, 1-22. (3) Seo, D.; Wang, T.; Dressman, H., Herderick, E. E., Iversen, E. S.; Dong, C.; Vata, K.; Milano, C. A.; Rigat, F., Pittman, J.; Nevins, J. R.; West, M., Goldschmidt-Clermont, P. J. Gene expression phenotypes of atherosclerosis. Arterioscler. Thromb. Vasc. Biol. 2004, 24, 1922-1927 (4) Tabibiazar, R.; Wagner, R. A.; Ashley, E. A.; King, J. Y.; Ferrara, R.; Spin, J. M.; Sanan, D. A.; Narasimhan, B.; Tibshirani, R.; Tsao, P. S.; Efron, B.; Quertermous, T. Signature patterns of gene expression in mouse atherosclerosis and their correlation to human coronary disease. Physiol. Genomics 2005, 22, 213-226. (5) Tabibiazar, R.; Wagner, R. A.; Deng, A.; Tsao, P. S.; Quertermous, T. Proteomic profiles of serum inflammatory markers accurately predict atherosclerosis in mice. Physiol. Genomics 2006, 25, 194 - 202. (6) Hanash, S. Disease proteomics. Nature 2003, 422, 226-232. (7) Gygi, S. P.; Rist, B.; Gerber, S. A.; Turecek, F.; Gelb, M. H.; Aebersold, R. Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nat. Biotechnol. 1999, 17, 994-999. (8) Ross, P. L.; Huang, Y. N.; Marchese, J. N.; Williamson, B.; Parker, K.; Hattan, S.; Khainovski, N.; Pillai, S.; Dey, S.; Daniels, S.; Purkayastha, S.; Juhasz, P.; Martin, S.; BartletJones, M.; He, F.; Jacobson, A.; Pappin, D. J. Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Mol. Cell. Proteomics 2004, 3, 1154– 1169.


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(9) von zur Muhlen, C.; Schiffer, E.; Sackmann, C.; Zürbig, P.; Neudorfer, I.; Zirlik, A.; Htun, N.; Iphöfer, A.; Jänsch, L.; Mischak, H.; Bode, C.; Chen, Y. C.; Peter, K. Urine Proteome Analysis Reflects Atherosclerotic Disease in an ApoE–/– Mouse Model and Allows the Discovery of New Candidate Biomarkers in Mouse and Human Atherosclerosis. Mol. Cell. Proteomics [Online] 2012, 11, 1-13. doi: 10.1074/mcp.M111.013847. (10) Zimmerli, L. U.; Schiffer, E.; Zürbig, P,; Good, D. M.; Kellmann, M.; Mouls, L.; Pitt, A. R.; Coon, J. J.; Schmieder, R. E.; Peter, K. H.; Mischak, H.; Kolch, W., Delles, C., Dominiczak, A. F. Urinary Proteomic Biomarkers in Coronary Artery Disease. Mol. Cell. Proteomics 2008, 7, 290 - 298. (11) von zur Muhlen, C.; Schiffer, E.; Zuerbig, P.; Kellmann, M.; Brasse, M.; Meert, N.; Vanholder, R. C.; Dominiczak, A. F.; Chen, Y. C.; Mischak, H.; Bode, C.; Peter, K. Evaluation of urine proteome pattern analysis for its potential to reflect coronary artery atherosclerosis in symptomatic patients. J. Proteome Res. 2009, 8, 335-35. (12) Ritorto, M. S. and Borlak, J. Combined serum and tissue proteomic study applied to a cMyc transgenic mouse model of hepatocellular carcinoma identified novel disease regulated proteins suitable for diagnosis and therapeutic intervention strategies. J. Proteome Res. 2011, 10, 3012-3030. (13) Yamada, M.; Satoh, M.; Seimiya, M.; Sogawa, K.; Itoga, S., Tomonaga, T.; Nomura, F. Combined proteomic analysis of liver tissue and serum in chronically alcohol-fed rats. Alcohol Clin. Exp. Res. [Online] 2013, 37, E79-E87. doi: 10.1111/j.1530-0277.2012.01883.x


36

Page 37 of 44

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(14) Zhang, S. H.; Reddick, R. L.; Piedrahita, J. A.; Maeda, N. Spontaneous hypercholesterolemia and arterial lesions in mice lacking apolipoprotein E. Science 1992, 258, 468 - 471. (15) Plump, A. S.; Smith, J. D.; Hayek, T.; Aalto-Setala, K.; Walsh, A.; Verstuyft, J. G.; Rubin, E. M.; Breslow, J. L. Severe hypercholesterolemia and atherosclerosis in apolipoprotein Edeficient mice created by homologous recombination in ES cells. Cell 1992, 71, 343-353. (16) Matsushima, Y.; Sakurai, T., Ohoka, A.; Ohnuki, T.; Tada, N., Asoh, Y.; Tachibana, M. Four strains of spontaneously hyperlipidemic (SHL) mice: phenotypic distinctions determined by genetic backgrounds. J. Atheroscler. Thromb. 2001, 8, 71-79. (17) Meir, K. S. and Leitersdorf, E. Atherosclerosis in the Apolipoprotein E–Deficient Mouse: A Decade of Progress. Arterioscler. Thromb. Vasc. Biol. 2004, 24, 1006-014. (18) Reddick, R. L.; Zhang, S. H.; Maeda, N. Atherosclerosis in mice lacking apo E. Evaluation of lesional development and progression. Arterioscler. Thromb. Vasc. Biol. 1994, 14, 141-147. (19) Nakashima, Y.; Plump, A. S.; Raines, E. W.; Breslow, J. L.; Ross, R. ApoE-deficient mice develop lesions of all phases of atherosclerosis throughout the arterial tree. Arterioscler. Thromb. Vasc. Biol. 1994, 14, 133-140. (20) Zhao, Y.; Kuge, Y.; Zhao, S.; Strauss, H. W.; Blankenberg, F. G.; Tamaki, N. Prolonged High-Fat Feeding Enhances Aortic 18F-FDG and 99mTc-Annexin A5 Uptake in Apolipoprotein EDeficient and Wild-Type C57BL/6J Mice. J. Nucl. Med. 2008, 49, 1707-1714. (21) Yokosuka, T.; Yoshinari, K.; Kobayashi, K.; Ohtake, A.; Hirabayashi, A.; Hashimoto, Y.; Waki, I.; Takao, T. ‘Information-Based-Acquisition’ (IBA) technique with an ion-trap/time-of-


37


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 38 of 44

flight mass spectrometer for high-throughput and reliable protein profiling. Rapid Commun. Mass Spectrom. 2006, 20, 2589-2595. (22) Tissot, J. D.; Sanchez, J. C.; Vuadens, F.; Scherl, A.; Schifferli, J. A.; Hochstrasser, D. F.; Schneider, P.; Duchosal, M. A. IgM are associated to Sp alpha (CD5 antigen-like). Electrophoresis 2002, 23, 1203-1206. (23) Rao, A. G. and Sodetz, J. M. Purification of functional subunits of the eighth component of human complement (C8) under nondenaturing conditions. Complement 1984, 1, 182-186. (24) Miyazaki, T.; Hirokami, Y.; Matsuhashi, N.; Takatsuka, H.; Naito, M. Increased Susceptibility of Thymocytes to Apoptosis in Mice Lacking AIM, a Novel Murine Macrophagederived Soluble Factor Belonging to the Scavenger Receptor Cysteine-rich Domain Superfamily. J. Exp. Med. 1999, 189, 413-422. (25) Mayr, M.; Chung, Y.- L.; Mayr, U.; Yin, X.; Ly, L.; Troy, H.; Fredericks, S.; Hu, Y.; Griffiths, J. R.; Xu, Q. Proteomic and Metabolomic Analyses of Atherosclerotic Vessels From Apolipoprotein E-Deficient Mice Reveal Alterations in Inflammation, Oxidative Stress, and Energy Metabolism. Arterioscler. Thromb. Vasc. Biol. 2005, 25, 2135-2142. (26) Suski, M.; Olszanecki, R.; Madej, J.; Totoń-Żurańska, J.; Niepsuj, A.; Jawień, J.; BujakGiżycka, B.; Okoń, K.; Korbut, R. Proteomic analysis of changes in protein expression in liver mitochondria in apoE knockout mice. J. Proteomics 2011, 74, 887-893. (27) Jing, L.; Parker, C. E.; Seo, D.; Hines, M. W.; Dicheva, N., Yu, Y.; Schwinn, D.; Ginsburg, G. S.; Chen, X. Discovery of biomarker candidates for coronary artery disease from an APOE-


38

Page 39 of 44

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


knock out mouse model using iTRAQ-based multiplex quantitative proteomics. Proteomics 2011, 11, 2763-2776. (28) Koenig, W. and Khuseyinova, N. Biomarkers of Atherosclerotic Plaque Instability and Rupture. Arterioscler. Thromb. Vasc. Biol. 2007, 27, 15-26. (29) Xu, J. and Shi, G.-P. Vascular wall extracellular matrix proteins and vascular diseases. Biochim. Biophys. Acta. [Online] 2014, 1842, 2106-2119. doi: 10.1016/j.bbadis.2014.07.008. (30) Hansson, G. K.; Libby, P.; Schönbeck, U.; Yan, Z.- Q. Innate and Adaptive Immunity in the Pathogenesis of Atherosclerosis. Circ. Res. 2002, 91, 281-291. (31) Speidl, W. S.; Kastl, S. P.; Huber, K.; Wojta, J. Complement in atherosclerosis: friend or foe? J. Thromb. Haemost. 2011, 9, 428-440. (32) Yasojima, K.; Schwab, C.; McGeer, E. G.; McGeer, P. L. Complement Components, but Not Complement Inhibitors, Are Upregulated in Atherosclerotic Plaques. Arterioscler. Thromb. Vasc. Biol. 2001, 21, 1214-1219. (33) Lewis, M. J.; Malik, T. H.; Ehrenstein, M. R.; Boyle, J. J.; Botto, M.; Haskard, D. O. Immunoglobulin M Is Required for Protection Against Atherosclerosis in Low-Density Lipoprotein Receptor–Deficient Mice. Circulation 2009, 120, 417-426. (34) Cimmino, G.; D'Amico, C.; Vaccaro, V.; D'Anna, M.; Golino, P. The missing link between atherosclerosis, inflammation and thrombosis: is it tissue factor? Expert Rev. Cardiovasc. Ther. 2011, 9, 517-523.


39


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 40 of 44

(35) Ambrose, J. A. Plaque Disruption and the Acute Coronary Syndromes of Unstable Angina and Myocardial Infarction: If the Substrate Is Similar, Why Is The Clinical Presentation Different? J. Am. Coll. Cardiol. 1992, 19, 1653-1658. (36) Uchida, Y.; Uchida, Y.; Sakurai, T.; Kanai, M.; Shirai, S.; Morita, T. Characterization of Coronary Fibrin Thrombus in Patients With Acute Coronary Syndrome Using Dye-Staining Angioscopy. Arterioscler. Thromb. Vasc. Biol. 2011, 31, 1452-60. (37) Didangelos, A.; Yin, X.; Mandal, K.; Baumert, M.; Jahangiri, M.; Mayr, M. Proteomics Characterization of Extracellular Space Components in the Human Aorta. Mol. Cell. Proteomics 2010, 9, 2048-2062. (38) Clutterbuck, A. L.; Smith, J. R.; Allaway, D.; Harris, P.; Liddell, S.; Mobasheri, A. High throughput proteomic analysis of the secretome in an explant model of articular cartilage inflammation. J. Proteomics 2011, 74, 704-715. (39) Sjöberg, A.; Önnerfjord, P.; Mörgelin, M.; Heinegård, D.; Blom, A. M. The Extracellular Matrix and Inflammation: FIBROMODULIN ACTIVATES THE CLASSICAL PATHWAY OF COMPLEMENT BY DIRECTLY BINDING C1q. J. Biol. Chem., 2005, 280, 32301-32308. (40) Arai, S.; Shelton, J. M.; Chen, M.; Bradley, M. N.; Castrillo, A., Bookout, A. L.; Mak, P. A.; Edwards, P. A.; Mangelsdorf, D. J.; Tontonoz, P. T.; Miyazaki, T. A role for the apoptosis inhibitory factor AIM/Spalpha/Api6 in atherosclerosis development. Cell Metab. 2005, 1, 201213. (41) Anderson, L. Candidate-based proteomics in the search for biomarkers of cardiovascular disease. J. Physiol. 2005, 563, 23-60.


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Table of Contents/ Abstract Figure


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Figure 1

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Figure 2


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Figure 3

Combined Plasma and Tissue Proteomic Study of ... - ACS Publications

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