Proteomic Profile of Human Aortic Stenosis - American Chemical Society

Article pubs.acs.org/jpr

Proteomic Profile of Human Aortic Stenosis: Insights into the Degenerative Process Tatiana Martín-Rojas,† Felix Gil-Dones,† Luis F. Lopez-Almodovar,‡ Luis R. Padial,§ Fernando Vivanco,*,∥,⊥,# and Maria G. Barderas†,# †

Department of Vascular Physiopathology, Hospital Nacional de Paraplejicos (HNP), SESCAM, Toledo, Spain Cardiac Surgery, Hospital Virgen de la Salud, Toledo, Spain § Department of Cardiology, Hospital Virgen de la Salud, Toledo, Spain ∥ Department of Immunology, IIS- Fundacion Jimenez Diaz, Madrid, Spain ⊥ Department of Biochemistry and Molecular Biology I, Universidad Complutense, Madrid, Spain ‡

S Supporting Information *

ABSTRACT: Degenerative aortic stenosis is the most common worldwide cause of valve replacement. While it shares certain risk factors with coronary artery disease, it is not delayed or reversed by reducing exposure to risk factors (e.g., therapies that lower lipids). Therefore, it is necessary to better understand its pathophysiology for preventive measures to be taken. In this work, aortic valve samples were collected from 20 patients that underwent aortic valve replacement (55% males, mean age of 74 years) and 20 normal control valves were obtained from necropsies (40% males, mean age of 69 years). The proteome of the samples was analyzed by quantitative differential electrophoresis (2D-DIGE) and mass spectrometry, and 35 protein species were clearly increased in aortic valves, including apolipoprotein AI, alpha-1-antitrypsin, serum albumin, lumican, alfa-1-glycoprotein, vimentin, superoxide dismutase Cu−Zn, serum amyloid P-component, glutathione S-transferase-P, fatty acid-binding protein, transthyretin, and fibrinogen gamma. By contrast, 8 protein species were decreased (transgelin, haptoglobin, glutathione peroxidase 3, HSP27, and calreticulin). All of the proteins identified play a significant role in cardiovascular processes, such as fibrosis, homeostasis, and coagulation. The significant changes observed in the abundance of key cardiovascular proteins strongly suggest that they can be involved in the pathogenesis of degenerative aortic stenosis. Further studies are warranted to better understand this process before we can attempt to modulate it. KEYWORDS: proteomics, human aortic valves, aortic stenosis

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calcification.8−10 Epidemiological studies have identified risk factors for degenerative AS that are similar to those of vascular atherosclerosis, such as smoking, male gender, hypertension, elevated cholesterol levels and renal failure.11,12 These data have led to the suggestion that like atherosclerosis, calcified aortic valve disease is a chronic inflammatory process. Although some animal models have supported this hypothesis,6 several trials have failed to support that a reduction in blood cholesterol slows the progress of degenerative AS, underlining the need to better understand this disease.13,14 Adopting a proteomic approach to study the pathogenesis of AS might provide useful information to understand the development of AS and help to define its possible relationship with coronary atherosclerosis. To date, no proteomic studies have investigated the difference between stenotic and

INTRODUCTION Degenerative aortic stenosis (AS) is a prevalent disease and is currently responsible for most aortic valve replacement procedures performed. Since degenerative AS increases with age, the aging process of the western population forecasts a significant increase in AS prevalence in future decades.1 To date, AS has been considered as a passive process secondary to calcium deposition in the worn-out aortic valves. Indeed, clinicians and researchers have mainly focused on surgical treatments for valvular heart disease, while the underlying mechanisms remained largely unknown.2 Recent descriptive studies of AS valves have helped define the hallmark features of aortic valve disease, including early atherosclerosis, cell proliferation and osteoblast expression.3−5 The initial aortic valve lesion, the so-called valve sclerosis, is related to infiltration and oxidation of lipoproteins,6,7 similar to that seen in atherosclerotic plaques, and it is associated with endothelial dysfunction, increased leukocyte adhesion/infiltration and © 2012 American Chemical Society

Received: June 16, 2011 Published: January 25, 2012 1537

dx.doi.org/10.1021/pr2005692 | J. Proteome Res. 2012, 11, 1537−1550

Journal of Proteome Research

Article

Table 1. Clinical Characteristics of the Individuals with ASa

a

no patient

age/sex

type of AS

HTN

diabetes

dyslipidemia

DIGE

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Mean

74/F 73/M 68/M 81/M 69/M 79/F 73/F 77/M 79/F 75/M 74/M 74/F 79/F 79/F 72/F 63/M 72//M 75/F 78/M 74/M 74 ± 0/45%F−55%M

Moderate Severe Severe Severe Severe Severe and Calcification Severe Moderate Moderate Severe Severe Severe Severe Severe and Calcification Severe Severe and Calcification Severe Moderate Severe Moderate

Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes yes Yes Yes Yes Yes 100.00%

No Yes No No Yes Yes Yes No Yes No Yes Yes Yes yes No no No Yes Yes Yes 60.00%

Yes No Yes Yes Yes No Yes No No Yes Yes No No No No Yes Yes Yes No No 50.00%

X X

WB

X X X X

IHC X X X X X

X X X

X X

X X

X X X X X X X X

X X X

X X

X

HTN, arterial hypertension; DIGE, quantitative differential electrophoresis; WB, Western blot; IHC, immunohistochemistry.

from all the patient subjects and relatives of control subjects prior to entry onto the study.

nonstenotic human valve proteins. Thus, to the best of our knowledge, we describe here the first proteomics study comparing AS valves with healthy aortic valves by quantitative differential electrophoresis (2D-DIGE) combined with mass spectrometry (MS). We identified 43 protein spots that were present with different abundances between AS and healthy valves, corresponding to antioxidant enzymes, structural and contractile proteins and proteins involved in inflammation. Moreover, we validated this candidate proteins15−17 by conventional techniques, such as Western blotting and Immunohistochemistry.

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Sample Preparation

AS valves were processed within a maximum of 2 h after surgery (Figure 1) having maintained the tissue at 4 °C in RPMI medium. The valves were washed 3 times in PBS to reduce blood contaminants, and the aortic valve leaflet was then ground into powder in liquid N2 with a mortar and 0.2 g of this powder was resuspended in 400 μL of protein extraction buffer (Tris 10 mM [pH 7.5], 500 mM NaCl, 0.1% Triton x-100, 1% β-mercaptoethanol, 1 mM PMSF).18,19 The homogenate was centrifuged at 21000× g (5840R Eppendorfs) for 15 min at 4 °C to precipitate the membranes and tissue debris, and the supernatant (E1) containing most of the soluble proteins, was collected and stored at −20 °C. The pellet was then solubilized in 7 M Urea, 2 M Thiourea, 4% CHAPS,20,21 and centrifuged again at 21000× g. This second supernatant (E2) was rich in hydrophobic proteins, mainly membrane proteins, such that the cellular debris and lipids were eliminated. The protein concentration was determined by the Bradford-Lowry method (Bio-Rad protein assay).22

EXPERIMENTAL PROCEDURES

Patient and Control Subject Selection

Heart valves with degenerative AS (n = 20) were obtained from patients of both sexes (55% male, 45% female), with an average age of 74 (±3.9) years, who underwent aortic valve replacement due to severe degenerative AS. All patients (100%) had hypertension, whereas 50% suffered hyperlipemia and 60% had diabetes mellitus. Coronary artery disease was present in 60% patients and surgery was indicated according to current practice guidelines. Patients with aortic regurgitation, mitral valve disease or any suspicion of rheumatic disease were not included in the study (Table 1). Control valves were obtained from necropsies. Before resecting the valve, a cardiac surgeon explored carefully the valve. Valves with calcifications or cusp restriction were excluded as control cases. If inserted saline in the aortic root remained in the closed valve, we ruled out significant regurgitation and took these aortic valves as a control case. These subjects did not die from cardiovascular illnesses and had no history of coronary artery disease or diabetes mellitus (n = 20, Table 2). This study was carried out in accordance with the recommendations of the Helsinki Declaration and it was approved by the ethics committee at the Hospital “Virgen de la Salud” (Toledo, Spain). Signed informed consent was obtained

Conventional Two-dimensional Electrophoresis

Conventional silver stained gels with 350 μg protein were performed to identify the protein spots which could not be identified throughout the 2D-DIGE restained gels. Protein extracts were diluted in rehydration buffer 7 M Urea, 2 M Thiourea, 4% CHAPS, 1−2% Ampholites and 1% TBP: BioRad,23 and they were applied to pH 4−7, 17 cm. IPG strips. After the IEF strips were equilibrated as described previously,23 and 12% SDS-PAGE was performed according to Laemmli24 on a Protean II system (Bio-Rad) at 25 mA/gel and at 4 °C. The gels were fixed overnight and stained using a Silver Staining kit (GE Healthcare), a protocol that is compatible with mass spectrometry. Finally, gels were then scanned with GS-800 Calibrated Densitometer (Bio-Rad). 1538



Article

Table 2. Clinical Characteristics of the Individuals whose Aortic Valves were Used as Controls (Without Aortic Stenosis)a no control

age/sex

cause of the death

HTN

DIGE

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Mean

64/M 51/F 47/H 47/F 47/F 34/M 64/F 78/F 42/F 67/M 74/M 87/F 48/F 55/M 87/F 76/M 80/F 49/F 74/M 77/F 69 ± 7.07/40%M−60%F

Bilateral acute bronchopneumonia Septic shock Sepsis Bacterial pneumonia Carcinogenesis Acute Pancreatitis Bacterial Sepsis Aspiration bronchopneumonia Lymphoma Renal insufficiency COPD Carcinoma Pulmonary thromboembolism Atypical pneumonia Cholelithiasis Carcinoma Atrial fibrillation Respiratory insufficiency Septic shock COPD

No Yes No No No Yes No No No No No No No No No No Yes No Yes Yes 25.00%

X

X X

WB

IHC

X

X X X

X X X X

X X X

X X X X

X X

X

X

X X X

X

X X X

X X X

a

COPD, chronic obstructive pulmonary disease; HTN, arterial hypertension; DIGE, quantitative differential electrophoresis; WB, Western blot; IHC, immunohistochemistry.

Figure 1. Schematic representation of the protocol to obtain the samples and the subsequent workflow, involving valve homogenization, protein extraction and 2D-DIGE analysis. Sixteen samples (8 controls and 8 patients) were analyzed by 2D-DIGE and 12 for validation (4 for Western blot and 8 for IHC).

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2-DE DIGE Separation

Proteomic Unit. Prior to identification process, the protein spots were automatically digested with an “Ettan Digester” (GE Healthcare) according to Shevchenko et al.,25 with minor modifications,18 gel plugs were subjected to reduction with 10 mM dithiothreitol (Sigma Aldrich) in 50 mM ammonium bicarbonate (99% purity; Scharlau) and alkylation with 55 mM iodoacetamide (Sigma Aldrich) in 50 mM ammonium bicarbonate. The gel pieces were then rinsed with 50 mM ammonium bicarbonate in 50% methanol (gradient, HPLC grade, Scharlau) and acetonitrile (gradient, HPLC grade, Scharlau) and dried in a Speedvac. Modified porcine trypsin (sequencing grade; Promega, Madison, WI) at a final concentration of 20 ng/μL in 20 mM ammonium bicarbonate was added to the dry gel pieces and the digestion proceeded at 37 °C overnight. Finally, 70% aqueous acetonitrile and 0.1% formic acid (99.5% purity; Sigma Aldrich) were added for peptide extraction. After overnight digestion at 37 °C the peptides were extracted with 60% acetonitrile (ACN) in 0.1% formic acid (99.5% purity; Sigma Aldrich). The samples were then dried in a Speedvac and resuspended in 98% water with 0.1% formic acid (FA) and 2% ACN. An aliquot of each digestion was mixed with an aliquot of the matrix solution (3 mg/mL α-cyano4-Hydroxycinnamic acid: Sigma Aldrich) in 30% ACN, 15% 2-propanol and 0.1% TFA, and this mixture was pipetted directly onto the stainless steel sample plate of the mass spectrometer 384 Opti-TOF 123 × 81 mm MALDI (Applied Biosystems) and dried at room temperature. The MALDI-MS/MS data were obtained in an automated analysis loop using a 4800 Plus MALDI TOF/TOF Analyzer (Applied Biosystems). Spectra were acquired in the reflector positive-ion mode with a Nd:YAG, 355 nm wavelength laser at a frequency of 200 Hz, and between 1000 and 2000 individual spectra were averaged. The experiments were acquired in a uniform mode with fixed laser intensity. For the MS/MS 1 kV analysis mode, precursors were accelerated to 8 kV in source 1, and they were selected at a relative resolution of 350 (fwhm) and with metastable suppression. Fragment ions generated by collision with air in a CID chamber were further accelerated at 15 kV in source 2. Mass data were analyzed automatically with the 4000 Series Explorer Software version 3.5.3 (Applied Biosystems). Internal calibration of MALDI-TOF mass spectra was performed using two trypsin autolysis ions with m/z = 842.510 and m/z = 2211.105. For the calibrations in the MS/MS mode, the fragment ion spectra obtained from Glub-fibrinopeptide were used (4700 Cal Mix, Applied Biosystems). MALDI-MS and MS/MS data were combined through the GPS Explorer Software (Version 3.6) to search a nonredundant protein database (Swiss-Prot 56.5) using the Mascot software (version 2.2: Matrix Science).26 The following parameters were employed: 50 ppm precursor tolerance; 0.6 Da MS/MS fragment tolerance; 1 missed cleavage permitted; and with carbamidomethyl cysteines and methionine oxidation as a modification. MALDI-MS (/MS) spectra and database search results were manually inspected using the aforementioned software. For combined MS and MS/MS data, identifications were accepted when Confidence Interval (C.I. %) of GPS software was 95% or higher. Since Protein Scores and Ion Scores from different searches cannot be directly compared, GPS software calculates this C.I. % to combine results from MS and MS/MS database searches. This coefficient value means that the probability that the observed match is a random event is lower than 5%. For PMF spectra, identifications were also

Proteins were labeled according to the manufactureŕs instructions (GE Healthcare, Piscataway, NJ) (table S1 Supporting Information), such that 50 μg of AS and aortic valve tissue protein extracts were labeled with 400 pmol of the N-hydroxysuccinimide Cy3 or Cy5 fluorescent cyanine dye on ice for 30 min. The labeling reaction was quenched with 0.2 mM lysine. All experiments involved an internal standard containing equal amounts of each protein extract labeled with 400 pmol of N-hydroxysuccinimide Cy2 dye. The internal standard, the AS and aortic valves, protein extracts were combined and run in a single gel (150 μg of total protein). Protein extracts were diluted in Rehydration Buffer (30 mM TRIS, 7 m Urea, 2 M Thiourea, 4% CHAPS) and applied to 24 cm pH 4−7 IPG strips. The first dimension was run in the IPGphor IEF II System (GE Healthcare) as follows: 500 V for 30 min, 1000 V for 1 h in a linear gradient to 200 V over 1 h, a linear gradient to 5000 V over 2 h, a linear gradient to 8000 V over 1 h, and 8000 V until 88000 v/h. After the first dimension, the strips were equilibrated in SDS-equilibration buffer (1.5 M Tris/HCl [pH 8.8], 6 M Urea, 87% Glycerol and 2% SDS), and then, the proteins were separated on 12% Acrylamide/Bisacrylamide gels using an Ettan Dalt Six device (GE-Healthcare). Image Acquisition and Analysis

After SDS-PAGE, the gels were scanned with a Typhoon 9400 fluorescence gel scanner (GE Healthcare, Piscataway, NJ) using appropriate individual excitation and emission wavelengths, filters and photomultiplier (PTM) values that are sensitive for each of the Cy3, Cy5 and Cy2 dyes (PTM values: 480, 490, and 500 nm, respectively). Relative protein quantification was performed on AS and healthy valves with DeCyder software v6.5 (GE Healthcare) and the multivariate statistical module EDA (Extended data analysis). The Differential in-gel analysis (DIA) module codetected the 3 images of a gel (the internal standard and the two samples), measured the spot abundance in each image, and expressed these values as Cy3/Cy2 and Cy5/Cy2 ratios. These DIA data sets were then analyzed using the Biological Variation Analysis module (BVA), which enabled the spot maps to be matched and the Cy3/Cy2 and Cy5/Cy2 ratios to be compared. Only protein spots with >1.5-fold differences in abundance were considered for the analysis. A statistical analysis was then carried out to determine the changes in protein species, with P-values below 0.05 accepted as significant when the Studentś t-test was applied. Finally, a multivariate analysis was performed by Principal Component Analysis (PCA) using the algorithm included in the EDA module of the DeCyder software (version 6.5) based on the spots that matched across all the gels. A pattern analysis hierarchical classification was obtained using the Pearson coefficient based on the spots present in 90% of all the gels. The gels were then restained with a silver staining kit (GE-Healthcare), as described above. Protein Identification by MALDI-TOF/TOF

2D-DIGE gels were silver stained after image digitalization, in order to manually pick protein spots present with different abundance. Those which could not be identified in these kinds of gels were obtained from 2-DE gels matched with the 2DDIGE images. The protein spots with differences in abundance were manually excised from 8 gels and identified at the HNP 1540



Article

Figure 2. Aortic stenosis tissue staining. (A) Haematoxilin & Eosin staining (H&E) of Aortic valves. (1) Normal aortic valve leaflet. (2) Moderate AS with a strong accumulation of lipids in the fibrosa layer. (3) Severe AS with displacement of the elastic membrane. Thickening and formation of calcium deposits (arrow) in the fibrosa layer. (B) Senescence analysis. (1) Blue color indicates β-galactosidase activity and cellular senescence. AS valves show a remarkable β-galactosidase activity, whereas no β-galactosidase activity is observed in the control aortic valve. (2) Microscopic analysis demonstrates that β-galactosidase-positive cells are localized on the surface of the leaflet at the aortic side (arrows and blue color) of the stenotic valve leaflet, whereas no β-galactosidase activity is observed in the control valve.

20 min. To ensure that equal amounts of aortic valve tissue was loaded onto the IPG strips (2-D Western blotting) or onto polyacrylamide gels (regular Western blotting), Ponceau S staining was performed on the transferred membranes. The membranes were then blocked for 1 h27 and then, were incubated overnight with the primary antibody in PBS-T 5% nonfat dry milk. Below, the membranes were incubated with the specific HRP-conjugated secondary antibody in PBS-T containing 5% of nonfat dry milk. Detection was performed by enhanced chemiluminescence (ECL, GE Healthcare) following the manufacturers’ instructions.

accepted when C.I. % of GPS software was 99% or higher. (Alternatively, the ppw files obtained from 4000 series explorer software (ABSciex) are included in Supporting Information. These ppw files have been extracted from GPS software. The ppw files are deposited in PRIDE, too.) Antibodies

The primary antibodies to probe Western blots included rabbit polyclonal antisera against Glutathione Peroxidase 3, amyloid P, HSP27, Alpha 1B glycoprotein, Superoxide dismutase 1, Calreticulin, Alpha 1 antitrypsin. In addition, monoclonal antibodies against Apoliprotein AI and SM22 alpha, as well as a goat polyclonal Fibrinogen gamma antiserum were used (all from Abcam). A rabbit polyclonal Fatty acid-binding protein antiserum (Lifespan Biosciences) and a rabbit polyclonal Lumican antiserum (SIGMA) were also used. For Immunohistochemistry, an additional monoclonal antibody against Vimentin (Abcam) and a goat polyclonal antiserum against Lumican (R&D Systems) were used.

Immunohistochemistry

Formalin-fixed sections (6-μm) from AS and control valves were decalcified in Shandon-TBD1 (Thermo Scientific) and embedded in OCT. The sections were blocked with 1% BSA in TBS Buffer with 0.1% Tween 20 and incubated for 1 h with the primary antibodies at 37 °C. The sections were then incubated with an IgG-HRP-conjugated secondary antibody (NORDIC Immunology) and the chromogenic reaction was developed using 3,3′diaminobenzidine. Sections were counterstained with hematoxilin prior to dehydration and coverslipping. As a negative control, the complete immunohistochemical procedure was performed on adjacent sections lacking the primary antibody.

Western Blotting

Protein samples obtained from AS and control valves were resolved by 12% SDS-PAGE using a Bio-Rad Miniprotean II electrophoresis unit run at a constant current of 25 mA/gel during 1 h. After SDS-PAGE, the proteins were transferred to a nitrocellulose membrane under a constant voltage of 15 V for 1541


1542

1732 Transgelin

Haptoglobin Haptoglobin Apolipoprotein A-I Apolipoprotein A-I Serum amyloid P Serum amyloid P Apolipoprotein A-I Glutathione peroxidase 3 Glutathione S-transferase P Transgelin, isoform CRA_c Glutathione S-transferase P Heat shock 27 kDa

193 752 545 547 546 554 558 568 575 582 590 584 583 593 602 610 614 746 750 758 762 803 804 966

1339 1383 1687 1691 1613 1693 1702 1749 1699 1737 1678 1787

protein name

Lumican Vimentin Alpha-1B-glycoprotein Alpha-1B-glycoprotein Serum albumin Serum albumin Alpha-1B-glycoprotein Alpha-1B-glycoprotein Alpha-1B-glycoprotein Serum albumin Serum albumin Serum albumin Serum albumin Serum albumin Serum albumin Serum albumin Serum albumin Alpha-1-antitrypsin Vimentin Alpha-1-antitrypsin Alpha-1-antitrypsin Fibrinogen gamma chain Fibrinogen gamma chain Calreticulin

spot no

TAGL_HUMAN

HPT_HUMAN HPT_HUMAN APOA1_HUMAN APOA1_HUMAN SAMP_HUMAN SAMP_HUMAN APOA1_HUMAN GPX3_HUMAN GSTP1_HUMAN TAGL_HUMAN GSTP1_HUMAN HSP27_HUMAN

LUM_HUMAN VIME_HUMAN A1BG_HUMAN A1BG_HUMAN ALBU_HUMAN ALBU_HUMAN A1BG_HUMAN A1BG_HUMAN A1BG_HUMAN ALBU_HUMAN ALBU_HUMAN ALBU_HUMAN ALBU_HUMAN ALBU_HUMAN ALBU_HUMAN ALBU_HUMAN ALBU_HUMAN A1AT_HUMAN VIME_HUMAN A1AT_HUMAN A1AT_HUMAN FIBG_HUMAN FIBG_HUMAN CALR_HUMAN

accession code

Down Down Up Up Up Up Up Down Up Down Up Down Down

−2.4012

Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Up Down

6.5

6.13 6.13 5.56 5.56 6.10 6.10 5.56 8.26 5.43 6.5 5.43 7.83

6.16 5.06 5.58 5.58 5.92 5.92 5.58 5.58 5.58 5.92 5.92 5.92 5.92 5.92 5.92 5.92 5.92 5.37 5.06 5.37 5.37 5.6 5.37 4.29

6.1

5.14 5.21 6.0 6.2 5.6 5.75 6.25 5.9 5.75 6.08 5.8 6.6

5 4.9 5.3 5.35 4.8 5.1 5.41 5.7 5.49 6.0 5.85 6.0 5.82 6.0 6.2 6.23 6.5 5.02 4.8 5.8 5.25 5.61 5.15 4.49

up/down pI theor. pI exp.

−1.7435 −1.9375 2.5134 1.6206 1.562 2.9514 2.5868 −1.8192 2.1109 −1.5872 1.7433 −1.9935

1.585 1.9018 1.8266 1.8284 1.5611 1.6519 1.5457 1.5317 1.8659 1.8659 1.6653 1.7707 1.6745 1.9458 1.8257 1.7941 1.7602 2.9606 3.1366 1.5042 1.7286 1.6867 1.7482 −1.6281

av. ratio

23.691

45.177 45.177 30.759 30.759 25.371 25.371 30.759 25.537 23.341 23.691 23.341 22.313

38.405 53.619 54.239 54.239 69.321 69.321 54.239 54.239 54.239 69.321 69.321 69.321 69.321 69.321 69.321 69.321 69.321 46.707 53.619 46.707 46.707 51.479 51.479 48.084

MW

Table 3. Altered Protein Species Identified by Mass Spectrometry (MALDI TOF-TOF)a MW

29

31 31 32.5 32.5 33 33 32 29 30 29 30 32

115 74.5 105.5 105.5 106 106 105.5 105.5 105.5 105.5 105.5 105.5 105.5 105.5 105.5 105.5 106 85.2 74.5 75 85.2 75 85.2 79.5

p-value

0.02055

0.01757 0.01829 0.02304 0.007406 0.03407 0.002002 0.005885 0.02862 0.01985 0.02955 0.00949 0.02893

0.03717 0.02175 0.001887 0.001271 0.007737 0.001892 0.003642 0.01701 0.004937 0.004636 0.01182 0.03754 0.004436 0.03196 0.003753 0.005465 0.005736 0.0008152 0.01133 0.02561 0.001521 0.02328 0.01859 0.01925

112

66 99 231 115 313 360 345 175 77 112 90 70

163 249 58 70 189 182 165 126 70 184 259 128 442 130 242 240 405 109 229 141 149 106 204 118

13

3 3 20 11 8 13 26 12 4 13 6 6

11 23 7 7 19 20 12 16 7 14 30 15 42 10 17 30 42 8 24 11 14 12 12 10

57

5 5 61 31 24 28 74 34 12 57 31 25

24 57 13 11 32 34 18 35 11 14 49 17 68 11 19 36 62 10 51 25 39 31 16 23

Mascot matched cover. %

Swiss-Prot

Contractil

Carrier Carrier Transport Transport Immune response Immune response Transport Antioxidant enzyme Antioxidant enzyme Contractil Antioxidant enzyme Chaperone

Structural Structural Unknown Unknown Transport Transport Unknown Unknown Unknown Transport Transport Transport Transport Transport Transport Transport Transport Inflammation Structural Inflammation Inflammation Coagulation Coagulation Chaperone

Swiss-Prot Extracellular matrix Cytoplasm Secreted Secreted Secreted Secreted Secreted Secreted Secreted Secreted Secreted Secreted Secreted Secreted Secreted Secreted Secreted Secreted Cytoplasm Secreted Secreted Secreted Secreted Cytoplasm, Endoplasmic reticulum Secreted Secreted Secreted Secreted Secreted Secreted Secreted Secreted Cytoplasm Cytoplasm Cytoplasm Cytosol, Endoplasmic reticulum Cytoplasm

Journal of Proteome Research Article


Swiss-Prot

Antioxidant enzyme Carrier Carrier Carrier Transport Transport 29 40 24 40 31 31 5 7 4 6 5 5

Extracellular space Secreted Secreted Secreted Cytoplasm, Nucleus Cytoplasm, Nucleus

IHC images were captured with an Olympus BX61 microscope and DP71 color camera. Capture settings were optimized and fixed for each validated protein and background correction and white balance were applied. For quantification of DAB staining we performed an orthonormal transformation of the RGB images by using an ImageJ plugin based on Ruifrok and Johnston’s28 method for color deconvolution. New vectors for hematoxilin and DAB separation were defined for each validated protein in order to account for subtle variations on staining properties. After separation, a threshold was set on the DAB image for each validated protein in order to eliminate background staining and the area above threshold was measured. Threshold values of each validated protein were held constant in the analysis of the images. The percentage of immunostained area was calculated as follows: (area of tissue stained with DAB/total tissue area) × 100. Statistical analysis of the data was performed with GraphPad Prism 4.0 software. Student’s t test was used to compare pathological and control tissue and differences were considered to be significant when p values were below 0.05.

MW

15.926 15.877 15.877 15.877 14.709 14.849

22 16.75 16.75 16.75 13 14.4

0.0315 0.004126 0.01304 0.00341 0.04459 0.0114

215 123 99 166 80 80

Senescence-associated β-Gal activity was examined in aortic valve tissues. Control and stenotic aortic leaflets were incubated for 24 h at 37 °C (no CO2) in freshly prepared β-gal staining solutions containing 40 mmol/L citric acid/sodium phosphate, 1 mg/mL 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-gal), 5 mmol/L potassium ferrocyanide, 5 mmol/L potassium ferricyanide, 150 mmol/L NaCl, 2 mmol/L MgCl2 adjusted to pH 6.0. After staining tissue samples were photographed. Smalls pieces of control and stenotic aortic leaflets were formalin-fixed, decalcified in Shandon-TBD1 (Thermo Scientific) and embedded in OCT for histological analysis. Sections (6 mm) were cut and fix with cold formalin (4 °C), slides were incubated in X-gal solution at 37 °C for 24 h (humidified chamber), washed in PBS and distilled water. Finally sections were counterstained and coverslipping.

5.92 5.73 5.73 5.4 6.3 6.35 5.70 5.52 5.52 5.52 6.59 6.29

■

MW, experimental molecular weight; pI Th, theoretical isoelectric point.

RESULTS We first classified the AS valves according to the results obtained from the histological hematoxilin/eosin analysis, which showed a prominent accumulation of lipid, cells and extracellular matrix in moderate lesions. Severe lesions were evident through the fragmentation of the subendothelial elastic lamina and the thickening of the fibrosa associated with the accumulation of calcium (Figure 2A). Senescence associated β-Gal activity was examined to see the differences between AS and control valve tissues. We observed an intensive β-Gal activity in AS valves which was completely absent in control valves (Figure 2B). These findings show that despite the origin of the control valves, autopsies of individuals who died of causes unrelated to cardiovascular disease, the cells remain in good condition with no sign of apoptosis or senescence. To maximize the number of proteins extracted, we have developed a method based on two sequential protein extractions.29 A strong lysis buffer is initially used in which most of the soluble proteins from human valves were extracted (E1) and the second extraction buffer was designed to recover the membrane and hydrophobic proteins (E2).18,29 This method is very reproducible for 2-DE and 2D-DIGE analysis.

a

Down Up Up Up Up Up −4.4855 2.2299 2.1627 2.2091 1.5956 1.7339

av. ratio accession code

1915 1986 1987 1993 2121 2122

SODE_HUMAN TTHY_HUMAN TTHY_HUMAN TTHY_HUMAN FABP4_HUMAN FABP4_HUMAN

protein name

Superoxide dismutase [Cu−Zn] Transthyretin Transthyretin Transthyretin Fatty acid-binding protein, adipocyte Fatty acid-binding protein, adipocyte

spot no

Table 3. continued

Article

β-Gal Activity

up/down pI theor. pI exp.

MW

p-value

Mascot matched cover. %

Swiss-Prot


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Figure 3. (A) Representative image of 2D-DIGE gel of human aortic valves. (B) Representative image of 2-DE silver stained gel of aortic valves. Protein spots present with different abundances in AS valves versus controls are highlighted. The spot numbers and protein names are shown.

2D-DIGE

conceivable that protein species can be regulated independently, generating regulatory networks more complicated than previously anticipated. It should be noted that the protein species corresponding to the same protein follow the same trend (all are increases or decreases in its abundance), (Figure 3 and Table 3 show the results of the 2D-DIGE proteomic analysis of AS versus control valves). A Principal Component Analysis (PCA) was used to reduce the complexity of the multidimensional data set and to obtain a clearer overview that may help to reveal trends within the data. In our study, this analysis efficiently discriminated the group of AS valves from the control group. The samples were separated perfectly by the first principal component (PC1). Control valves are scattered in the right part of the plot, and AS valves are located in the left area (Figure 4A). These data indicated that the alterations of the proteins and their protein species identified in this study might be suitable to distinguish between AS patients and control subjects. We also performed another PCA between patients (severe), patients (moderate) and controls with the same software. In this new analysis, we could see that the moderate and severe patients were not separated in independent groups, although both were perfectly separated from the control group (data not show). The same results were obtained in a Hierarchical analysis, whereby the groups of AS valves and control valves were perfectly separated into two clusters (Figure 4B).

We selected a group of 8 control valves and 8 AS samples to carry out the proteomic analysis by 2D-DIGE (Tables 1 and 2). Gel images were imported to the DeCyder Differential Analysis Software that detected 2156 spots per gel, of which 906 spots matched in all the gels studied. Reproducibility was tested comparing the variation within the different gels in the same group. The t test statistical analysis did not show significative differences (p value ≤ 0.05, ratio >1.5 or ratio < −1.5). Statistical analysis of the data in DeCyder software revealed changes in the abundance of 43 protein spots at p < 0.05, considering only the spots present in every internal standard: 35 up-regulated in AS, 8 down-regulated in AS. FDR analysis demonstrated that the majority of them showed the lowest calculated q-values (Table S4 Supporting Information). These 43 protein spots were excised from silver stained gels, digested with trypsin and analyzed by MALDI-TOF/TOF. The ensuing database search of the 43 protein spots identified all of them. In the 2D-DIGE pattern, it can be observed that a single protein is represented by several spots which represent different protein species and each of these different protein species of the same protein are altered identically (up or down) in every case. Thus, the 5 protein species of Alpha-1B-glycoprotein are up-regulated, and the same happens with the 3 protein species of Transthyretin (Table 3), etc. However, quantitatively there are remarkable differences. For example, two different protein species of α1-antitrypsin (spot 762 and spot 746 (Table 3)) have very different average ratios, 1.72 and 2.96, respectively. Likewise, different protein species of apolipoprotein A-I (spot 1687 and spot 1691) are both upregulated but they have average ratios of 2.51 and 1.6, respectively. These data indicate a differential regulation of each protein species. Thus, it is

Functional Classification of the Proteins Differentially Expressed

For each of the 43 protein spots identified, the theoretical molecular mass (MW) and isoelectric point (pI) were recovered, together with the accession number, sequence coverage and score; 1544



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Figure 4. (A) Principal Component Analysis of the samples studied. The first principle component (PC1) perfectly separated both study groups. Ellipses surrounding related samples are displayed only to emphasize the distribution of each group in the plot. (B) Hierarchical analysis also separates both groups and the altered spots as up or downregulated. (C) Protein spots as key for the separation between patients and controls. Aortic stenosis patients: apolipoprotein AI (spot 1702 and spot 1687), serum amyloid P (spot 1693), transthyretin (spot 1987) and alpha-1-antitrypsin (spot 746) Control group: haptoglobin (spots 1383 and 1339), transgelin (spot 1737) and glutathione peroxidase 3 (spot 1749).

p = 0.049; p = 0.0045, p = 0.024; p = 0.0163, respectively: (Figure 5A). By contrast, the amount of the Hsp 27 (27 kDa) appeared to diminish in AS valves when analyzed in Western blots (p = 0.03; Figure 5A). Three proteins more were also analyzed in 2D-Western blots. In Figure 5B, 2D Western blots of three proteins, glutathione peroxidase 3, lumican and transgelin are shown. These results confirmed those obtained in the proteomic analysis with the DeCyder Differential Analysis Software.

the main function of the identified proteins are also included (Table 3). Accordingly, we classified these proteins into six different groups based on their function (Uniprot database): (a) Antioxidant enzymes: Superoxide dismutase (Cu−Zn), Glutathione S-transferase P, Glutathione peroxidase 3, haptoglobin. (b) Structural and contractile: Lumican, Transgelin, Vimentin. (c) Inflammation: Serum amyloid P-component, Alpha-1antitrypsin, Fibrinogen gamma chain. (d) Transport: Transthyretin, Apoliprotein A1, Serum albumin Fatty acid binding protein. (e) Chaperone: HSP27, Calreticulin. (f) Other: Alpha-1B-glycoprotein.

Immunostaining Analysis

Immunohistochemical studies were also performed using different antibodies against these specific proteins to determine their distribution in frozen sections of AS and control valves (Figures 6 and 7). The results revealed that the abundance of HSP27 is decreased in the endothelium layer of AS valves when compared to control valves (p = 0.0126). Cells expressing vimentin were found in the spongiosa and ventricularis layers where this protein was more strongly expressed in AS valves than in control valves (p = 0.0016). By contrast, apolipoprotein AI is more strongly stained in the endothelium and in the fibrosa layer of AS valves than in control valves where this protein appeared expressed in endothelium exclusively (p = 0.038). Cells expressing serum amyloid P-component, Alpha-1B-glycoprotein, alpha 1 antitrypsin (Figure S4, B Supporting Information), fibrinogen gamma (Figure S4, E Supporting Information) and fatty

Western Blot Analysis

To confirm the proteomic results, a group of 9 proteins was analyzed in 1D-Western blots. Western blots were probed with antibodies against: apolipoprotein AI (25.3 kDa), P-component (25 kDa), fatty acid-binding protein adipocyte (14.8 kDa), alpha-1B-glycoprotein (51.9 kDa), alpha 1 antitrypsin (46.07 kDa), superoxide dismutase 1 (Cu−Zn (25.8 kDa), vimentin (53.6 kDa) and fibrinogen gamma (51.4 kDa). All these proteins were more abundant in the AS valves than in the control valves (p = 0.0003; p = 0.0017; p = 0.04; p = 0.0039; 1545



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Figure 5. Verification of the differences observed by 2D-DIGE in Western blots. (A) 1-D SDS-PAGE Validation; on the left, a band of HSP27, decreased in AS patients vs control subjects. Apo AI Western blot detected of a band overexpressed in AS patients with respect to control subjects. The same result was obtained for the rest of the validated proteins that were compared in AS and control subjects: Serum Amyloid P-component, Superoxide Dismutase-1 and FABP4. On the right the same results were confirmed in 1-D Western blots. A band corresponding with Vimentin was augmented in AS vs Control subjects. The anti human Alpha-1-antitrypsin antibody detected a band in AS patients that were less abundant in control subjects. A band corresponding to Fibrinogen was elevated in AS patients with respect to control subjects. Finally, an alpha 1 glycoprotein band was overexpressed in AS patients. (B) In the bottom of the panel, 2-D Western blot show differences in the expression of Lumican. A slight difference was seen in several protein species in 2-D Western blots (arrows in the figure), and the p value after densitometry is shown.

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acid-binding protein adipocyte were found in the endothelium and fibrosa layer. These proteins were all more strongly stained in AS valves than in control valves (p = 0.0051; p = 0.0087; p = 0.05; p = 0.001; p = 0.0001, respectively; Figure 6). Thus, the results obtained by immunohistochemistry confirmed the 2D-DIGE results. With this analysis we have obtained the tissue location of the altered proteins (Figure 7).

DISCUSSION

To the best of our knowledge, this is the first study to compare the proteome of degenerative AS tissue with normal heart valve tissue. The analysis carried out here reveals the altered abundance of 35 protein species in AS valves (apoliprotein AI, alpha-1-antitrypsin, serum albumin, lumican, alpha-11546



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Figure 6. Immunohistochemical verification of the differences observed with 2D-DIGE. We detect the same result on the tissue as in Western blots. Hsp 27 was more weakly expressed in AS valves than in control valves. The rest of the proteins (Serum amyloid P-component, Apo AI, FABP4, vimentin, alpha 1B glycoprotein) were elevated in AS patients vs control subjects. Arrows indicate the layers where these proteins are over stained.

disease. Indeed, the development of new biomarkers and therapies will require a clear comprehension of the pathogenic mechanisms of disease onset and progression. A total of 2156 spots covering a broad range of molecular mass (10−120 kDa) were analyzed and 43 of them showed an altered abundance in valves from degenerative AS patients when compared with those from control individuals. The majority of the altered proteins could be classified in functional groups directly associated with cardiovascular pathology: inflammation, transport, structural. The use of fluorescent labeling and the DeCyder analysis software considerably increases the reliability in the quantitative measurement of AS differential analysis compared to traditional methods.29 The proteins considered to be involved in inflammation and the immune response (SAP, Alpha-1-Antitrypsin) are thought to participate in atherosclerosis lesions30,31 and inflammation.31,32 Fibrinogen is a protein directly implicated in the coagulation cascade and it has been shown to be implicated in ischemic cardiopathy and ischemic recurrence.33−35 The abundance of these 3 proteins follows a similar trend to that demonstrated in previous studies, highlighting the inflammatory elements of AS, such as subendothelial thickening, disruption of the basement membrane, accumulation of intraextracellular lipids, and calcium and cellular

glycoprotein, vimentin, superoxide dismutase (Cu−Zn), serum amyloid P-component, glutathione S-transferase-P, fatty acidbinding protein, transthyretin and fibrinogen gamma) and the decreased abundance of another 8 protein species in AS valves (transgelin, haptoglobin, glutathione peroxidase 3, HSP27, calreticulin). All these proteins play a significant role in important biological processes in the cardiovascular system, such a fibrosis, coagulation, inflammation. Therefore, our data pave the way for a better understanding of AS pathophysiology and degenerative process. Some of the proteins identified in this work appeared in plasma too, but it is important to note that all of them were validated by IHC and these, are distributed along the different layers (endothelium, elastic, fibrosa and ventricularis) which exclude the possibility of plasma contamination. In recent years, great efforts have been made to understand the physiological and pathological mechanisms implicated in degenerative AS. Calcification of the native aortic cups is a complex process, and it appears to be related to inflammation and ossification.6 Nevertheless, the mechanism underlying degenerative valve heart disease still remains largely unclear, especially when compared with our understanding of the mechanisms underlying heart failure and atherosclerotic 1547



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Figure 7. Schematic drawing of the valvular aortic stenosis lesion. The lesion is characterized by the displacement of subendothelial elastic lamina associated with the accumulation of lipids, macrophages, T-lymphocytes, proteins and calcium. There is an important accumulation of mineral calcium in the fibrosa layer. On the right, we indicate the localization of the altered proteins validated by IHC comparing patients vs control subjects.

infiltration with macrophages.6 This finding supports the idea that AS is an inflammatory disease, possibly with similar origin as atherosclerosis. All the transport proteins found in this study are up-regulated (apoliprotein AI, transthyretin, serum albumin), which suggests they may be involved in protecting the aortic valve tissue from the damage36 caused by an increase of LDL-cholesterol, lipoprotein (a)37 and triglycerides.38−44 This damage tends to be localized to the aortic surface of the leaflets that most frequently suffer from more mechanical strain and less shear stress. With respect to the structural proteins, the proteomics analysis demonstrated an increase in Lumican and Vimentin levels, and a decrease in Transgelin. Based on these data, we might consider that the cells undergo structural remodeling, both in terms of the intermediate cytoskeletal filaments and the extracellular matrix.45,46 Furthermore, Lumican and Vimentin are implicated in macrophage adhesion in cellular inflammation47,48 and in atherosclerosis progression, as well as in other cardiovascular diseases. The information obtained in this analysis is concordant with the data reported previously,47,48 suggesting that these proteins are involved in inflammation, which again support our initial theory, and the immune response. It is thought that oxidation of low density lipoprotein in the blood contributes to heart disease. The enhancement of glutathione S-transferase and superoxide dismutase in AS opens

the possibility of a protective role in this pathology, due to the fact that both proteins modulate oxidative stress and increase cell resistance to oxidative vascular injury.49 These proteins are also involved in the detoxification of harmful substances.50 Considering the central role of the carrier and chaperone proteins in the cell and body fluids, we found several proteins of this class deregulated in AS and control valves. Each carrier protein is designed to recognize only one substance or one group of very similar substances, and carry it (or them) in the blood or across cell membranes. Our results revealed a decreased HSP27, haptoglobin and Calreticulin. The implication of these proteins in several cardiovascular diseases is well established51 and it has been suggested that they could also play a key role in the development of AS. Additional information and possible implications of each of the altered protein species in AS is discussed in Supporting Information. These data support similarity of AS with atherosclerotic process based in changes produced in the abundance of several proteins (HSP27, lumican, alpha 1 antitrypsin, glutathione S transferase, superoxide dismutase, transthyretin) that have been described previously as protein variations in proteomic studies of atherosclerosis.47 1548



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CONCLUSIONS We have shown here for the first time that the abundance of 43 protein species is altered in degenerative AS valves in comparison with control valves. Some of them have been suggested to play an important role in the pathophysiology of heart disease and atherosclerosis. Therefore, this protein profile, or any of the individual protein species, could serve as a focal point for future mechanistic studies. In addition, some of the protein species associated with this disorder may be candidate prognostic biomarkers for clinical trials.

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

Supplemental tables and figures. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Dr. Fernando Vivanco, Department of Immunology, ISSFundacion Jimenez Diaz, Madrid, Avda Reyes Católicos 2, 28040 Madrid, Spain. E-mail: [email protected]. Telephone: 0034 915498446. Author Contributions #

These authors contributed equally to this work

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by grants from the Instituto de Salud Carlos III (FIS PI070537, PI080970), Fondo de Investigación Sanitaria de Castilla la Mancha (FISCAM, PI2008/08), Fondo de Investigación Sanitaria de Castilla la Mancha (FISCAM PI2008/28) and Fondos Feder-Redes Temáticas de Investigación Cooperativa (RD06/0014/1015). We thank the Proteomics Unit HNP for assistance with the protein identification and, and the Microscopy Unit HNP for assistance with the image analysis. We would also like to thank Gloria Alvarez-Llamas and Fernando de la Cuesta for their helpful suggestions with the proteomic analysis, and Carmen Bermudez for her technical support to this work.

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ABBREVIATIONS 2-DE, two-dimensional electrophoresis; 2D-DIGE, two dimensional fluorescence difference gel electrophoresis; ACN, acetonitrile; AS, aortic stenosis; β-Gal, beta-galactosidase; BSA, Bovine serum albumin; BVA, biological variation analysis; COPD, chronic obstructive pulmonary disease; DAB, 3,3′diaminobenzidine; DIA, differential in-gel analysis; EDA, extended data analysis; FA, formic acid; FDR, false discovery rate; HRP, horseradish peroxidase; HTN, arterial hypertension; HSP, heat shock protein; IHC, immunohistochemistry; LDL, low density lipoprotein; Lp(a), lipoprotein a; MALDI, matrixassisted laser desorption/ionization; MS/MS, tandem mass spectrometry; MS, mass spectrometry; OCT, optimal cutting temperature; PBS, phosphate buffer saline; PCA, Principal Component Analysis; PTM, photomultiplier, also post-translational modification; RGB, red, green, blue; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; TFA, trifluoroacetic acid; TOF, time-of-flight; WB, Western blot; X-Gal, 5-bromo-4-chloro-3indolylbeta-D-galactopyranoside 1549



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Proteomic Profile of Human Aortic Stenosis - American Chemical Society

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