Use of Proteomics to Discover Novel Markers of Cardiac Allograft Rejection Svetlana Borozdenkova,† Jules A. Westbrook,‡ Vaksha Patel,‡ Robin Wait,§ Islam Bolad,† Margaret M. Burke,| Alexander D. Bell,| Nicholas R. Banner,† Michael J. Dunn,*,⊥ and Marlene L. Rose† National Heart and Lung Institute, Imperial College School of Medicine, Heart Science Centre, Harefield Hospital, Harefield, United Kingdom, Proteome Sciences plc, Institute of Psychiatry, Kings College, London, United Kingdom, Kennedy Institute of Rheumatology Division, Imperial College School of Medicine, London, United Kingdom, Department of Pathology, Harefield Hospital, Harefield, United Kingdom, and Department of Neuroscience, Institute of Psychiatry, Kings College, London, United Kingdom Received August 5, 2003
Endomyocardial biopsy remains the most reliable method of detecting rejection following cardiac transplantation. Despite numerous attempts to detect rejection using a blood assay, none have proved reliable enough to replace the biopsy. Here, we have investigated the hypothesis that proteomics has the potential to reveal many molecules which are upregulated in the heart during rejection, some of which may serve as novel blood markers of rejection. Initially, sequential cardiac biopsies (33 in total) from 4 patients were analysed by two-dimensional gel electrophoresis according to whether they showed rejection (n ) 16) or no rejection (n ) 17); over 100 proteins were found to be upregulated by between 2- and 50-fold during rejection. Of these, 13 were identified and were found to be cardiac specific or heat shock proteins. Two of these (RB-crystallin, tropomyosin) were measured by ELISA in the sera of 17 patients followed for 3 months after their transplants. Mean levels of RB-crystallin and tropomyosin were significantly higher in sera associated with biopsies showing 1A (p ) 0.007) or all grades of rejection (p ) 0.022) compared to no rejection. These studies demonstrate that proteomics is a powerful method that can be used to identify novel serum markers of human cardiac allograft rejection. Keywords: heart • transplantation • transplant rejection • rejection • allograft • markers of rejection
1. Introduction Routine histological analysis of endomyocardial biopsies (EMB) remains the gold standard for the detection of acute rejection following cardiac transplantation.1 At our institution, routine surveillance EMBs are taken at frequent intervals, such that every patient has a minimum of 12 biopsies in the first six months after transplantation. Additional biopsies are taken when clinically indicated. A positive biopsy is followed by a repeat biopsy one to two weeks later to ensure that antirejection therapy has been successful. The biopsy procedure is invasive, is potentially unpleasant for the patient, and is associated with a small risk of complications; it is also laborintensive and costly. Therefore, it would be of considerable * To whom correspondence should be addressed. Michael J. Dunn, Professor of Proteomics, Department of Neuroscience, Box P045, Institute of Psychiatry, De Crespigny Park, London SE5 8AF United Kingdom. Tel: +44-207-848-5110. Fax: +44-207-848-5109. E-mail:
[email protected]. † National Heart and Lung Institute, Imperial College School of Medicine, Heart Science Centre, Harefield Hospital. ‡ Proteome Sciences plc, Institute of Psychiatry, Kings College. § Kennedy Institute of Rheumatology Division, Imperial College School of Medicine. | Department of Pathology, Harefield Hospital. ⊥ Department of Neuroscience, Institute of Psychiatry, Kings College.
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benefit to have a reliable noninvasive method to detect acute rejection. In theory, there are a number of ways of noninvasively monitoring heart function such as magnetic resonance imaging,2 signal averaged electrocardiogram,3 and specialized echocardiographic indices,4-6 but none of these has replaced the cardiac biopsy as the gold standard. A peripheral blood test would be an ideal test in terms of simplicity and cost. There are two approaches; one is to exploit what is known about immune activation, and the second is to look for markers of graft damage. There have been numerous attempts to find signs of immune activation in peripheral blood. These have included measuring blood levels of IL-2,7,8 soluble IL-2R,7,9 IL-6,9,10 IL7,11 IL-8,10 TNFR,7,8 IFNγ,7 soluble ICAM-1,12 soluble Major Histocompatibility Complex antigens, activated T cells,13 T cell populations, and cytoimmunological monitoring.14-17 There have also been attempts to look for markers of cardiac damage in the blood, the most notable of which was release of troponin T.18 None of these methods have proved reliable enough to replace the cardiac biopsy procedure. Here, we have used a proteomic approach to ask which proteins are upregulated in 10.1021/pr034059r CCC: $27.50
2004 American Chemical Society
Proteomics and Markers of Rejection
the endomyocardial biopsy during acute rejection. The advantage of proteomics is that is displays thousands of proteins at once, computer analysis can be used to identify those proteins that are differentially upregulated during acute rejection, and the proteins of interest can be identified by mass spectrometry. Unlike the hypothesis driven approach, which looks for known molecules, proteomics reveals associations between molecules and rejection, which have not previously been described. Proteomics thus has the potential to uncover many novel markers of disease processes such as cardiac rejection. Indeed, a recent paper has used a proteomics approach based on mass spectral fingerprinting of serum to identify cardiac peptides associated with myocardial infarction.35
2. Materials and Methods 2.1 Patients. Between 1998 and 2000, 17 adult cardiac transplant patients at Harefield Hospital were prospectively recruited into the study. Twelve patients were male, five were female, and their ages ranged from 35 to 55 years. Explant diagnoses were dilated cardiomyopathy (n ) 11), ischaemic heart disease (n ) 2), hypertrophic cardiomyopathy (n ) 1), congenital heart disease (n ) 2), giant cell myocarditis (n ) 1). Each patient underwent routine endomyocardial biopsy at weekly intervals to 6 weeks, two weekly intervals to 3 months, and monthly until 6 months. Additional biopsies were taken if acute rejection was suspected clinically. During each biopsy procedure, a minimum of 4 endomyocardial samples were obtained for routine histological analysis (n ) 19). Extra endomyocardial samples were collected during each biopsy procedure for this study during the first 3 months, representing a total of 129 biopsies. Serum (129 samples) was taken at the time of each biopsy procedure and frozen at -20 °C until use, and in addition pre-transplant serum (17 samples) were stored. Local ethical permission (Hillingdon Health Authority, UK) was obtained in accordance with the Helsinki Declaration of 1975 and written informed consent was obtained from each patient. 2.2 Histological Diagnosis of Acute Allograft Rejection. A total of 129 endomyocardial biopsies were reviewed. All had been routinely processed to paraffin wax, ribbons of sections 5-mm thick cut, and selected ribbons stained with haematoxylin-eosin. All biopsies were reviewed independently by two histopathologists (ADB, MMB) who were blinded to the results of the serum analysis. The changes of acute rejection were graded according to the Working Formulation of the International Society for Heart and Lung Transplantation.19 For the purpose of this study, acute rejection was defined by histological and not clinical criteria. Thus, biopsies were said to be showing rejection if they achieved a grade of 1A or 1B (mild acute rejection) on two consecutive occasions within a 2 week period, or single episodes of grades 2, 3A, 3B, or 4. 2.3 Identification of Heart Proteins using Proteomics. 2.3.1 Radiolabeling of Biopsy Samples. Biopsies were radiolabeled on the day of collection with [35S]-methionine (Redivue, Amersham Biosciences) to reveal those proteins that were newly synthesized. Each biopsy was placed in the well of a standard 96-well plate and incubated for 20 h at 37 °C in 100 µL of methionine-free Hanks Balanced Salt Solution (HBSS) containing 225 mCi [35S]-methionine. Empty wells surrounding the biopsies were filled with HBSS to prevent drying-out during the incubation period. Following incubation, each biopsy was rapidly rinsed in 0.35 M sucrose to minimize contamination with salts that interfere with isoelectric focusing (IEF). The radiolabeled biopsies were stored frozen at -70 °C (between 1
research articles and 5 days) until processed for two-dimensional polyacrylamide gel electrophoresis (2-DE). 2.3.2 Preparation of Radiolabeled Proteins. The biopsy samples were crushed while still frozen between two cooled metal blocks. The resulting powder was collected into 1.5 mL microcentrifuge tubes, then resuspended and solubilized in 200 µL of lysis buffer containing 9.5 M urea (GibCo), 1% DTT (Sigma), 2% CHAPS (Calbiochem), and 0.8% Pharmalyte pH 3-10 (Amersham Biosciences, Amersham, UK). Samples were centrifuged in an micro-centrifuge and the resulting supernatants collected. The solubilized protein samples were assayed for radioactivity by scintillation counting. Briefly, radiolabeled proteins were precipitated with 10% TCA and the addition of 1 mg/mL BSA as a “seeding” for precipitation for 1 h on ice. The resulting protein precipitates were collected under vacuum on glass fiber filters, washed three times with ice-cold 10% TCA, once with ice-cold acetone, and then allowed to air-dry. The filters were then placed in tubes containing scintillant (Packard) and the radioactivity measured using a scintillation counter (Packard Tri-Carb). 2.3.3 Two-Dimensional Gel Electrophoresis (2-DE). Firstdimension isoelectric focusing (IEF) was performed using 18 cm immobilized pH gradient (IPG) strips (Amersham Biosciences), with pH ranges 4-7 L (linear) and 3-10 NL (nonlinear), using an in-gel rehydration method.20 Biopsy protein samples, containing between 2.5 × 105 and 106 cpm (typically between 20 and 50 mg protein) were diluted to a total volume of 450 µL with rehydration solution containing 8M urea, 0.5% CHAPS, 0.2% DTT, and 0.2% Pharmalyte pH 3-10 prior to rehydration overnight in a reswelling tray (Amersham Biosciences). The strips were focused at 0.05 mA/IPG strip for 60 kVh at 20°C. After IEF, the strips were equilibrated in 1.5M Tris pH 8.8 buffer containing 6 M urea, 30% glycerol, 2% SDS and 0.01% Bromophenol blue, with the addition of 1% DTT for 15 min, followed by the same buffer with the addition of 4.8% iodoacetamide for 15 min. Second-dimension SDS-PAGE was performed using 22 cm 12% T, 2.6% C separating polyacrylamide gels without a stacking gel using a Hoefer DALT system. The second-dimension separation was carried out overnight at 20 mA/gel at 10°C and was stopped as the Bromophenol blue dye front reached the bottom of the gels. 2.3.4 Protein Visualization, Densitometry and Computer Analysis. On completion of electrophoresis, 2-D gels were fixed overnight in 50% methanol, 10% acetic acid and then soaked for 24 h in a solution containing 3% glycerol, 30% methanol that renders gels amenable to drying without cracking. Gels were dried down under vacuum onto 3 MM paper (Whatman) using a Savant SpeedGel SG210D gel dryer for 1 h at 62°C, followed by a further 1 h at 66°C. Dried 2-D gels were then exposed to BioMax-MR autoradiography film (Kodak) for between 6 and 12 days at room temperature. The developed films were scanned at 100 µm resolution using a Molecular Dynamics Personal Densitometer SI (Amersham Biosciences). Autoradiographic images were categorized according to the IPG pH range and EMB-determined rejection status for each patient and analysed using PDQuest v6.1 software (Bio-Rad). Briefly, spots were detected by a semi-automated method and then carefully checked in order to remove artifacts. Protein spots were then ‘landmarked’ between the individual 2-D gel profiles in order to match the images together. Comparative analysis was performed for sequential 2-D protein profiles for each patient, and images derived from biopsies collected at a time when no rejection was diagnosed were grouped and compared Journal of Proteome Research • Vol. 3, No. 2, 2004 283
research articles to images derived from biopsies collected at the time when rejection was diagnosed (regardless of grade of rejection). The results of the nonrejecting group of patients were used as the baseline with which to compare proteins which showed either increased or decreased radioactivity in the rejection group. 2.3.5 Protein Identification. For micro-preparative 2-DE, aliquots of nonradiolabeled heart protein sample containing a total of 400 µg total protein were added to reswelling solution and used to rehydrate 3-10 NL and 4-7 L IPGs in the same way as described above (see Section 2.3.3). The resulting 2-D gels were stained using a modified version of the PlusOne silver staining kit (Amersham Biosciences) that is compatible with mass spectrometry.21 Proteins were excised from the gels, digested with trypsin and identified by peptide mass profiling using MALDI-TOF mass spectrometry (TofSpec 2E, Micromass, Manchester, UK) essentially as described in ref 21. The resulting peptide mass fingerprints were searched against a local copy of the NCBI nonredundant database (http:// www3.ncbi.nlm.nih.gov) using the Protein Probe search engine (Micromass). Where necessary, additional searches were performed using the programs Mascot or ProFound. One missed cleavage per peptide was allowed, and an initial mass tolerance of 50 ppm was used in all searches. Cysteines were assumed to be carbamidomethylated, but other potential modifications were not considered in the first pass search. 2.4 Enzyme-Linked Immunoassays. Immunoassay for RBcrystallin and tropomyosin (TM-1) was done by competitive enzyme linked immunoassay (ELISA), using an in house method. Briefly, 96-well microtiter plates were coated for 2 h at 37 °C with purified antigen either TM-1 (from Sigma) at 2.5µg /mL or recombinant RB-crystallin (from Clinical Chemistry Laboratory, R&D, University Geneva Hospital) at 2.5µg/mL. After being coated with protein, the plates were blocked with 5% dried milk in PBS. This was followed by addition of patients’ samples (diluted 1:3 in PBS) or protein standards in the presence of antibody (rabbit anti-crystallin diluted 1/100 in PBS, from BabCo, Richmond CA, USA or rabbit anti-tropomyosin diluted 1/40 000 in PBS from Cymbus Biotechnology, Hampshire) for 1 h. The wells were washed and the amount of antibody bound to the plate was quantitated by addition of horseradish peroxidase (HRP) labeled secondary goat antirabbit Ig (1/1000, from Dako) for 1 h. After washing again, the substrate buffer was added (Lumiglow from Amersham) and the luminescent signal measured by MicroBeta Trilux (Wallac, Finland). Myosin light chain (MLC-1) was detected by sandwich ELISA. Briefly, the plates were coated with monoclonal mouse antibody to MLC-1 (diluted 1/5,000 in PBS, from BioSpecific, Emeryville, CA) and blocked by 5% milk. Patients’ serum or standards (MLC-1 from BioSpecific) were added for 1 h, plates were washed and polyclonal goat anti-MLC-1 antibody (at 1/400 in PBS, from BioSpecific) was added for 1 h. After washing again, HRP labeled swine anti-goat Ig was added and allowed to incubate for 1 h. Substrate (Lumiglow from Amersham) was added, and the reaction was read by MicroBeta Trilux, (Wallac). The results were expressed as ng/mL, mean ( SD. 2.5 Statistics. Differences between means of protein concentrations from ELISAs were analyzed using SPSS for Windows, independent samples ‘t’ test.
3. Results 3.1 Identification of Proteins Associated with Acute Rejection. Using the experimental approach described above, it was 284
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Figure 1. Autoradiographic image of [35S]-methionine labeled cardiac proteins separated by 2-DE using a pH 3-10 NL IPG in the first dimension. The scale at the left indicates protein Mr × 10-3.
Figure 2. Autoradiographic image of [35S]-methionine labeled cardiac proteins separated by 2-DE using a pH 4-7 L IPG in the first dimension. The scale at the left indicates protein Mr × 10-3.
possible to obtain reproducible 2-D gel profiles of radiolabeled heart biopsy proteins using both pH 3-10 NL (Figure 1) and pH 4-7 (Figure 2) IPG strips. As described in Section 2.3.4, to minimize problems of inter-individual heterogeneity, it was decided to analyze sequential biopsies from individual patients. By grouping 2-D gel profiles into “no-rejection” and “rejection” groups it was then possible by quantitative computer analysis
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Proteomics and Markers of Rejection Table 1. Clinical Characteristics of Patients Analysed by Quantitative Computer Analysisa
Table 2. Quantitative Analysis of Protein Changes Detected in 2-D Gels Using pH 3-10 NL IPG
HLA mismatch data pt. no.
ind. tx.
A
B
DR
DQ
PRA
Lym-XM
blood grp.
pt. no.
no. bx (NR/R)a
1 2 3 4
IHD IHD DCM VDCM
1 1 2 2
2 2 2 1
2 2 1 1
2 1 1 1
ND NEG NEG NEG
NEG ND NEG NEG
O+ B+ A+ A-
1 2 3 4
7 (3/4) 6 (2/4) 10 (7/3) 10 (5/5)
a HLA ) Human Leukocyte Antigen; pt. no. ) patient number; ind. tx. ) indication for transplantation; DCM ) dilated cardiomyopathy; IHD ) ischaemic heart disease; VDCM ) viral DCM; PRA ) panel reactive antibody test; Lym. XM ) lymphocytic crossmatch test; ND ) not done.
increased
decreased
2-fld
3-fld
4-fld
5-fld
2-fld
3-fld
4-fld
5-fld
143 112 84 180
69 87 35 94
39 42 15 61
19 24 10 47
183 104 184 222
98 34 85 113
56 15 46 75
35 12 26 52
a No. bx. (NR/R) refers to the number of biopsy gel images of no-rejection (NR) and rejection (R, all grades) conditions that were used for the comparative analysis.
Table 3. Quantitative Changes Detected in 2-D Gels Using pH 4-7 L IPG increased
decreased
pt. no.
no. bx (NR/R)a
2-fld
3-fld
4-fld
5-fld
2-fld
3-fld
4-fld
5-fld
1 2 3 4
7 (2/5) 4 (1/3) 8 (5/3) 9 (5/4)
101 81 106 122
30 26 47 46
20 11 24 21
11 4 14 11
79 61 182 132
20 22 97 62
11 14 55 35
8 9 39 21
a No. bx. (NR/R) refers to the number of biopsy gel images of no-rejection (NR) and rejection (R, all grades) conditions that were used for the comparative analysis.
Figure 3. Autoradiographic images of [35S]-methionine labeled cardiac proteins separated by 2-DE using pH 3-10 NL IPG in the first dimension. The series of 2-D gels are of sequential biopsies taken from a patient at 26 (A), 34 (B), 39 (C), 53 (D), and 75 (E) days post-transplantation.
to look for differences in the radiolabeling (“synthesis”) of proteins associated with rejection status. This detailed quantitative analysis was done on 33 biopsies obtained from 4 transplant patients whose clinical data is shown in Table 1. An example of 2-D gels of five sequential biopsies from one patient to illustrate examples of the types of change that can be observed is shown in Figure 3. Here, it can be seen that protein spots X, Y, and Z are barely detectable on 2-D gel profiles at days 26, 34, and 39 following transplantation (Figure 3A-C), but their synthesis is upregulated subsequently so that they are detected at much higher abundance at days 53 and 76 (Figure 3 D,E). The quantitative data for the 2-D gel analysis for these four patients using pH 3-10 NL and pH 4-7 L gradients is shown in Tables 2 and 3, respectively.
At the 2-fold level, it can be seen that many proteins displayed apparently altered levels of synthesis in association with rejection. For example, for patient 1, more than 100 proteins showed increased and decreased levels on both types of 2-D gels used (Tables, 1, 2). Not unexpectedly, the number of proteins showing increased or decreased levels of synthesis was significantly reduced using more stringent fold-change criteria. Thus, at the 5-fold level, in patient 1, there were 19 proteins increased and 35 proteins decreased 5-fold using pH 3-10 NL 2-DE (Table 2), and 11 proteins increased and 8 proteins decreased 5-fold using pH 4-7 L 2-DE (Table 3). An initial panel of 13 proteins showing a 2-5-fold change in response to rejection in all four patients were selected for identification from the pH 3-10 NL 2-D gel profiles. These proteins are indicated in Figure 4 and their identities as identified using peptide mass profiling by MALDI-TOF MS are detailed in Table 4. It can be seen that 11/13 proteins were identified; 8 are cardiac specific proteins, 2 are stress proteins (hsp27 and RB-crystallin) and 1 was an enzyme, peroxisomal enoyl-CoA hydratase. All of the proteins except myosin light chain-1 (MLC-1) were upregulated during rejection; MLC-1 was decreased during rejection. Three proteins, the heart specific proteins tropomysion-1 (TM-1) and MLC-1 and the stress protein RB-crystallin were selected for further study. 3.2 Sequential Enzyme Linked Immunoassays. ELISAs were used to determine the concentrations of RB-crystallin, TM-1, and MLC-1 in pre-transplant serum and serum taken at the same time as biopsies. Sera from 17 patients were analyzed, these 17 included the 4 patients whose biopsies had been analyzed in great detail (as above). The sensitivities of the assays were 6, 8, and 1 ng/mL for RB-Crystallin, TM-1 and MLC-1, respectively. Of the 129 sets of biopsies collected in the first three months following transplantation, 5 were excluded for analysis (1 was confirmed as recurrent giant cell myocarditis, one was cytomegalovirus (CMV) induced myocarditis, and 3 were ‘inadequate’ due to scarring), leaving 124 paired serum and myocardial samples available for analysis. Of these, 77 biopsies showed no rejection, 36 showed grade Journal of Proteome Research • Vol. 3, No. 2, 2004 285
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Table 4. Identities of Potential Rejection Marker Proteins spot no.
1 2 3 4 5 6 7 8 9 10 11 12 13
protein identity
SWISS-PROT name
SWISS-PROT accession no.
myosin regulatory light chain 2, ventricular/cardiac muscle isoform tropomyosin 1 R chain (fragment) troponin C, slow skeletal and cardiac muscles actin, R cardiac actin, R cardiac (fragment) heat shock 27 kDa protein myoglobin peroxisomal enoyl-CoA hydratase unidentified unidentified tropomyosin 1 R chain R-crystallin B chain myosin light chain 1
MLRV_HUMAN
P10916
TPM1_HUMAN TPCC_HUMAN ACTC_HUMAN ACTC_HUMAN HS27_HUMAN MYG_HUMAN ECHP_HUMAN
P09493 P02590 P04270 P04270 P04792 P02144 Q08426
TPM1_HUMAN CRAB_HUMAN MLE1_HUMAN
P09493 P02511 P05976
or post-transplant serum samples. We did not compare nonrejection with grades 2, 3, and 4, because of low numbers of these grades (n ) 8). Interestingly, a single biopsy taken at the same time the patient was confirmed as having CMV myocarditis disease (diagnosed by CMV inclusion bodies on the biopsy) was associated with a high level of RB-crystallin (1000 ng/mL) but not TM-1 (
