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Clinical and Translational Research

Proteomic Profiling of Acute Cardiac Allograft Rejection

Kienzl, Katrin1; Sarg, Bettina2; Golderer, Georg3; Obrist, Peter4; Werner, Ernst R.3; Werner-Felmayer, Gabriele3; Lindner, Herbert2; Maglione, Manuel1; Schneeberger, Stefan1; Margreiter, Raimund1; Brandacher, Gerald1,5

Author Information
doi: 10.1097/TP.0b013e3181b119b1

Abstract

Prevention of allograft rejection continues to be a major challenge in solid organ transplantation, even though improvements in pharmacologic therapy have produced outstanding short-term patient and graft survival rates. Thus, in heart transplantation, current immunosuppressive drugs give a 1-year graft survival rate of 85% (1). However, these excellent short-term graft survival rates have not been accompanied by improved long-term outcomes because the 5-year graft survival rate still ranges between 60% and 70% (1). The major reason for such impaired long-term results is chronic allograft dysfunction, referred to as cardiac allograft vasculopathy in cardiac transplantation. In addition to several nonimmunologic factors including ischemia-reperfusion injury, drug toxicity, and donor and recipient characteristics, the development of chronic allograft dysfunction is determined mainly by the number of acute rejection episodes, their severity, timing of occurrence, and histopathologic expression pattern (2).

Therefore, the diagnosis of acute cardiac allograft rejection before the onset of clinical symptoms and the manifestation of histopathologic changes is of crucial importance in optimizing immunosuppressive therapy and hence to prevent progression to chronic allograft dysfunction. To date, endomyocardial biopsy remains the most sensitive and specific means of reliably diagnosing acute rejection. Although endomyocardial biopsy is usually considered a safe procedure, it is associated with significant morbidity, especially when performed as a routine surveillance biopsy at frequent intervals in the first year posttransplant. Potential complications include arterial puncture, vasovagal reactions and prolonged bleeding during catheter insertion, arrhythmias and conduction abnormalities, pneumothorax and intracardial fistula or biopsy-induced tricuspid regurgitation, and even perforation of the heart (3–5). Hence, a noninvasive biomarker for acute rejection that allows frequent immunologic monitoring of the graft would be of considerable value.

As allograft rejection is a complex immunologic process that remains to be fully elucidated, successful characterization of biomarkers is entirely dependent on global expression analysis. Intragraft gene expression profiles using quantitative transcriptional profiling by means of gene chip arrays have been shown to be a reliable tool for evaluating the alloimmune response both in animal transplant models and in humans (6, 7). Recently, proteomic technology has emerged as a powerful means of protein expression profiling, enabling insights into posttranscriptional regulation (8–12). In addition, data obtained from such studies lay the groundwork and may be translated for developing further noninvasive diagnostic modalities. However, as yet, only few groups have applied this novel technique for biomarker detection in solid organ transplantation.

Here, we report on the use of proteomic technology using two-dimensional difference gel electrophoresis (2D-DIGE) and LC-nanospray-tandem mass spectrometry (MS/MS) in murine heart transplants to identify a diagnostic molecular signature and specific biomarkers of acute cardiac allograft rejection.

MATERIALS AND METHODS

Animal Experiments

Two fully allogeneic strains of inbred male mice C57BL/10 (H2b) and C3H/He (H2k) were obtained from Harlan-Winkelmann Co. (Borchen, Germany).

All animals received human care in compliance with the “Principles of Laboratory Animal Care” formulated by the National Society for Medical Research and the “Guide for the Care and Use of Laboratory Animals” prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH Publication No. 86-23, revised 1985). All experiments were approved by the Austrian Ministry of Education, Science and Culture (BMBWK-66.011/0151-BrGT/2006).

Heart Transplantation

Cervical heterotopic heart transplantation was performed with a modified cuff technique for revascularization as previously described (13, 14). Cardiac allograft survival was determined by daily palpation and binocular inspection, with complete cessation of heart beats indicating severe rejection that was confirmed by histology.

Experimental Groups, Sample Collection, and Preparation

Four experimental groups (n=6 per group) were studied: untreated allogeneic animals (C57BL/10 to C3H/He) and syngeneic animals (C3H/He to C3H/He) with graft explantation of day 6 posttransplant, and allogeneic and syngeneic survivals serving as controls.

The transplants were harvested on postoperative day 6 or on cessation of graft function for intragraft protein expression analysis and histopathologic evaluation. Tissue samples were frozen immediately in liquid nitrogen and stored at −80°C until further analysis for protein expression or were fixed in formalin and embedded in paraffin until evaluated by hematoxylin and eosin staining or immunohistochemistry.

Frozen heart tissue samples were homogenized in lysis buffer (8 M urea, 4% CHAPS, 30 mM TRIS/HCl, pH 8.3) (15), centrifuged at 16,000g for 10 min, and the supernatant was used for protein analysis (Bradford assay, Bio-Rad, Hercules, CA).

2D-DIGE

CyDye Labeling

Equal amounts of individual samples from each group were pooled. These pooled samples were minimally labeled with CyDye DIGE Fluors (GE Healthcare) as previously described (15, 16). Briefly, protein (50 μg) was labeled with 400 pmol of cyanine dye. The labeling reaction was incubated for 30 min on ice in the dark. The reaction was quenched by addition of 1 μL 10 mM lysine followed by incubation on ice for a further 10 min. Equal volumes of 2× lysis buffer (as above plus 2% dithiothreitol (DTT) and 2% pharmalyte pH 3–11) were added to each of the labeled protein samples.

Samples of each group were alternately labeled with Cy3 and Cy5, thus accounting for possible intensity differences between the dyes. A pooled internal standard containing all samples included in the experiment (17, 18) was labeled with Cy2. The differently labeled samples and the pooled internal standard were mixed, and rehydration buffer (8 M urea, 2% CHAPS, 0.5% immobilized pH gradient (IPG)-Buffer pH 3–11, 0.002% bromphenol blue) was added to bring the volume up to 340 μL before isoeletric focusing. Each sample was analyzed in triplicate.

Protein Separation and Detection by 2D Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis

Nonlinear IPG strips 18-cm long, pH 3 to 11 NL (GE Healthcare) were rehydrated with CyDye-labeled samples (150 μg protein in 340 μL rehydration solution per strip) overnight at room temperature using a reswelling tray (GE Healthcare). Isoeletric focusing was performed using the IPGphor II apparatus (GE Healthcare) for a total of 40,000 Vh at 20°C and 50 μA per strip.

Before sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the strips were equilibrated for 15 min in 50 mM Tris-HCl (pH 8.8), 6 M urea, 30% glycerol, 2% SDS supplemented with 1% DTT to maintain the fully reduced state of proteins, and then by 15 min in equilibration buffer supplemented with 2.5% iodoacetamide to prevent reoxidation of thiol groups during electrophoresis. The strips were loaded and run on 12.5% SDS polyacrylamide gels (Laemmli) manually cast using the Ettan-Dalt-six system (GE Healthcare). Gels were run at 15°C and 0.5 W per gel for 1.5 hr, followed by 3 W per gel until the bromophenol blue dye front reached the end of the gels.

Imaging

Labeled proteins separated in the 2-D gel were visualized using a Typhoon 9410 scanner (GE Healthcare). Images were scanned as follows: Cy2: 488 nm excitation, 520 nm BP (band pass) 40 emission filter; Cy3: 532 nm laser, 580 nm BP 30 emission filter; and Cy5: 633 nm laser, 670 nm BP 30 emission filter.

DIGE Analysis

Gel analysis was performed using DeCyder 2D 6.5 software (GE Healthcare) applying the biological variation analysis module to allow the comparison of the spot ratios between all gels by matching the internal standard images of all gels. Relevant spots were selected by narrowing the filter settings, such that differences in the spot volume between the proteins from the various samples were more than or equal to 1.5 with a t test confidence more than or equal to 95% (P≤0.05).

Selected protein spots were excised from 2D gels using the Ettan Spot Picker with a 2-mm diameter picking head and stored at −20°C ready for identification by MS.

Mass Spectrometry

In-Gel Digestion

The protein spot of interest was transferred to a 0.25-mL polyethylene sample vial and washed with two changes of 50 μL 100 mM NH4HCO3, pH=8.0 (15 min at 30°C). The supernatant was discarded, and the gel spot was shrunk by dehydrating twice in 50 μL of 50% vol/vol acetonitrile/100 mM NH4HCO3 (15 min at 30°C) and dried in a vacuum centrifuge. Then, 50 μL of 10 mM DTT in 100 mM NH4HCO3 was added (30 min at 56°C), the supernatant was removed and 50 μL acetonitrile added. After 10-min incubation, the acetonitrile was replaced and 50 μL of 55 mM iodoacetamide in 100 mM NH4HCO3 was added. After 20-min incubation at room temperature in the dark, the spot was washed with 50 μL 100 mM NH4HCO3 for 5 min and shrunk by adding 150 μL acetonitrile for 15 min. After removing the supernatant, the gel piece was dried in a vacuum centrifuge. The gel spot was swollen in a digestion buffer containing 100 mM NH4HCO3 and 0.05 μg/μL trypsin (Sigma, proteomics grade) at 4°C for 20 min. Digestion took place overnight at 37°C. Peptides were extracted by adding 50 μL 10 mM NH4HCO3 (37°C, 15 min) and 50 μL acetonitrile (37°C, 15 min). After collecting the supernatant, 100 μL 10% formic acid/20% acetonitrile/20% 2-propanol was added to the gel pieces (37°C, 10 min). The supernatants were combined, dried down to approximately 5 μL, and stored at −20°C.

LC-Nanospray-MS/MS

Protein digests were analyzed using a LCQ ion trap instrument (ThermoFinnigan, San Jose, CA) equipped with a nanospray interface according to Ott et al. (19). MS/MS spectra were searched against a murine database (ipi.mouse.fasta from ftp.ebi.ac.uk) using SEQUEST (LCQ BioWorks; ThermoFinnigan). Specific cleavage sites for trypsin (KR) were selected with three missed cleavage sites allowed. The identified peptides were further evaluated using charge state versus cross-correlation number (Xcorr). The criteria for positive identification of peptides were Xcorr more than 1.5 for singly charged ions, Xcorr more than 2.0 for doubly charged ions, and Xcorr more than 2.5 for triply charged ions. Peptide tolerances of ±2 Da and MS/MS tolerances of ±1 Da were allowed.

Histopathology and Immunohistochemistry

Immunohistochemistry was performed using the pyruvate kinase, PKM antibody (c-term), a purified rabbit polyclonal antibody (Abgent), and the sheep anti-nonselenium gluthathione peroxidase antibody (Chemicon), as described previously (20). Briefly, 5-μm sections were cut from paraffin-embedded tissue blocks, mounted on adhesive-coated glass slides, deparaffinized, and rehydrated. Endogenous peroxidase was blocked with methanol containing 3% hydrogen peroxide over 20 min. After washing in Tris buffer, slides were incubated for 30 min at room temperature with the primary antibody (anti-PKM2, rabbit polyclonal antibody, dilution 1:100; anti-NSGP antibody, dilution 1:100). As a second step, slides were treated with an enzyme-linked antibody using the Envision DAKO ChemMAT Detection Kit followed by peroxidase/chromogene diaminobenzidin (DAB). Finally, slides were counterstained with Mayer’s hemalaun solution. Immunohistochemistry was evaluated by a pathologist (P. Obrist) using light microscopy.

Western Blot

Protein extracts from individual samples were separated on 12% polyacrylamide gels using Tris-Glycine SDS-PAGE. The amount of protein loaded into each well was 20 μg. After being transferred to polyvinylidene fluoride membranes (Hybond-P, Amersham Biosciences), the membranes were blocked in PBS 0.1% Tween, 5% (w/v) skim milk powder, and 10% horse serum for 2 hr and subsequently probed with the specific antibody (anti-peroxiredoxin 6 antibody, 1:500; anti-pyruvate kinase isozyme 2 (PKM2) antibody, 1:100) overnight at 4°C, visualized by goat anti-rabbit IgG horseradish peroxidase-conjugated secondary antibody (PIERCE), and developed using the ECL-PLUS Western blot analysis kit (Amersham Biosciences). To monitor the amount of protein loaded, membranes were stripped and reprobed with mouse anti-actin antibody (CHEMICON). Recording and visualization were performed with a Typhoon 9410 Laser scanner (Amersham Biosciences). Protein abundance was quantified using ImageQuant 5.2 software (Amersham Biosciences).

Statistical Analysis

The Mann-Whitney U test and Student’s t test for comparison of all groups were used for statistical analysis of all data; P values less than 0.05 were taken to indicate significant differences. All statistical analyses and tests were performed with the SPSS statistical package (SPSS 11.0 for windows, Chicago, IL).

RESULTS

Graft Survival and Histologic Evaluation

In untreated animals, allografts were rejected on approximately days 8 and 9 posttransplantation, with a mean graft survival of 8.83±0.17 days, whereas all syngeneic grafts showed indefinite survival (>100 days; P<0.0001, Student’s t test; Fig. 1).

FIGURE 1.
FIGURE 1.:
Kaplan Meyer survival graph. Median graft survival of untreated allogeneic grafts was 8.83±0.17 days, when compared with syngeneic controls (P<0.0001, Student’s t test).

Histologic evaluation of the allografts in the control group with graft retrieval on the day of graft failure showed aggressive diffuse polymorphonuclear infiltration and focal necrosis of the transplant indicating acute and severe rejection (International Society for Heart and Lung Transplantation [ISHLT]-score IV) (21). As expected, untreated allografts harvested on day 6 posttransplant, namely shortly before graft loss, yielded ISHLT scores of IIIB with diffuse inflammatory infiltrates and necrosis. However, all grafts at this time were beating and showed homogeneous contractility but slightly decreased frequency, when compared with syngeneic grafts. Syngeneic grafts retrieved on days 6 and 100 posttransplant revealed minimal infiltrates (ISHLT score IB). There were no histopathologic signs of chronic rejection in syngeneic controls on day 100.

Quantitative Changes in Cardiac Proteome Profile on Acute Allograft Rejection

To characterize the molecular signature of acute cardiac allograft rejection, we compared intragraft protein expression of untreated allogeneic transplants and syngeneic grafts on day 6 posttransplant by means of 2D-DIGE. Quantitative analysis of the 2D-DIGE gels (Fig. 2) revealed a total of 95 protein spots with a significant change in abundance of more than 1.5-fold, when comparing rejecting grafts with syngeneic controls. Expression was regulated at more than twofold in 35 proteins, four of which showed a more than fourfold change in abundance. Of the 95 proteins differentially expressed, the majority (58 proteins) was upregulated, whereas 36 proteins were downregulated during acute graft rejection.

FIGURE 2.
FIGURE 2.:
Two-dimensional difference gel electrophoresis. Two-dimensional gel image showing differentially expressed spots when comparing allogeneic and syngeneic tissue samples on day 6 posttransplant. Localization and identity of the protein spots with highest altered regulation are depicted.

MS/MS Identification of Proteins Associated With Acute Rejection

A panel of 35 spots was selected for protein identification using MS/MS. Of the 35 analyzed spots, 12 contained more than one protein; these spots were thus considered not to have been clearly identified and excluded from further analysis. Six spots could not be identified at all. The remaining 17 proteins were identified unambiguously. Table 1 reports the nature of each clearly identified protein, SwissProt entry, its theoretical molecular weight and pI, % sequence coverage for the identified protein, and differences in protein abundance expressed as the average ratio of allogeneic versus syngeneic samples (for detailed MS/MS reference see Table, Supplemental Digital Content 1, https://links.lww.com/TP/A88).

TABLE 1
TABLE 1:
Potential biomarkers of acute cardiac allograft rejection

The majority of altered proteins were found to be enzymes. In particular, aconitate hydratase and fumarate hydratase, both enzymes of the tricarboxylic acid cycle, were significantly decreased in rejecting transplants, namely 1.8- and 1.6-fold, respectively. Three proteins were functionally related to energy metabolism: pyruvate kinase isozyme M2 and glyceraldehyde-3-phosphate dehydrogenase were both significantly increased in allogeneic transplants, namely 1.7- and 4.8-fold, respectively, whereas creatine kinase was downregulated by 1.9-fold in rejecting grafts. Peroxiredoxin 6, a peroxidase that plays an important role in antioxidant defense, displayed a more than 4-fold increase during acute rejection. Finally, expression of protein disulfide isomerase A3 precursor was upregulated 1.6-fold on allograft rejection.

Two differentially expressed proteins are cardiac tissue-specific proteins. Myosin heavy chain was independently identified twice, showing a significant increase on acute rejection ranging from 1.9- to 2.7-fold in all of the four spots. Troponin T displayed a 1.6-fold increase in expression in allogeneic grafts, when compared with the syngeneic controls.

Three proteins have been reported to be structural proteins. Vimentin that is known to be an intermediate filament protein showed a 1.7-fold downregulation on acute allograft rejection. Skeletal muscle LIM protein 3, a stress sensor in both cardiac and skeletal muscle, displayed a 1.56-fold decrease in rejecting grafts as well. In contrast, the actin-binding protein coronin 1A was found to be upregulated more than threefold in response to acute rejection.

Furthermore, serum albumin precursor significantly increased on allograft rejection. And finally, the secretory proteins fibrinogen alpha and beta chain precursor that are involved in platelet aggregation were upregulated 3.3- and 2.9-fold, respectively in allogeneic heart transplants, when compared with syngeneic grafts.

Western Blot Analysis and Immunohistochemistry

To confirm the 2D-DIGE data from the pooled samples, we assessed the expression level of selected proteins in individual samples using Western blot analysis based on commercial antibody availability. Immunoblotting for peroxiredoxin 6 and pyruvate kinase isozyme M2 revealed significant overexpression of both proteins in allogeneic untreated transplants on day 6 posttransplant, when compared with the syngeneic controls (2.48-fold change, P=0.0043 for peroxiredoxin 6; 14.78-fold change, P=0.0022 for pyruvate kinase isozyme M2, Mann-Whitney U test; Fig. 3). To test for longitudinal expression of the differentially expressed proteins, Western blot analysis for peroxiredoxin 6 and pyruvate kinase isozyme M2 was also performed on postoperative days 2 and 4 and compared with syngeneic controls. Expression levels for peroxiredoxin 6 were almost identical in allogeneic and syngeneic animals on postoperative days 2 and 4 showing a 0.9- and 0.8-fold change, respectively. Pyruvate kinase isozyme M2 displayed a gradual increase in protein expression in allogeneic animals during the time course as reflected by a 4.7-fold change on postoperative day 2 and 2.0-fold change on postoperative day 4.

FIGURE 3.
FIGURE 3.:
Expression level of peroxiredoxin 6 and pyruvate kinase isozyme M2 (PKM2) in allogeneic and syngeneic grafts on day 6 posttransplant (Western blots). Protein extracts of individual cardiac transplants were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analyzed by immunoblotting for peroxiredoxin 6 and pyruvate kinase isozyme M2. Protein loading was assessed by immunoblotting for β-actin. Peroxiredoxin 6 and PKM2 are both significantly overexpressed in grafts with acute rejection, when compared with syngeneic controls (2.48-fold change, P=0.0043 for peroxiredoxin 6; 14.78-fold change, P=0.0022 for pyruvate kinase isozyme M2, Mann-Whitney U test). Representative samples are shown.

In addition, to determine the main cellular source of peroxiredoxin 6 and pyruvate kinase isozyme M2 during acute allograft rejection immunohistochemical analysis was performed. Immunohistochemical staining thereby again confirmed overexpression of both proteins in the acutely rejecting grafts as compared with the syngeneic samples (Fig. 4). Staining for peroxiredoxin 6 and for pyruvate kinase isozyme M2 was found to be predominantly positive in infiltrating mononuclear cells.

FIGURE 4.
FIGURE 4.:
Immunohistochemical staining of peroxiredoxin 6 and pyruvate kinase isozyme M2 in cardiac grafts on day 6 posttransplant. Representative sections from allogeneic and syngeneic transplants are shown (200×). Peroxiredoxin 6 and PKM2 both yielded significant overexpression in acutely rejecting cardiac transplants, when compared with syngeneic controls. Both proteins are predominantly expressed in infiltrating mononuclear cells. (A) Peroxiredoxin 6, allogeneic. (B) Peroxiredoxin 6, syngeneic. (D) Pyruvate kinase isozyme M2, allogeneic. (E) Pyruvate kinase isozyme M2, syngeneic. Control panels, secondary antibody alone for peroxiredoxin 6 (C) and PKM2 (F).

DISCUSSION

To our knowledge, this is the first study to report on the use of proteomic technology for identifying potential biomarkers of acute rejection in murine cardiac allografts. We could demonstrate that proteomics (2D-DIGE) is a powerful method for studying the molecular signature of acute cardiac allograft rejection. A set of 95 proteins was shown to be differentially expressed in acutely rejecting heart transplants as compared with syngeneic controls. Characterization of selected protein spots finally revealed 17 proteins that were unambiguously identified as potential surrogate parameters of acute cardiac allograft rejection. In particular, identified proteins like peroxiredoxin 6 and pyruvate kinase isozyme M2 are novel indicators of acute rejection and may become useful surrogate markers for monitoring alloimmune response.

Proteomic technology complements functional genomic approaches such as microarrays for the characterization of global expression profiles. Gene chips have proven to be an exciting investigative tool for generating genome-wide profiles of mRNA expression. They allow identification and characterization of the interplay among multiple gene pathways. Microarray analysis of graft rejection often yields a multiple of regulated genes, when compared with proteomic analysis (22–24). This discrepancy in the vast amount of genes that are regulated on allograft rejection and the few proteins that are eventually found to be differentially expressed might be due to the limitations that are specific for DNA microarrays: there is no strict linear relationship between genes and the protein complement or proteome of a cell. The existence of an open reading frame in genomic data does not necessarily imply the existence of a functional gene and, most importantly, there is often poor correlation between mRNA abundance in a cell or tissue and the quantity of the corresponding functional protein. In addition, protein modifications such as isoforms and posttranslational modifications that are not apparent from the DNA sequence can be determined only by proteomic means (6, 8, 9, 25).

To date only few groups have applied proteomic technology in the transplant setting. There is one study by Borozdenkova et al. (26) that reports on the use of proteomics for detecting biomarkers of cardiac allograft rejection and there is the study by De Souza et al. (27) who have focused on the proteomic identification of proteins that protect allografts from cardiac allograft vasculopathy, which is a major long-term complication of heart transplantation. These groups, too, applied the core technology of choice for proteomic analysis, namely two-dimensional gel electrophoresis followed by protein identification by mass spectrometry (28). Nevertheless, limitation of conventional 2D-PAGE is the high degree of gel-to-gel variation in spot patterns, which makes it difficult to distinguish biological from experimental variation. For this reason, we applied fluorescent 2D-DIGE that offers the unique possibility of internal standardization, reduces the number of gels that must be run, has a wide dynamic range for quantitation and has demonstrated high sensitivity and reproducibility (15, 17, 29). Thus, in line with the data presented here, Borozdenkova et al. identified various proteins to be regulated during acute cardiac allograft rejection that constituted cardiac-specific proteins (myosin, tropomyosin, troponin C, actin, and myoglobulin) and stress proteins (hsp27 and αB-crystallin). Thus, the most likely markers of acute cardiac rejection obtained by means of proteomic technology seem to be markers of damage or stress and not of commonly known pathways of lymphocyte activation.

The majority of clinical studies published so far have focused on analysis of the urinary proteome to detect noninvasive biomarkers of acute renal allograft rejection (30–32). Compared with the present work, these studies applied a pure diagnostic or pattern proteomic approach that refers to high-throughput methods that rely on a pattern of peaks to differentiate between two conditions, whereas the biomarker discovery approach used here aims to identify the proteins that are differentially expressed (28). This might permit a better understanding of the molecular and immunological basis of the rejection process.

The current study determined a more than fourfold increase in expression following acute allograft rejection for peroxiredoxin 6, and overexpression of this protein was confirmed by Western blot analysis and immunohistochemistry. Peroxiredoxin 6 is a member of a new family of antioxidant enzymes. The reduction of peroxidized membrane phospholipids, which facilitates repair of damaged cell membranes, is believed to account for the unique antioxidant effects of peroxiredoxin 6 (33–35). In a recent study, Nagy et al. (36) demonstrated a nonredundant role of peroxiredoxin 6 in cardioprotection because hearts of Prdx6−/− mice were susceptible to ischemia-reperfusion injury, had reduced postischemic ventricular recovery and increased myocardial infarct size, and exhibited a greater number of apoptotic cardiomyocytes, when compared with wild-type hearts. Our findings, therefore, suggest that overexpression of peroxiredoxin 6 in the acutely rejecting cardiac transplant constitutes a protective mechanism for limiting the oxidative stress caused by the alloimmune response.

Pyruvate kinase isozyme M2 displayed a 1.7-fold overexpression in acutely rejecting heart transplants. Pyruvate kinase catalyzes the last step in glycolysis thus enabling the generation of ATP under hypoxic conditions. There are four isozymes of pyruvate kinase; the M2 isoform is expressed in fetal tissues and is progressively replaced by the other three isoforms as cells differentiate (37). McDowell et al. (38) demonstrated that plasma concentration of dimeric M2 pyruvate kinase increased significantly in patients with chronic cardiac failure, probably to maintain a balance between energy production and the supply of glycolytic intermediates to sustain synthetic processes. Therefore, the upregulation of M2 pyruvate kinase in acute allograft rejection as demonstrated in the present work seems to reflect the status of the chronically failing heart transplant. In contrast to our findings, Maneus et al. (39) observed a marked decrease in pyruvate kinase activity in rejecting rat heart transplants, although not in the specific M2 isoform, thus implying a marked decrease in the glycolytic pathway after acute cardiac rejection.

Another identified protein that showed a more than twofold increase in expression in response to acute cardiac allograft rejection was serum albumin precursor. Vascotto et al. (40) demonstrated upregulation of human serum albumin after reperfusion in human orthotopic liver transplantation, whereas Pan et al. (41) found albumin to be decreased in a model of spontaneous tolerance in rat orthotopic liver transplantation. Furthermore, Kaiser et al. (42) identified a 1.83-kDa peptide from albumin as being excreted at a high concentration in the urine of patients with graft-versus-host disease after allogeneic hematopoietic stem-cell transplantation. Funding et al. (43) reported increased albumin concentrations in aqueous humor of patients with corneal rejection. These studies indicate that albumin constitutes a surrogate marker of rejection, although its increase after allograft rejection and reperfusion is probably due to an unspecific inflammatory reaction. Furthermore, independent identification of albumin as a key protein in these immunologically different posttransplant settings validates proteomics as a reliable and reproducible means of identifying surrogate markers in the transplantat setting. However, the results of our study still remain to be further verified in human cardiac transplant recipients. A respective study to test for M2 pyruvate kinase, peroxiredoxin 6, and albumin serum levels in heart transplant patients is currently underway. Another limitation of the current study is that proteomic analysis was performed at rather late and advanced stages of acute rejection such as postoperative day 6. Future studies including earlier time points with lower rejection scores and the use of immunosuppressive agents such as cyclosporine-A might be more likely to best mimic the human situation.

In this study, we demonstrate that proteomics is a powerful and valuable tool for identifying molecular signatures and might provide new insights into the pathophysiology of alloimmune response. Identified proteins like peroxiredoxin 6 and pyruvate kinase isozyme M2 constitute novel biomarkers of acute cardiac allograft rejection that deserve further testing in clinical studies.

ACKNOWLEDGMENT

The authors thank Petra Loitzl and Renate Kaus for expert technical assistance.

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Keywords:

Proteomics; Heart transplantation; Biomarker

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