Molecular Vision 2014; 20:1192-1202
Received 13 March 2014 | Accepted 27 August 2014 | Published 27 August 2014
1Department of Ophthalmology, Hando Hospital, Ansan, Korea; 2Department of Bioengineering and Institute for Bioengineering and Biopharmaceutical Research, Hanyang University, Seoul, Korea
Correspondence to: Taiyoun Rhim, Department of Bioengineering and Institute for Bioengineering and Biopharmaceutical Research, Hanyang University, Seoul, Korea; Phone: +82-2-2220-2347; FAX: +82-2-2220-4347; email: firstname.lastname@example.org
Purpose: To identify differentially expressed proteins in the pterygium compared to healthy conjunctiva using a proteomic analysis.
Methods: Pterygial and healthy conjunctival tissues were obtained from 24 patients undergoing pterygium excision. Total proteins of the pterygia and healthy conjunctiva were analyzed with one-dimensional electrophoresis, and protein bands of interest were excised and subjected to liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-MS/MS) using Thermo’s Finnigan ProteomeX workstation LTQ linear ion trap MS/MS. Using bioinformatics, differentially expressed proteins were classified, and three proteins closely involved in the response to oxidative stress were selected for further validation. Differential expression of these proteins was confirmed with western blot and immunohistochemistry.
Results: A web-based gene ontology program, DAVID, was used to classify 230 proteins that were differentially expressed in pterygial tissues. Among these genes, we chose three proteins, aldehyde dehydrogenase, dimeric NADP-preferring (ALDH3A1), protein disulfide-isomerase A3 (PDIA3), and peroxiredoxin-2 (PRDX2), that were significantly upregulated in pterygium and further increased in recurrent pterygium. Immunohistochemistry and western blot analysis confirmed that these three proteins were mainly detected in the basal epithelial layer, and their expression was significantly increased in the pterygium compared to normal conjunctiva.
Conclusions: This study reported increased expression of ALDH3A1, PDIA3, and PRDX2 in pterygia using a proteomic approach. These proteins are presumed to have a protective role against oxidative stress-induced apoptosis. This result is consistent with the hypothesis that oxidative stress is a significant factor in the pathogenesis of pterygia.
Pterygium refers to a wing-shaped fibrovascular tissue thought to be formed by the overgrowth of transformed conjunctival tissue into the cornea. The lesion is characterized by centripetal growth of a leading edge of altered limbal epithelial cells, followed by squamous cell metaplasia with goblet cell hyperplasia. Activated stromal fibroblasts, neovascularization, and extracellular matrix remodeling are also present [1-4].
Ultraviolet (UV) radiation damage, chronic irritation, and/or inflammation are hypothesized to play roles in the pathogenesis of pterygium. Patients with increased ultraviolet (UV) exposure, such as those living close to the equator or those with sunlight-related conditions, have an increased risk of pterygia formation [3-5]. Previous studies focused primarily on the clinical characteristics and surgical management of the disease, and therefore, its pathogenesis remains incompletely understood. However, considerable progress in this area has been achieved, and recent evidence implicates antiapoptotic mechanisms, oxidative stress, immunological mechanisms, various cytokines, and extracellular matrix (ECM) modulation as possible causative factors [3,6-10]. Regardless of the inciting agent, inflammatory mediators appear to be associated with pterygia. Interleukin (IL-6), IL-8, and tumor necrosis factor-α are increased in pterygia and induced after UV-B exposure in cultured pterygia epithelial cells . Cyclooxygenase-2 is overexpressed in pterygia, contributing to a further increase in the inflammatory cascades . Immunological mechanisms also likely contribute to the development of pterygia. An increase in mast cells, lymphocytes, plasma cells, dendritic cells, and CD4+ and CD8+ T cells in pterygia has also been documented . The contribution of inflammatory mediators, immunological mechanisms, angiogenic stimulation, and ECM modulation are likely secondary in nature after actual pterygia formation .
In this study, we adopted a proteomics technique for identifying the proteins that were differentially expressed in pterygium compared to normal conjunctiva. Proteomics has expanded the opportunities to identify disease-specific proteins involved in many diseases, including ophthalmic diseases such as dry eye [14,15], cataract [16,17], keratoconus , myopia , and pterygia .
As in previous studies, hundreds of protein molecules were newly identified as candidate biomarkers of pterygia. To narrow our interests, we focused on proteins that show increased expression in primary pterygium and are further increased in recurrent pterygium. Among those proteins, we further validated increased expression of three proteins known to have roles related to oxidative stress, aldehyde dehydrogenase, dimeric NADP-preferring (ALDH3A1), protein disulfide-isomerase A3 (PDIA3), and peroxiredoxin-2 (PRDX2), using western blot analysis and immunohistochemistry.
Between May 2012 and Apr 2013, 24 subjects, 10 males and 14 females, with mean age 49±5.2 (38-62) years old who underwent pterygium excision in Hando general hospital (Ansan, Korea) were included in the study. Samples of healthy conjunctiva were obtained from the same patients who underwent pterygium excision. Pterygium tissues and remnants from the autologous upper bulbar conjunctiva grafts were collected immediately at the time of surgery and frozen in liquid nitrogen. This study protocol was approved by the institutional review board of our hospital and it conformed to the guidelines provided in the ARVO statement on human subjects in ophthalmic and vision research. Written informed consent to participate in this research was obtained from each patient.
Pterygia tissues were homogenized using Tissue-Ruptor (Qiagen, Valencia, CA) with PBS containing 1 mM phenylmethylsulfonyl fluoride (PMSF, Sigma-Aldrich, CA). Proteins were quantified with bicinchoninic acid (BCA) assay (Pierce, Cramlington, UK). Fifty micrograms of proteins were separated with gradient sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE; 4–20% polyacrylamide gels, Novex gel, Invitrogen, Carlsbad, CA). After Coomassie staining, the protein bands of interest were excised and subjected to in-gel trypsin digestion as previously described . Briefly, after destaining in 75 mM ammonium bicarbonate/40% ethanol (1:1), the gels were subjected to reducing (5 nM dithiothreitol [DTT] in 25 mM ammonium bicarbonate) and alkylating (55 mM iodoacetamide) conditions, each at room temperature for 30 min. After dehydration with acetonitrile, 20 μg/mL sequencing-grade trypsin (Roche Applied Science) containing 25 mM ammonium bicarbonate (pH 8.5) was added, and the slice was incubated at 37 °C overnight. The in-gel tryptic peptides were eluted with 0.1% formic acid. Liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-MS/MS) analysis was performed using Thermo’s Finnigan ProteomeX workstation LTQ linear IT MS (Thermo Electron, San Jose, CA) equipped with a nanospray ionization (NSI) source (San Jose, CA).
Data-dependent acquisition mode (m/z 300–1800) was enabled, and each survey MS scan was followed by five MS/MS scans with the 30 s dynamic exclusion option on. The spray voltage was 1.9 kV and the temperature of the ion transfer tube was set at 1957C. The normalized collision energy was set at 35% . MS/MS data were searched based on the IPI human protein database (version 3.29) using the SEQUEST algorithm (Thermo Electron). Scaffold ver. 01_07_00 was used to validate MS/MS-based peptides and protein identification.
Gene ontology analysis was performed using Integrated Discovery (DAVID) Functional Annotation Tool. Through DAVID, proteins with gene symbols were grouped according to biologic process.
Protein extracts were obtained from pterygia and normal conjunctiva tissue and were lysed by sonication in PBS containing 1 mM PMSF. Proteins were resolved with 12.5% SDS–PAGE and transferred to a polyvinylidene ﬂuoride (PVDF) membrane (Millipore, Milan, Italy). The membrane was blocked for 1 h in a 5% skim milk solution and then incubated with mouse monoclonal antibodies against ALDH3A1 (1:2,000 dilution, AbFrontier, Seoul, Korea), PDIA3 (1:5,000 dilution, AbFrontier), and PRDX2 (1:2,000 dilution, AbFrontier) for 2 h at room temperature. The unbound primary antibodies were removed with one 15-min and two 5-min washes in PBS containing 0.1% (v/v) Nonidet P40. Then the membrane was incubated with horseradish peroxidase-conjugated goat anti-mouse immunoglobulin (IgG) secondary antibody (Ab Frontier) for 2 h at room temperature. The target protein was detected with enhanced chemiluminescence (ECL) solution (Amersham Biosciences, Cleveland, OH) using X-ray film.
The tissue was fixed with 4% (v/v) buffered paraformaldehyde for 1 day and then washed in tap water for 1 day. The tissue was dehydrated with a series of graded ethanol and embedded in paraffin. Sections were cut to a thickness of 4 µm and deparaffinized. Then, the sections were hydrated using a series of decreasing concentrations of ethanol. The sections were incubated in blocking buffer for 1 h at room temperature to block non-specific binding. The primary antibodies used were against ALDH3A1 (1:200 dilution, AbFrontier), PDIA3 (1:400 dilution, AbFrontier), and PRDX2 (1:200 dilution, AbFrontier). The sections were incubated in primary antibody solution for 1 h at room temperature. All samples were incubated for 30 min with universal biotinylated secondary antibodies at room temperature. Samples were then washed twice, and final incubation was performed for 30 min using streptavidin-peroxidase. Signals were developed using 3,3-diaminobenzidine for 5 min and counterstained with Mayer’s hematoxylin (Dako, Glostrup, Denmark). Negative controls were prepared by leaving out the primary antibody.
Pooled crude extracts of eight individuals from each group (normal conjunctiva, pterygium, and recurrent pterygia) were run on an SDS–PAGE gel, and the gel was stained with Coomassie brilliant blue G-250 (Figure 1). The stained SDS–PAGE gel was cut into 15 pieces according to molecular weight and subjected to in-gel digestion. Digested peptides were then fractionated and identified with LC-MS/MS. All the experiments were replicated twice. In the first experiment, 346 proteins were differentially expressed in the three groups. Among these, 249 protein expression patterns were replicated in the second experiment.
First, we analyzed the pterygia-specific proteins by comparing normal conjunctiva with primary and recurrent pterygium. The expression of 153 proteins was downregulated (Appendix 1), and 77 proteins were upregulated in pterygia (Appendix 2).
Using a web-based gene ontology program, DAVID, 230 proteins that were up- or downregulated in pterygia tissues were classified. Molecular functions were attributed to 230 proteins using GOTERM_MF_FAT from the DAVID database. The related gene ontology categories were merged into a cluster using the DAVID functional clustering module. The major relevant categories were as follows: structural molecule activity, nucleotide binding, cytoskeletal protein binding, peptidase activity, guanyl ribonucleotide binding, structural constituent of eye, and oxidoreductase activity (Table 1). Interestingly, a group of proteins closely related to the structural molecule and peptidase activity were either up- or downregulated in pterygium. These data suggest that tissue rearrangement is strongly involved in pterygia formation and growth.
The number of different proteins in the pterygium tissues was still too high to be validated. To narrow the possible candidate proteins, we further analyzed the proteomics data. Instead of comparing two groups (normal conjunctiva and pterygia), we compared proteome from three groups: normal conjunctiva, primary pterygium, and recurrent pterygium. Several fundamental profile patterns could be identified from these analyses, and we focused on increasing or decreasing patterns: upregulated in primary pterygium and further increased in recurrent pterygium or downregulated in primary pterygium and further decreased in recurrent pterygium. We thought that if the change in expression remained or increased in recurrent pterygium, the molecule should be considered to have a critical role in pterygia formation or growth. In this context, 40 proteins were differentially expressed in primary and recurrent pterygia. Among them, we chose three proteins, ALDH3A1, PDIA3, and PRDX2, for further validation. As shown in Table 2, the expression of all three proteins was significantly increased in pterygium and further increased in recurrent pterygium. Interestingly, all three proteins have some roles related to oxidative stress. PDIA3 and ALDH3A1 were classified into cell redox homeostasis (GO:0045454) and oxidation-reduction process (GO:0055114) groups, respectively. PRDX2 was also associated with cellular response to oxidative stress (GO:0034599).
To validate the proteomic analysis data, we performed a western blot analysis of homogenates from normal conjunctiva and pterygium (100 μg of protein/lane). Our data showed that expression of the ALDH3A1, PDIA3, and PRDX2 proteins was significantly increased in the pterygia tissue compared to normal conjunctiva. This finding was consistent with the proteome analysis data (Figure 2). Using immunohistochemistry, upregulation of ALDH3A1, PDIA3, and PRDX2 expression in pterygia was also detected. As shown in Figure 3, all three proteins were mainly detected in the epithelial layer, and their intensities were increased in pterygium tissues compared to normal conjunctiva. Interestingly, these proteins were preferentially localized to the basal layer of the pterygial epithelium. These results confirmed that these three proteins were expressed at lower levels in normal conjunctiva and were present at considerably higher levels in the pterygium tissue.
The mechanism of pterygia formation is not yet fully understood, and its origin remains controversial. Despite many publications showing the influence of UV radiation in the pathogenesis, the role of oxidative stress and the antioxidant system is still unclear. The presence of oxidative stress in pterygium has been proved by finding an increase in 8-hydroxydeoxyguanosine (8-OHdG), malonedialdehyde (MDA), and decreased activity of superoxide dismutase, catalase, and glutathione peroxidase [7,23].
In this study, a proteomic approach was used to identify putative differences in pterygium compared to healthy conjunctiva. The expression of 153 proteins was downregulated (Appendix 1), and 77 proteins were upregulated in pterygia (Appendix 2). Our results revealed that proteins associated with an increase in extracellular matrix, structural, and mitotic proteins and those involved in tissue invasion were significantly affected. A remarkable change was also found in the group of proteins associated with oxidative stresses and protection against them. Among them, we confirmed increased expression of ALDH3A1, PDIA3, and PRDX2 in pterygial epithelium.
ALDH3A1 is an abundant corneal crystalline in mammals, and it has been known to contribute to corneal defense by playing a multifunctional role in the protection of the cornea, and perhaps the entire eye, against UV-induced oxidative stress [24,25]. ALDH3A1 is a member of the ALDH superfamily of proteins that catalyze the NAD(P)-dependent oxidation of a wide range of endogenous and exogenous aldehydes. The central roles and mechanisms by which ALDH3A1 protects against UV-induced damage are thought to include the following: 1) metabolism of the toxic aldehydes, 2) direct absorption of UV light, 3) antioxidant function either directly through the scavenging of free radicals or indirectly through the production of NADPH, 4) maintaining corneal refractive and transparent properties as a corneal crystalline, 5) chaperon-like activity, and 6) cell cycle regulation [25-28]. DNA is one of the major targets of UV-induced oxidative stress, and modified DNA, if left unchecked, may result in genetic mutation or cell death. Cell cycle control by ALDH3A1 resonates with the antiapoptotic and cell growth regulating roles of the lens alpha-crystallins [29,30]. Despite the upregulated expression of ALDH3A1 in pterygial tissue, we noted decreased expression of other crystalline proteins (alpha-crystallin A chain, alpha-crystallin B chain, beta-crystallin A3, beta-crystallin B1, beta-crystallin B2, and beta-crystallin S) in pterygia. This result suggests specific roles of ALDH3A1 in pterygia compared to other crystallins that have structural roles in conjunctiva.
PDIA3, another biomarker in our study, is a member of the endoplasmatic reticulum (ER) stress signaling pathway also known as unfolded protein response (UPR). The expression level of PDIA3 increases in response to cellular stress due to its function as a chaperone [31,32]. Recently published data connected PDIA3 to the apoptotic process and demonstrated PDIA3 had an antiapoptotic effect in the melanoma cell line A375 after ER stress was induced . PDIA3 possibly plays a role in the malignant transformation of prostate and cervical cancer [34,35]. The expression of this gene is induced during neoplastic transformation possibly leading to redox-dependent modulation of cancer-relevant regulatory factors [36,37]. These previous findings suggest that the observed increase of PDIA3 in pterygium is most likely due to elevated cellular stress, which may be induced by oxidative damage. In addition to a role in the ER stress pathway, PDIA3 has recently gained attention due to its function as a component of the peptide-loading complex of the major histocompatibility complex (MHC) class I pathway . In Pdia3-deficient mice, this complex is impaired and negatively influences presentation of antigenic peptides . This may help tumors escape from immune surveillance with cytotoxic T cells. It has been proposed that pterygium and neoplasia have common features, raising the possibility that pterygium is a neoplastic-like growth disorder. Whether PDIA3 plays a similar role in pterygium as in tumor formation is still unclear.
We also reported increased expression of PRDX2 in pterygium. This result confirmed the recent findings of Bautista-de Lucio et al. ; they also conducted a proteomic approach to identify disease-specific protein expression and identified upregulated expression of PRDX2 in the pterygium. PRDX2 is a cytoplasmic enzyme that reduces intracellular reactive oxygen species (ROS) levels. It has been described that PRDX2 overexpression protects significantly from peroxide-induced apoptosis and necrosis, while downregulation of this enzyme promotes injurious effects of oxidative stress in cardiomyocytes . A recent report found that pterygia express high levels of cellular proliferation proteins and low levels of apoptosis markers, suggesting that there is a disruption in the equilibrium among proliferation and apoptosis favoring cell proliferation . The antiapoptotic role of this enzyme has been demonstrated in red blood cells and pancreatic cells as well [42,43]. Many isoforms of the peroxiredoxin family have been implicated in the survival of several types of ocular cells [44-47]. These data support the proposal that PRDX2 could be involved in inhibiting apoptosis in the pterygium.
Another important aspect of this study is the immunohistochemical localization of ALDH3A1, PDIA3, and PRDX2 in pterygia. Interestingly, all three proteins are preferentially localized in the pterygium basal epithelium. Previous studies have reported that the expression of mutant p53 and other important apoptosis-related proteins, bax and bcl-2, was noted predominantly in the basal epithelial layer of pterygia [10,41]. Immunohistochemical studies demonstrated that apoptotic cells are found mainly confined to basal epithelial cells in pterygia [10,41], leading the chance that ALDH3A1, PDIA3, and PRDX2 are involved in regulating apoptosis and cell proliferation in pterygia. The antiapoptotic role of these three proteins has been demonstrated in various tissues [24,28,33,40,42,43]. Further studies are required to establish the functional roles of these proteins in pterygia formation.
Limited investigation regarding differences in the pathogenesis of primary and recurrent pterygia has been performed. We focused on proteins that show graded patterns of overexpression in primary and recurrent pterygia compared to normal conjunctiva. We presumed that these proteins might have more critical roles in pterygia formation and recurrence. We also focused on the difference in proteomic data between primary and recurrent pterygium, because the mechanisms that determine the genesis and the recurrence of a pterygia may not be the same. Interestingly, our result showed that periostin is a pathognomic marker for recurrent pterygium, and it was not expressed in primary pterygium or in normal conjunctiva. This finding confirms Kuo et al.’s transcriptome study [48,49]. It seemed reasonable to assume that recurrent pterygia possess molecular changes related to their recurrence.
In this study, we reported increased expression of ALDH3A1, PDIA3, and PRDX2 in pterygia using a proteomic approach. All three proteins are presumed to have significant roles related to oxidative stress. Our results are consistent with the hypothesis that oxidative stress is a significant factor in the pathogenesis of pterygia. We hypothesized that UV exposure and resultant oxidative stress leads to increased ALDH3A1, PDIA3, and PRDX2 levels, which provide resistance to UV-induced apoptosis and a hyperproliferative state.
This work was supported by the research fund of Hanyang University (HY-2009–419). Boram Lee was partly supported by NRF-2012M3A9D1054451.