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Quantitative proteomic analysis of mice corneal tissues reveals angiogenesis-related proteins involved in corneal neovascularization
Biochimica et Biophysica Acta 1864 (2016) 787–793
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Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbapap
Quantitative proteomic analysis of mice corneal tissues reveals angiogenesis-related proteins involved in corneal neovascularization Minqian Shen a,1, Yimin Tao b,c,1, Yifan Feng a, Xing Liu b,c, Fei Yuan a,⁎, Hu Zhou b,c,⁎⁎ a
Department of Ophthalmology, Zhongshan Hospital, Fudan University, Shanghai 200032, China Department of Analytical Chemistry and CAS Key Laboratory of Receptor Research, Shanghai Institute of MateriaMedica, Chinese Academy of Sciences, Shanghai 201203, China SIMMUOMICS Laboratory, Joint Research Laboratory of Translational “OMICS” between Shanghai Institute of Materia Medica, Chinese Academy of Sciences, China and University of Ottawa, Canada b c
a r t i c l e
i n f o
Article history: Received 30 October 2015 Received in revised form 22 March 2016 Accepted 1 April 2016 Available online 2 April 2016 Keywords: Alkali burn Corneal neovascularization Mice Proteomics
a b s t r a c t Corneal neovascularization (CNV) was induced in Balb/c mice by alkali burns in the central area of the cornea with a diameter of 2.5 mm. After fourteen days, the cornea from one eye was collected for histological staining for CNV examination, while the cornea from the other eye of the same mouse was harvested for proteomic analysis. The label-free quantitative proteomic approach was applied to analyze five normal corneal tissues (normal group mice n = 5) and five corresponding neovascularized corneal tissues (model group mice n = 5). A total of 2124 proteins were identified, and 1682 proteins were quantified from these corneal tissues. Among these quantified proteins, 290 proteins were significantly changed between normal and alkali burned corneal tissues. Of these significantly changed proteins, 35 were reported or predicted as angiogenesis-related proteins. Then, these 35 proteins were analyzed using Ingenuity Pathway Analysis Software, resulting in 26 proteins enriched and connected to each other in the protein-protein interaction network, such as Lcn-2, αB-crystallin and Serpinf1 (PEDF). These three significantly changed proteins were selected for further Western blotting validation. Consistent with the quantitative proteomic results, Western blotting showed that Lcn-2 and αB-crystallin were significantly up-regulated in CNV model, while PEDF was down-regulated. This study provided increased understanding of angiogenesis-related proteins involved in corneal vascular development, which will be useful in the ophthalmic clinic of specifically target angiogenesis. © 2016 Elsevier B.V. All rights reserved.
Statement of significance of the study
Abbreviations: CNV, corneal neovascularization; VEGFs, vascular endothelial growth factors; FGFs, fibroblast growth factors; PDGFs, platelet-derived growth factors; MMPs, matrix metalloproteinases; sVEGFRs, soluble VEGF receptors; PEDF, pigment epitheliumderived factor; TSPs, thrombospondins; iTRAQ, isobaric tags for relative and absolute quantitation; ARVO, Association for Research in Vision and Ophthalmology; SCX, strong cation exchanger; DTT, 1,4-Dithiothreitol; IAA, Iodoacetamide; CHAPS, 3-[(3cholamidopropyl)dimethyl-ammonio]-1-propane sulfonate; LC-MS, liquid chromatograph-mass spectrometer; UPLC, ultra performance liquid chromatography; ESI, electrospray ionization; HCD, high-energy collision-induced dissociation; FDR, false discovery rate; DAVID, Database for Annotation, Visualization and Integrated Discovery; HCA, hierarchical clustering analysis; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; IPA, Ingenuity Pathways Analysis; BCA, bicinchonininic acid; PVDF, polyvinylidene fluoride; HRP, horseradish peroxidase; Lcn-2, lipocalin-2; LFQ, label-free quantitation; HCA, hierarchical clustering analysis; HTRA1, HtrA serine peptidase 1; HIF1α, hypoxia-inducible factor 1α; Erk, extracellular signal-regulated kinase. ⁎ Correspondence to: F. Yuan, Department of Ophthalmology, Zhongshan Hospital, Fudan University, No. 180 Fenglin Road, Shanghai 200032, China. ⁎⁎ Correspondence to: H. Zhou, Department of Analytical Chemistry and CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, No.555, Zuchongzhi Road, Shanghai 201203, China. E-mail addresses: [email protected] (F. Yuan), [email protected] (H. Zhou). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bbapap.2016.04.001 1570-9639/© 2016 Elsevier B.V. All rights reserved.
Corneal neovascularization (CNV) is a sight-threatening condition that is commonly associated with corneal inflammatory, infectious, degenerative and traumatic disorder. However, the molecular mechanisms of CNV are not fully understood and the prognosis is poor. In this study, label-free quantitative proteomics was employed to compare corneas from CNV model with those from normal group, resulting in a total of 2124 proteins identified and 1682 proteins quantified. Of which, 290 proteins were significantly changed between normal and alkali burned corneal tissues with p b 0.05 and fold change N 1.5, including 111 up-regulated and 179 down-regulated proteins in CNV model. Our study showed that glycolysis/gluconeogenesis pathway, regulation of actin cytoskeleton and focal adhesion pathways were the main pathways involved in CNV. Among these significantly changed proteins, 35 proteins were reported or predicted as angiogenesis-related proteins, and 26 proteins were enriched and connected to each other in the protein-protein interaction networks using Ingenuity Pathway Analysis Software. Three proteins associated with CNV were selected and further validated by Western blot. Our study provided increased understanding of angiogenesis-related proteins involved in corneal vascular
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development, which will be useful in the ophthalmic clinic to specifically target angiogenesis.
1. Introduction Corneal neovascularization (CNV) is a sight-threatening condition that is commonly associated with corneal inflammatory, infectious, degenerative and traumatic disorder. Angiogenesis is highly regulated by a delicate balance of naturally occurring pro- and anti-angiogenic factors to maintain ‘angiogenic privilege’ in cornea. Neovascularization occurs when the balance between angiogenic and antiangiogenic factors tend to angiogenic molecules [1]. The angiogenesis factors include vascular endothelial growth factors (VEGFs), fibroblast growth factors (FGFs), platelet-derived growth factors (PDGFs), matrix metalloproteinases (MMPs), and angiopoietins; while the anti-angiogenic factors include soluble VEGF receptors (sVEGFRs), pigment epithelium-derived factor (PEDF), angiostatin, and thrombospondins (TSPs) [2]. Although many regulatory factors have been identified, the molecular mechanisms of CNV are not fully understood and the prognosis is poor. To facilitate the research in the pathogenesis of CNV and to find new clues for developing new therapeutic agents, proteomic technology was applied to profile the protein expression in corneas. Although proteomics studies of normal and disease cornea have been reported in human and mice [3–7], limited studies have been performed on neovascularization following alkali injury [8,9]. Alkali injuries to the cornea are a common approach to model CNV [10]. Parikh et al. performed a proteomic analysis by 1D SDS-PAGE, iTRAQ and mass spectrometry of changes in the proteome of corneal tissue following alkali exposure [8]. Only 15 proteins were identified and had relative quantitation. Wu et al. analyzed recovery following alkali treatment and the protective effect of hyaluronic acid by 2D gel electrophoresis coupled to mass spectrometry; however, this study was performed using culture cells [9]. In this study, a gel-free and label-free quantitative proteomics approach was employed to compare mice corneas following alkali injury versus control to find differentially expressed proteins associated with neovascularization. Three proteins associated with CNV were selected and further validated by Western blot. This study may provide new insight to understand the pathogenesis of CNV and new therapeutic targets. 2. Materials and methods 2.1. Animals Six- to 8-week-old BALB/c mice were obtained from the Shanghai Jiuting Animal Center, Chinese Academy of Sciences and maintained in accordance with the recommendations of the National Institutes of Health (NIH) Guidelines for the Care and Use of Laboratory Animals. All experiments on animals adhered to the ARVO Statements for the Use of Animals in Ophthalmic and Vision Research and were approved by the Animal Ethics Committee of Zhongshan Hospital, Fudan University. 2.2. Induction of corneal neovascularization All mice were anesthetized via pentobarbital sodium (10 mg/mL, 50 mg/kg) by intraperitoneal injection and bilateral topical anesthesia via drops of 0.5% proparacaine hydrochloride (Alcaine eye drops, Alcon Inc., Fort Worth, TX, USA). A circular piece of filter paper with a diameter of 2.5 mm either soaked in 1 M NaOH was placed on the central cornea of the right eye for 30 s immediately followed by a rinse with 30 mL 0.9% saline solution for 30 s, and the untreated eyes were served as controls. Fourteen days following the treatment, the animals were sacrificed, and their eyes were collected.
2.3. Histology Fourteen days after alkali burns, the eyeballs were removed, fixed in 4% paraformaldehyde in 0.1 M phosphate buffer for 24 h, paraffin embedded, and sagittal sectioned at a thickness of 5 μm for routine histological processing. After staining with hematoxylin and eosin, vessels and cell infiltration were examined by light microscopy. Images were captured using a digital camera attached to a Carl Zeiss microscope. 2.4. Centrifugal proteomic reactor 5 individual corneas from alkali-burned group were compared with 5 individual corneas from untreated group. Proteins from corneal tissues were extracted using the lysis buffer (8 M Urea, 4% CHAPS, 50 mM NH4HCO3) by beads-beating at 4 °C·The resulting protein samples were processed by the centrifugal proteomic reactor with minor modifications [11]. Briefly, 20 μg of protein from each sample was diluted and acidified by 1.2 mL of 1% formic acid, mixed with 10 μL of SCX slurry by vigorous vortexing (for 1 min), and the protein samples were absorbed to SCX slurry at such a low pH. The samples was then centrifuged (12,000 rpm, 5 min), and the resulting pellet (containing SCX beads and proteins) was washed 1% formic acid. The samples were reduced by mixing with 50 μL of 150 mM NH4HCO3, 50 mM DTT (shaking at 600 rpm, 56 °C, 15 min), and after reduction the DTT in the sample was diluted using 1.2 mL 1% formic acid and followed by centrifugation. The samples were then subjected to alkylation by mixing with 50 μL of 150 mM NH4HCO3, 100 mM IAA in darkness (20 min, room temperature), and the reaction was stopped by adding 1.2 mL of 0.5% formic acid containing 0.4 μg trypsin. After centrifugation, the resulting pellet was dissolved in 50 μL of 1 M NH4HCO3, and followed by trypsin digestion on a shaker at 600 rpm at 37 °C for 4 h. Finally, 300 μL of 25% NH3H2O and 50% acetonitrile was added to elute the peptides out of the SCX slurry. The digested samples were subjected to nano-LCMS/MS analysis using high energy collision induced dissociation (Orbitrap Elite). 2.5. LC-MS/MS The LC-MS/MS was performed as previous described [12]. Briefly, samples were acidified with formic acid to a final concentration of 0.1% (v/v) and loaded on a 75 μm × 150 mm fused silica column packed in-house with 3-μm ReproSil-Pur C18 beads (120 Å; Dr. Maisch GmbH, Ammerbuch, Germany) using an Easy nano-UPLC 1000 (Thermo Electron, Waltham, MA). The peptides were eluted using a gradient (5–80% acetonitrile with 0.1% formic acid) at a flow rate of 300 nL/min over 240 min period into an nano-ESI Velos Pro-Orbitrap Elite mass spectrometer (Thermo Electron, Waltham, MA). MS/MS spectra were acquired in a data-dependent mode with survey scans acquired at a resolution of 60,000 (m/z 400). Up to the 10 most abundant precursor ions with charge N = 2 from the survey scan were selected with an isolation window of 2.0 Da and fragmented by HCD. 2.6. Data analysis MaxQuant (http://maxquant.org/, Version 1.3.0.5) was used to generate peak lists from raw files, and Andromeda was used to search the protein sequence database using the following parameters: carbamidomethyl (C) was set as a fixed modification, while oxidation (M, +15.99492 Da) was set as a variable modification. Acquired MS/MS spectra were searched against the decoyed mouse International Protein Index protein sequence database (version 3.87, 59,534 protein sequences; European Bioinformatics Institute) augmented with the reversed sequence of each entry in the forward database. The precursor mass tolerances for the first and main searching were set at 20 and 6 ppm respectively, and the fragment mass tolerance for HCD MS/MS spectra was set at 20 ppm. Trypsin/P was selected as the digestive
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enzyme with two potential missed cleavages. Only fully tryptic peptides ranked first were accepted in our database searching results. The false discovery rate (FDR) of peptides and proteins was controlled at b1%. MaxQuant software was used to calculate the label-free quantitation intensity for each protein. The protein LFQ intensity lists of the 10 corneal tissue samples were processed using Perseus software (version 1.2.0.16). The LFQ intensity values were logarithmized (Log2), and the missing value was imputed with random numbers from a normal distribution (width = 0.3, shift = 1.8). Only the proteins detected in N = 3 individual corneal tissues either from normal group or from alkali-burned group were selected as quantified proteins. The p-value of the protein between two cohorts was calculated using Student's t-test by Perseus software, and p b 0.05 and fold change N 1.5 was set as a criterion for the significant change. The differentially expressed proteins were annotated using the Database for Annotation, Visualization and Integrated Discovery (DAVID, v6.7) with the whole murine genome as the background [13]. To investigate the significantly changed proteins pattern in the specific pathway, the proteins were located to cellular components and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway database using DAVID Bioinformatics Resources. The significantly changed proteins were imported to Ingenuity Pathways Analysis software (IPA, version 8.5, Ingenuity Systems, Redwood City, CA), and these proteins were classified by molecular function annotation. Then the proteins with angiogenesis function were selected to generate the protein interaction network using the “Build➔Connect” process in the “Path Designer” module in IPA software. 2.7. Western blotting analysis Fourteen days after alkali burns, mice were sacrificed and their corneas were enucleated for western blotting examinations. Normal corneas served as control group. Corneas were placed into RIPA lysis buffer (Beyotime, Shanghai, China) containing protease inhibitor and phosphatase inhibitors. Protein concentrations were determined by BCA protein assay kit (Beyotime, Shanghai, China). Lysate samples were electrophoretically separated on 15% SDS-PAGE, transferred onto PVDF membrane, blocked with 5% nonfat milk for 1 h and incubated in primary antibody overnight at 4 °C. Antibodies to the following proteins were purchased from Abcam (Cambridge, MA, USA): PEDF (ab180711, ~ 46 kDa, with 1:500 dilution), Lcn-2 (ab63929, ~ 23 kDa, with 1:250 dilution), αB-crystallin (ab13496, ~21 kDa, with 1:1000 dilution). Membranes were washed and incubated with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (1:3000 dilution; ZSGB-BIO, Beijing, China), then detected by chemiluminescence western blotting detection reagents (Pierce ECL and SuperSignal, Thermo Fisher Scientific, Rockford, IL, USA). Each gel was loaded with
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molecular weight markers including proteins molecular weight from 10 to 250 kDa (Bio\\Rad). Each target protein was normalized to β-actin, and relative values were expressed as the protein of interest divided by the loading control. The results were expressed as the mean ± SD. Student's t-test was used for comparisons. p b 0.05 was used as a criterion for statistical significance. 3. Results 3.1. Alkali burn-induced corneal neovascularization altered the corneal proteome Corneal alkali burn is a common approach to model corneal injury and recovery [10]. Following the injury, the area affected by CNV increased over time, progressing most quickly at day 7 and reaching maximal length at day 14 (Fig. 1A). The histological changes following alkali burn included edema of the whole cornea, heavy infiltration and neovessels in corneal stroma (Fig. 1B). To explore the potential pathogenesis of CNV and novel targets for further investigation, we compared the proteomes between the normal corneal tissues and the neovascularized corneal tissues from mice 14 days following alkali burns using label-free quantitative proteomic analysis. In our labelfree quantitative proteomics analysis, the mass tolerance for peptide precursor ions and product ions was set at 6 and 20 ppm, respectively, and the false discovery rates at both peptide and protein levels were controlled to be b1%. Using these strict filtration criteria, a total of 16,126 unique peptides were identified and assigned to 2124 proteins. Moreover, the variation of the retention times across different LC − MS/MS runs were b 1 min, and the variations in global peptide intensities were statistically insignificant, demonstrating the high degree of consistency and reproducibility of this platform. As shown in Supplementary Table 1, the number of MS/MS scans, percentage of identified MS/MS scans, unique peptide sequences and protein groups had relative standard deviations lower than 10% across the 10 LC-MS/MS runs. Therefore, the identification results indicated that the data sets were suitable for comparative label-free analysis. Moreover, the results from the different experiments were also suitable for relative quantitation of proteins. As shown in Fig. 2A, a box plot analysis was applied to compare the Log2LFQ intensity distribution of the 10 individual samples. The mean of Log2LFQ intensity of each individual sample was nearly identical, which indicated that the results of LFQ analysis had no biases toward different samples. As shown in Fig. 2B, the correlation coefficient between two LC-MS/MS runs was higher than 0.79, which demonstrated that relative label-free quantitation was highly reproducible between each sample inside each cohort or between the LC-MS/MS runs from different cohorts. The system variation of our label-free proteomic
Fig. 1. Alkali burn-induced corneal neovascularization. (A) Front view and side view of corneas with or without corneal neovascularization under slit lamp. (B) Hematoxylin-eosin staining of corneas with or without corneal neovascularization. The scale bar represents 20 μm.
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Fig. 2. Box plots of the protein LFQ intensity (log 2) for each sample (A) and scatter plots and correlation between two samples (B). Hierarchical clustering analysis (C) of the 2124 proteins identified by label-free quantitative proteomics from the chemical-burned group (n = 5 mice) and normal group (n = 5 mice), and N: normal corneal tissues; C: chemical-burned corneal tissues.
analysis was well controlled in our experiment procedures, so the resulting data was convincing and can be used for further analysis. Hierarchical clustering analysis (HCA) of LFQ intensity for the 2124 identified proteins revealed clear separation of the chemical-burned group from the control group (Fig. 2C). 3.2. Analysis of 290 significant changed proteins The details for the 1682 quantified proteins were provided in Supplementary Table 2. The protein-level volcano plots of log 2 ratio and the p value between the normal group and chemical-burned group (N and C) were shown in Fig. 3A, and they were very similar to the normal distribution. Among these proteins, 290 proteins (p b 0.05 and fold change N 1.5) were significantly changed with 111 proteins up-regulated and 179 proteins down-regulated in the CNV model. GeneOntology (GO) was utilized to predict the cellular components annotation of these changed proteins using DAVID Bioinformatics
Resources (david.abcc.ncifcrf.gov). As shown in Fig. 3B, most changed proteins were located in cytoplasm, and about 11% (33 proteins) were located in mitochondrion. In addition, 11% (32 proteins) changed proteins were involved in cytoskeleton. The DAVID functional annotation tool was used to map up-regulated or down-regulated proteins to KEGG pathways with enrichment pvalue b 0.05 (Table 1). The up-regulated proteins were mainly involved in focal adhesion, ECM-receptor interaction, glycolysis/gluconeogenesis and oxidative phosphorylation pathways. Glycolysis pathway played an important role in angiogenesis [14]. Glucose, glutamine and alanine transformations sustain nucleotide and fatty acid synthesis. In contrast, hypoxic cells slow down their proliferation, enhance glycolysis to produce ATP and reject lactate which is recycled as fuel innormoxic cells. Thus, glucose is spared for biosynthesis and/or for hypoxic cell function. Emerging studies suggested that, in addition to growth factors, vascular metabolism also regulated angiogenesis and was a viable target for manipulating the microvasculature. Although there were 6 proteins
Fig. 3. (A) Volcano plot of the 1682 quantified proteins from the corneal tissues (the logarithmic ratio of protein LFQ intensities in the C/N tissues were plotted against negative logarithmic p values of the t-test performed from five replicates). (B) Cellular component annotation by Gene Ontology of the 290 significantly changed proteins. N: normal corneal tissues; C: chemical-burned corneal tissues.
M. Shen et al. / Biochimica et Biophysica Acta 1864 (2016) 787–793 Table 1 Enriched KEGG pathways from the significantly changed 290 proteins. Enriched pathways
Enriched p-Value proteins
Enriched pathways of up-regulated proteins in neovascularized corneal tissue mmu04510: focal adhesion 8 7.80E-04 mmu04512: ECM-receptor interaction 6 4.40E-04 mmu05012: Parkinson's disease 6 3.60E-03 mmu00010: glycolysis/gluconeogenesis 5 1.90E-03 mmu03320: PPAR signaling pathway 5 3.20E-03 mmu04540: gap junction 5 4.40E-03 mmu00190: oxidative phosphorylation 5 1.80E-02 mmu04260: cardiac muscle contraction 4 2.30E-02 Enriched pathways of down-regulated proteins in neovascularized corneal tissue mmu04810: regulation of actin cytoskeleton 10 1.40E-03 mmu04510: focal adhesion 9 3.10E-03 mmu00980: metabolism of xenobiotics by cytochrome P450 7 1.60E-04 mmu00982: drug metabolism 7 3.20E-04 mmu05412: arrhythmogenic right ventricular 7 3.20E-04 cardiomyopathy (ARVC) mmu04512: ECM-receptor interaction 6 3.70E-03 mmu04530: tight junction 6 2.70E-02 mmu00010: glycolysis/gluconeogenesis 5 1.00E-02 mmu05414: dilated cardiomyopathy 5 2.80E-02 mmu00350: tyrosine metabolism 4 1.30E-02 mmu00480: glutathione metabolism 4 2.70E-02 mmu00670: one carbon pool by folate 3 1.70E-02 mmu00360: phenylalanine metabolism 3 3.10E-02 mmu00340: histidine metabolism 3 3.90E-02
enriched in Parkinson's disease, these enriched proteins were mainly involved in oxidative phosphorylation. The down-regulated proteins were mainly involved in regulation of actin cytoskeleton and focal adhesion pathways. It indicated that endothelial cells could generate mechanical force in their cytoskeleton and exert it on their extracellular matrix adhesions feed back to modulate cell sensitivity to soluble angiogenic factors, and thereby control vascular development. Our study showed that glycolysis/gluconeogenesis pathway, regulation of actin cytoskeleton and focal adhesion pathways were the main pathways involved in CNV. 3.3. Angiogenesis-related protein analysis and verification by Western blotting Chemical burns resulted in progressive ocular surface inflammation. Even with appropriate management, they still may lead to corneal opacification, neovascularization and visual loss [15]. All of significantly changed angiogenesis-related proteins were listed in Table 2 (p b 0.05, fold change N 1.5). These 35 significantly changed proteins were further analyzed by protein-protein interaction network analysis using Ingenuity Pathway Analysis Software. As shown in Fig. 4A, 26 proteins were enriched and connected to each other in the protein-protein interaction network. Serine protease HTRA1 (ratio C/N: 40.08, p-value: 2.76E-6), which may regulate retinal angiogenesis, was up-regulated in CNV model with N 40-fold increase. The inflammation related proteins, S100 A8 (ratio (C/N): 9.3, p-value: 2.63E-3), S100 A9 (ratio (C/N): 16.4, p-value: 4.40E-4) and S100 A4 (ratio (C/N): 26.77, p-value: 1.68E-3) were also increased in CNV model respectively. The upregulation of αB-crystallin (ratio (C/N): 8.06, p-value: 2.15E-2) and Lcn-2 (ratio (C/N): 2.84, p-value: 4.27E-2) in CNV model was shown in our dataset. Of which, 9 proteins, including CD44 (ratio (C/N): 0.62, p-value: 2.5E-2), G6PD (ratio (C/N): 0.57, p-value: 7.44E-4), FBLN1 (ratio (C/N): 0.63, p-value: 4.59E-3), PAK1 (ratio (C/N): 0.37, p-value: 2.99E-3), SERPINB5 (ratio (C/N): 0.30, p-value: 3.84E-5), SERPINF1(PEDF) (ratio (C/N): 0.56, p-value = 6.39E-3), THBS4 (ratio (C/N): 0.32, p-value: 3.15E-3), TGFBI (ratio (C/N): 0.34, p-value: 6.95E-5) and TGM2 (ratio (C/N): 0.46, p-value: 1.84E-3), were significantly down-regulated in CNV model group. These proteins were further filtered by literature search on cornea-related research field. PEDF
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Table 2 List of the 35 significantly changed angiogenesis-related proteins. IPI number
Gene symbol
IPI00128040 IPI00124096 IPI00222556 IPI00129240 IPI00230768 IPI00403938 IPI00138274 IPI00320420 IPI00470178 IPI00884460 IPI00131223 IPI00308852 IPI00266899 IPI00115941
HTRA1 S100 A4 S100 A9 VTN S100 A8 TNC CRYAB CLU CSRP2 PRKCA THBS2 DAB2 FKBP1A LCN2
Protein name
Serine protease HTRA1 Protein S100-A4 Protein S100-A9 Vitronectin Protein S100-A8 Isoform 1 of Tenascin Alpha-crystallin B chain Clusterin Cysteine and glycine-rich protein 2 Protein kinase C alpha type Thrombospondin-2 Isoform p96 of Disabled homolog 2 Peptidyl-prolyl cis-trans isomerase Neutrophil gelatinase-associated lipocalin IPI00118413 THBS1 Thrombospondin-1 IPI00975193 FN1 Fibronectin 1 IPI00108041 STIM1 Stromal interaction molecule 1 IPI00227299 VIM Vimentin IPI00121209 APOA1 Apolipoprotein A-I IPI00223446 LAMA4 Laminin subunit alpha-4 IPI00322748 FBLN1 Isoform D of Fibulin-1 IPI00410802 CD44 CD44 antigen isoform a precursor IPI00318595 ERAP1 Endoplasmic reticulum aminopeptidase 1 IPI00606550 Ighg2b Isoform 1 of Ig gamma-2B chain C region IPI00228385 G6PD Glucose-6-phosphate 1-dehydrogenase IPI00323035 FBLN5 Fibulin-5 IPI00331088 SERPINF1 Pigment epithelium-derived factor IPI00853896 Tmsb4x Isoform Long of Thymosin beta-4 IPI00229475 JUP Junction plakoglobin IPI00126861 TGM2 Protein-glutamine gamma-glutamyltransferase 2 IPI00132993 PAK1 Serine/threonine-protein kinase PAK 1 IPI00122528 TGFBI Transforming growth factor-beta-induced protein ig-h3 IPI00130445 THBS4 Thrombospondin-4 IPI00229963 SERPINB5 Serpin B5 IPI00121378 ALCAM CD166 antigen
Ratio p-Value (C/N) 40.08 26.77 16.37 15.06 9.31 8.75 8.06 5.49 4.64 4.51 4.45 4.23 3.12 2.84
2.76E-06 1.68E-03 4.40E-04 3.67E-02 2.63E-03 4.21E-08 2.15E-02 1.94E-06 2.15E-03 3.81E-02 1.22E-03 1.32E-02 2.64E-02 4.27E-02
2.48 2.42 2.35 1.83 1.55 1.51 0.63 0.62 0.62
1.08E-03 2.12E-03 2.19E-02 6.99E-03 4.73E-02 7.88E-03 4.59E-03 2.50E-02 4.00E-02
0.61 1.82E-02 0.57 7.44E-04 0.56 0.56 0.52 0.47 0.46
9.93E-04 6.39E-03 4.78E-03 1.97E-05 1.84E-03
0.37 2.99E-03 0.34 6.95E-05 0.32 3.15E-03 0.30 3.84E-05 0.08 1.65E-04
N: normal corneal tissues; C: chemical-burned corneal tissues.
played an important role in anti-angiogenesis and in the physiology and morphology of the transparent lens [16]. Yang et al. reported that decreased expression of PEDF in primary human lens epithelial cells resulted in an increase of αB-crystallin expression, and αB-crystallin is a critical protein for maintaining lens clarity and potential role in angiogenesis [16]. The overexpression of Lcn-2 could up-regulate mesenchymal markers, including vimentin and fibronectin, and significantly increase cell motility and invasiveness [17]. In addition, Lcn-2 was found to be associated with epidermal growth factor-induced epithelio-mesenchymal transition (EMT) and angiogenesis process [17,18]. Thus, we selected these three angiogenesis-related factors (PEDF, αB-crystallin and Lcn-2) from by label-free quantitative proteomic results (Fig. 4B), for further verification by Western blotting analysis. The protein expression of Lcn-2 and αB-crystallin was detected to statistically increase in alkali-burned corneal tissues, while the PEDF protein expression was found to statistically decrease in chemical burned corneal tissues (Fig. 4C). 4. Discussion In this study, we induced corneal neovessels successfully by alkali burns, which is a common animal model for CNV [10]. CNV progressed over time and reached maximal length at day 14. Label-free quantitative proteomic approach was employed to analyze corneal tissue, resulting
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Fig. 4. (A) Angiogenesis-related protein interaction network was generated by Ingenuity Pathways Analysis. (B) Box plot for the LFQ intensity of αB-crystallin, Lcn2 and Serpinf1 (PEDF) in label-free quantitative proteomic analysis. (C) Western blotting of αB-crystallin, Lcn 2, and Serpinf1 (PEDF) in normal corneas and corneas with corneal neovascularization. β-actin was used as a loading control. Data are presented as a ratio of the proteins toβ-actin. (*p b 0.05 vs. normal corneas; n = 3). N: normal corneal tissues; C: chemical-burned corneal tissues.
in 2124 proteins identified and 1682 proteins quantified. Of these quantified proteins, 290 were differentially expressed in CNV model, and these significant changed proteins may be related to the pathogenesis of CNV. In our study, angiogenesis-related factors were analyzed. PEDF has been found to be the most potent endogenous inhibitor of angiogenesis [19]. It has been shown that PEDF can enhance development and survival of photoreceptors, protect ocular neurons against cell death, and inhibit the growth of ocular blood vessels [20]. PEDF can inhibit neovascularization in cornea and neutralizing antibodies to PEDF can stimulate the invasion of new vessels into the cornea [19]. However, the expression of PEDF in ischemic retina is controversial. It was reported that up-regulation of VEGF in combination with down-regulation of PEDF modulated the formation of ocular new vessels in ischemic retinal diseases [21–23], while others found that VEGF and PEDF factors were both increased [24–26]. αB-crystallin, a member of the super-family of small heat shock proteins, is well known as a molecular chaperone. In recent years, it has been proved that αB-crystallin is associated with tumor angiogenesis, metastasis formation and poor prognosis [27–30]. Data from previous studies implicated that αB-crystallin can modulate VEGF expression by stimulating VEGF secretion [30,31], protecting VEGF for proteolytic degradation and maintaining intracrine VEGF signaling [29]. αBcrystallin can also increase vascular survival during tube morphogenesis [27]. Furthermore, recent observations suggested the importance of αBcrystallin in retinal disorders. It has been found that αB-crystallin was expressed in proliferative diabetic retinopathy membranes [32] and in the retinal pigment epithelium cells of patients with early and advanced age-related macular degeneration [33]. In addition, αB-crystallin has been described as a new regulator of leukocyte recruitment [34]. Interestingly, it has been reported that αB-crystallin was also expressed in mammalian corneas and cultured corneal cells [35]. In this study, we found that αB-crystallin was changed significantly in normal and neovascularized corneal tissues through label-free proteomics test and was verified by western blotting. According to the above, we suggested that αB-crystallin was involved with CNV. However, it has been proposed that the development of corneal cellular haze during woundhealing resembles the development of oxidative stress-induced opacity in the crystalline lens [36]. Thus, we wondered whether αB-crystallin was related with corneal repair. Further experiments need to be performed to explore the dynamic changes of αB-crystallin during the pathophysiological processes of CNV, and to confirm the relationship between αB-crystallin and VEGF.
Neutrophil gelatinase-B associated lipocalin (Lcn-2or NGAL) is a 25kD protein of the lipocalin superfamily. Lcn-2 is siderophore-binding antimicrobial protein that is up-regulated in epithelial tissues during inflammation and seems to play an important role in this process [37].Lcn-2 is up-regulated in a number of human cancers. Lcn-2 could regulate hypoxia-inducible factor 1α (HIF-1α) through extracellular signal-regulated kinase (Erk) and induce VEGF through HIF-1α and promote angiogenesis [38]. In addition, the over-expression of Lcn-2 protein could bind to MMP-9, inhibit MMP-9 degradation and increase MMP-9 activity. In turn, MMP-9 activity promotes cancer progression by degrading the basement membranes and extracellular matrix, liberating VEGF, and thus enabling angiogenesis, invasion and metastasis. Lcn-2 may also play a role in tear film. The aqueous humor Lcn-2 levels increased in eyes with idiopathic acute anterior uveitis [39]. Microarray and protein analysis of human pterygium showed that Lcn-2 increased in the pterygium basal epithelial cells. Due to its collocation with MMPs including MMP-9, Lcn-2 may augment the MMPs driven dissolution of Bowman's layer and invasion by limbal basal epithelial cells [40]. In our study, Lcn-2 was up-regulated in alkali-burned corneal tissues, which was in accordance with mass spectrometry-based proteomics results. Mechanisms and mediators of pathologic angiogenesis are thought to differ from physiological angiogenesis, since the latter does not usually carry an inflammatory component [41]. The increment of Lcn-2 may attribute to inflammatory response of cornea and then contribute to alkali burn-induced CNV. 5. Conclusion In summary, we compared the cornea protein expression from 14 days after alkali burned mice corneas and normal ones, resulting in 2124 proteins identified and 1682 proteins quantified. A total of 290 proteins were changed significantly, from which three angiogenesisrelated proteins were selected for validation. Although the effects of these factors during the pathophysiological process of CNV are not fully understood, investigation of molecules relevant in vascular development will be useful in the ophthalmic clinic to specifically target angiogenesis, and treat or prevent corneal neovascularization. Dynamic changes of these proteins during the process of CNV will be analyzed in further research to understand the detailed information during angiogenesis process. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.bbapap.2016.04.001.
M. Shen et al. / Biochimica et Biophysica Acta 1864 (2016) 787–793
Conflict of interest statement The authors have declared no conflict of interest. Transparency document The Transparency document associated with this article can be found, in online version. Acknowledgements This study was supported by The National Natural Science Foundation of China (grant No.21375138, to H.Z.; grant No. 81470637, to F.Y.; grant No. 31300681, to Y.T.), “one hundred talent program” of Chinese Academy of Sciences (to H.Z.), National Science and Technology Major Project of the Ministry of Science and Technology of China (grant No. 2013ZX09507001, to H.Z.) References [1] J.H. Chang, E.E. Gabison, T. Kato, D.T. Azar, Corneal neovascularization, Curr. Opin. Ophthalmol. 12 (2001) 242–249. [2] W. Stevenson, S.F. Cheng, M.H. Dastjerdi, G. Ferrari, R. Dana, Corneal neovascularization and the utility of topical VEGF inhibition: ranibizumab (Lucentis) vs bevacizumab (Avastin), Ocul. Surf. 10 (2012) 67–83. [3] T.F. Dyrlund, E.T. Poulsen, C. Scavenius, C.L. Nikolajsen, I.B. Thogersen, H. Vorum, J.J. Enghild, Human cornea proteome: identification and quantitation of the proteins of the three main layers including epithelium, stroma, and endothelium, J. Proteome Res. 11 (2012) 4231–4239. [4] R. Joseph, O.P. Srivastava, R.R. Pfister, Differential epithelial and stromal protein profiles in keratoconus and normal human corneas, Exp. Eye Res. 92 (2011) 282–298. [5] K. Nielsen, H. Vorum, P. Fagerholm, K. Birkenkamp-Demtroder, B. Honore, N. Ehlers, T.F. Orntoft, Proteome profiling of corneal epithelium and identification of marker proteins for keratoconus, a pilot study, Exp. Eye Res. 82 (2006) 201–209. [6] E.T. Poulsen, T.F. Dyrlund, K. Runager, C. Scavenius, T.P. Krogager, P. Hojrup, I.B. Thogersen, K.W. Sanggaard, H. Vorum, J. Hjortdal, J.J. Enghild, Proteomics of Fuchs' endothelial corneal dystrophy support that the extracellular matrix of Descemet's membrane is disordered, J. Proteome Res. 13 (2014) 4659–4667. [7] O.P. Srivastava, D. Chandrasekaran, R.R. Pfister, Molecular changes in selected epithelial proteins in human keratoconus corneas compared to normal corneas, Mol. Vis. 12 (2006) 1615–1625. [8] T. Parikh, N. Eisner, P. Venugopalan, Q. Yang, B.L. Lam, S.K. Bhattacharya, Proteomic analyses of corneal tissue subjected to alkali exposure, Invest. Ophthalmol. Vis. Sci. 52 (2011) 1819–1831. [9] C.L. Wu, H.C. Chou, J.M. Li, Y.W. Chen, J.H. Chen, Y.H. Chen, H.L. Chan, Hyaluronic acid-dependent protection against alkali-burned human corneal cells, Electrophoresis 34 (2013) 388–396. [10] M. Yoshizuka, Y. Katsume, S. Fujimoto, Y. Ogoh, Dynamic changes of the corneal epithelium and stroma after alkali burning, Kurume Med. J. 28 (1981) 23–27. [11] H. Zhou, F. Wang, Y. Wang, Z. Ning, W. Hou, T.G. Wright, M. Sundaram, S. Zhong, Z. Yao, D. Figeys, Improved recovery and identification of membrane proteins from rat hepatic cells using a centrifugal proteomic reactor, Mol. Cell. Proteomics 10 (2011) (O111 008425). [12] Y. Tao, L. Fang, Y. Yang, H. Jiang, H. Yang, H. Zhang, H. Zhou, Quantitative proteomic analysis reveals the neuroprotective effects of huperzine A for amyloid beta treated neuroblastoma N2a cells, Proteomics 13 (2013) 1314–1324. [13] W. Huang da, B.T. Sherman, R.A. Lempicki, Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources, Nat. Protoc. 4 (2009) 44–57. [14] K. De Bock, M. Georgiadou, S. Schoors, A. Kuchnio, B.W. Wong, A.R. Cantelmo, A. Quaegebeur, B. Ghesquiere, S. Cauwenberghs, G. Eelen, L.K. Phng, I. Betz, B. Tembuyser, K. Brepoels, J. Welti, I. Geudens, I. Segura, B. Cruys, F. Bifari, I. Decimo, R. Blanco, S. Wyns, J. Vangindertael, S. Rocha, R.T. Collins, S. Munck, D. Daelemans, H. Imamura, R. Devlieger, M. Rider, P.P. Van Veldhoven, F. Schuit, R. Bartrons, J. Hofkens, P. Fraisl, S. Telang, R.J. Deberardinis, L. Schoonjans, S. Vinckier, J. Chesney, H. Gerhardt, M. Dewerchin, P. Carmeliet, Role of PFKFB3-driven glycolysis in vessel sprouting, Cell 154 (2013) 651–663. [15] L.F. Martin, E.M. Rocha, S.B. Garcia, J.S. Paula, Topical Brazilian propolis improves corneal wound healing and inflammation in rats following alkali burns, BMC Complement. Alternat. Med. 13 (2013) 337. [16] J. Yang, L. Luo, X. Liu, M.I. Rosenblatt, B. Qu, Y. Liu, Y. Liu, Down regulation of the PEDF gene in human lens epithelium cells changed the expression of proteins vimentin and alphaB-crystallin, Mol. Vis. 16 (2010) 105–112. [17] J. Yang, D.R. Bielenberg, S.J. Rodig, R. Doiron, M.C. Clifton, A.L. Kung, R.K. Strong, D. Zurakowski, M.A. Moses, Lipocalin 2 promotes breast cancer progression, Proc. Natl. Acad. Sci. U. S. A. 106 (2009) 3913–3918.
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Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbapap
Quantitative proteomic analysis of mice corneal tissues reveals angiogenesis-related proteins involved in corneal neovascularization Minqian Shen a,1, Yimin Tao b,c,1, Yifan Feng a, Xing Liu b,c, Fei Yuan a,⁎, Hu Zhou b,c,⁎⁎ a
Department of Ophthalmology, Zhongshan Hospital, Fudan University, Shanghai 200032, China Department of Analytical Chemistry and CAS Key Laboratory of Receptor Research, Shanghai Institute of MateriaMedica, Chinese Academy of Sciences, Shanghai 201203, China SIMMUOMICS Laboratory, Joint Research Laboratory of Translational “OMICS” between Shanghai Institute of Materia Medica, Chinese Academy of Sciences, China and University of Ottawa, Canada b c
a r t i c l e
i n f o
Article history: Received 30 October 2015 Received in revised form 22 March 2016 Accepted 1 April 2016 Available online 2 April 2016 Keywords: Alkali burn Corneal neovascularization Mice Proteomics
a b s t r a c t Corneal neovascularization (CNV) was induced in Balb/c mice by alkali burns in the central area of the cornea with a diameter of 2.5 mm. After fourteen days, the cornea from one eye was collected for histological staining for CNV examination, while the cornea from the other eye of the same mouse was harvested for proteomic analysis. The label-free quantitative proteomic approach was applied to analyze five normal corneal tissues (normal group mice n = 5) and five corresponding neovascularized corneal tissues (model group mice n = 5). A total of 2124 proteins were identified, and 1682 proteins were quantified from these corneal tissues. Among these quantified proteins, 290 proteins were significantly changed between normal and alkali burned corneal tissues. Of these significantly changed proteins, 35 were reported or predicted as angiogenesis-related proteins. Then, these 35 proteins were analyzed using Ingenuity Pathway Analysis Software, resulting in 26 proteins enriched and connected to each other in the protein-protein interaction network, such as Lcn-2, αB-crystallin and Serpinf1 (PEDF). These three significantly changed proteins were selected for further Western blotting validation. Consistent with the quantitative proteomic results, Western blotting showed that Lcn-2 and αB-crystallin were significantly up-regulated in CNV model, while PEDF was down-regulated. This study provided increased understanding of angiogenesis-related proteins involved in corneal vascular development, which will be useful in the ophthalmic clinic of specifically target angiogenesis. © 2016 Elsevier B.V. All rights reserved.
Statement of significance of the study
Abbreviations: CNV, corneal neovascularization; VEGFs, vascular endothelial growth factors; FGFs, fibroblast growth factors; PDGFs, platelet-derived growth factors; MMPs, matrix metalloproteinases; sVEGFRs, soluble VEGF receptors; PEDF, pigment epitheliumderived factor; TSPs, thrombospondins; iTRAQ, isobaric tags for relative and absolute quantitation; ARVO, Association for Research in Vision and Ophthalmology; SCX, strong cation exchanger; DTT, 1,4-Dithiothreitol; IAA, Iodoacetamide; CHAPS, 3-[(3cholamidopropyl)dimethyl-ammonio]-1-propane sulfonate; LC-MS, liquid chromatograph-mass spectrometer; UPLC, ultra performance liquid chromatography; ESI, electrospray ionization; HCD, high-energy collision-induced dissociation; FDR, false discovery rate; DAVID, Database for Annotation, Visualization and Integrated Discovery; HCA, hierarchical clustering analysis; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; IPA, Ingenuity Pathways Analysis; BCA, bicinchonininic acid; PVDF, polyvinylidene fluoride; HRP, horseradish peroxidase; Lcn-2, lipocalin-2; LFQ, label-free quantitation; HCA, hierarchical clustering analysis; HTRA1, HtrA serine peptidase 1; HIF1α, hypoxia-inducible factor 1α; Erk, extracellular signal-regulated kinase. ⁎ Correspondence to: F. Yuan, Department of Ophthalmology, Zhongshan Hospital, Fudan University, No. 180 Fenglin Road, Shanghai 200032, China. ⁎⁎ Correspondence to: H. Zhou, Department of Analytical Chemistry and CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, No.555, Zuchongzhi Road, Shanghai 201203, China. E-mail addresses: [email protected] (F. Yuan), [email protected] (H. Zhou). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bbapap.2016.04.001 1570-9639/© 2016 Elsevier B.V. All rights reserved.
Corneal neovascularization (CNV) is a sight-threatening condition that is commonly associated with corneal inflammatory, infectious, degenerative and traumatic disorder. However, the molecular mechanisms of CNV are not fully understood and the prognosis is poor. In this study, label-free quantitative proteomics was employed to compare corneas from CNV model with those from normal group, resulting in a total of 2124 proteins identified and 1682 proteins quantified. Of which, 290 proteins were significantly changed between normal and alkali burned corneal tissues with p b 0.05 and fold change N 1.5, including 111 up-regulated and 179 down-regulated proteins in CNV model. Our study showed that glycolysis/gluconeogenesis pathway, regulation of actin cytoskeleton and focal adhesion pathways were the main pathways involved in CNV. Among these significantly changed proteins, 35 proteins were reported or predicted as angiogenesis-related proteins, and 26 proteins were enriched and connected to each other in the protein-protein interaction networks using Ingenuity Pathway Analysis Software. Three proteins associated with CNV were selected and further validated by Western blot. Our study provided increased understanding of angiogenesis-related proteins involved in corneal vascular
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development, which will be useful in the ophthalmic clinic to specifically target angiogenesis.
1. Introduction Corneal neovascularization (CNV) is a sight-threatening condition that is commonly associated with corneal inflammatory, infectious, degenerative and traumatic disorder. Angiogenesis is highly regulated by a delicate balance of naturally occurring pro- and anti-angiogenic factors to maintain ‘angiogenic privilege’ in cornea. Neovascularization occurs when the balance between angiogenic and antiangiogenic factors tend to angiogenic molecules [1]. The angiogenesis factors include vascular endothelial growth factors (VEGFs), fibroblast growth factors (FGFs), platelet-derived growth factors (PDGFs), matrix metalloproteinases (MMPs), and angiopoietins; while the anti-angiogenic factors include soluble VEGF receptors (sVEGFRs), pigment epithelium-derived factor (PEDF), angiostatin, and thrombospondins (TSPs) [2]. Although many regulatory factors have been identified, the molecular mechanisms of CNV are not fully understood and the prognosis is poor. To facilitate the research in the pathogenesis of CNV and to find new clues for developing new therapeutic agents, proteomic technology was applied to profile the protein expression in corneas. Although proteomics studies of normal and disease cornea have been reported in human and mice [3–7], limited studies have been performed on neovascularization following alkali injury [8,9]. Alkali injuries to the cornea are a common approach to model CNV [10]. Parikh et al. performed a proteomic analysis by 1D SDS-PAGE, iTRAQ and mass spectrometry of changes in the proteome of corneal tissue following alkali exposure [8]. Only 15 proteins were identified and had relative quantitation. Wu et al. analyzed recovery following alkali treatment and the protective effect of hyaluronic acid by 2D gel electrophoresis coupled to mass spectrometry; however, this study was performed using culture cells [9]. In this study, a gel-free and label-free quantitative proteomics approach was employed to compare mice corneas following alkali injury versus control to find differentially expressed proteins associated with neovascularization. Three proteins associated with CNV were selected and further validated by Western blot. This study may provide new insight to understand the pathogenesis of CNV and new therapeutic targets. 2. Materials and methods 2.1. Animals Six- to 8-week-old BALB/c mice were obtained from the Shanghai Jiuting Animal Center, Chinese Academy of Sciences and maintained in accordance with the recommendations of the National Institutes of Health (NIH) Guidelines for the Care and Use of Laboratory Animals. All experiments on animals adhered to the ARVO Statements for the Use of Animals in Ophthalmic and Vision Research and were approved by the Animal Ethics Committee of Zhongshan Hospital, Fudan University. 2.2. Induction of corneal neovascularization All mice were anesthetized via pentobarbital sodium (10 mg/mL, 50 mg/kg) by intraperitoneal injection and bilateral topical anesthesia via drops of 0.5% proparacaine hydrochloride (Alcaine eye drops, Alcon Inc., Fort Worth, TX, USA). A circular piece of filter paper with a diameter of 2.5 mm either soaked in 1 M NaOH was placed on the central cornea of the right eye for 30 s immediately followed by a rinse with 30 mL 0.9% saline solution for 30 s, and the untreated eyes were served as controls. Fourteen days following the treatment, the animals were sacrificed, and their eyes were collected.
2.3. Histology Fourteen days after alkali burns, the eyeballs were removed, fixed in 4% paraformaldehyde in 0.1 M phosphate buffer for 24 h, paraffin embedded, and sagittal sectioned at a thickness of 5 μm for routine histological processing. After staining with hematoxylin and eosin, vessels and cell infiltration were examined by light microscopy. Images were captured using a digital camera attached to a Carl Zeiss microscope. 2.4. Centrifugal proteomic reactor 5 individual corneas from alkali-burned group were compared with 5 individual corneas from untreated group. Proteins from corneal tissues were extracted using the lysis buffer (8 M Urea, 4% CHAPS, 50 mM NH4HCO3) by beads-beating at 4 °C·The resulting protein samples were processed by the centrifugal proteomic reactor with minor modifications [11]. Briefly, 20 μg of protein from each sample was diluted and acidified by 1.2 mL of 1% formic acid, mixed with 10 μL of SCX slurry by vigorous vortexing (for 1 min), and the protein samples were absorbed to SCX slurry at such a low pH. The samples was then centrifuged (12,000 rpm, 5 min), and the resulting pellet (containing SCX beads and proteins) was washed 1% formic acid. The samples were reduced by mixing with 50 μL of 150 mM NH4HCO3, 50 mM DTT (shaking at 600 rpm, 56 °C, 15 min), and after reduction the DTT in the sample was diluted using 1.2 mL 1% formic acid and followed by centrifugation. The samples were then subjected to alkylation by mixing with 50 μL of 150 mM NH4HCO3, 100 mM IAA in darkness (20 min, room temperature), and the reaction was stopped by adding 1.2 mL of 0.5% formic acid containing 0.4 μg trypsin. After centrifugation, the resulting pellet was dissolved in 50 μL of 1 M NH4HCO3, and followed by trypsin digestion on a shaker at 600 rpm at 37 °C for 4 h. Finally, 300 μL of 25% NH3H2O and 50% acetonitrile was added to elute the peptides out of the SCX slurry. The digested samples were subjected to nano-LCMS/MS analysis using high energy collision induced dissociation (Orbitrap Elite). 2.5. LC-MS/MS The LC-MS/MS was performed as previous described [12]. Briefly, samples were acidified with formic acid to a final concentration of 0.1% (v/v) and loaded on a 75 μm × 150 mm fused silica column packed in-house with 3-μm ReproSil-Pur C18 beads (120 Å; Dr. Maisch GmbH, Ammerbuch, Germany) using an Easy nano-UPLC 1000 (Thermo Electron, Waltham, MA). The peptides were eluted using a gradient (5–80% acetonitrile with 0.1% formic acid) at a flow rate of 300 nL/min over 240 min period into an nano-ESI Velos Pro-Orbitrap Elite mass spectrometer (Thermo Electron, Waltham, MA). MS/MS spectra were acquired in a data-dependent mode with survey scans acquired at a resolution of 60,000 (m/z 400). Up to the 10 most abundant precursor ions with charge N = 2 from the survey scan were selected with an isolation window of 2.0 Da and fragmented by HCD. 2.6. Data analysis MaxQuant (http://maxquant.org/, Version 1.3.0.5) was used to generate peak lists from raw files, and Andromeda was used to search the protein sequence database using the following parameters: carbamidomethyl (C) was set as a fixed modification, while oxidation (M, +15.99492 Da) was set as a variable modification. Acquired MS/MS spectra were searched against the decoyed mouse International Protein Index protein sequence database (version 3.87, 59,534 protein sequences; European Bioinformatics Institute) augmented with the reversed sequence of each entry in the forward database. The precursor mass tolerances for the first and main searching were set at 20 and 6 ppm respectively, and the fragment mass tolerance for HCD MS/MS spectra was set at 20 ppm. Trypsin/P was selected as the digestive
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enzyme with two potential missed cleavages. Only fully tryptic peptides ranked first were accepted in our database searching results. The false discovery rate (FDR) of peptides and proteins was controlled at b1%. MaxQuant software was used to calculate the label-free quantitation intensity for each protein. The protein LFQ intensity lists of the 10 corneal tissue samples were processed using Perseus software (version 1.2.0.16). The LFQ intensity values were logarithmized (Log2), and the missing value was imputed with random numbers from a normal distribution (width = 0.3, shift = 1.8). Only the proteins detected in N = 3 individual corneal tissues either from normal group or from alkali-burned group were selected as quantified proteins. The p-value of the protein between two cohorts was calculated using Student's t-test by Perseus software, and p b 0.05 and fold change N 1.5 was set as a criterion for the significant change. The differentially expressed proteins were annotated using the Database for Annotation, Visualization and Integrated Discovery (DAVID, v6.7) with the whole murine genome as the background [13]. To investigate the significantly changed proteins pattern in the specific pathway, the proteins were located to cellular components and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway database using DAVID Bioinformatics Resources. The significantly changed proteins were imported to Ingenuity Pathways Analysis software (IPA, version 8.5, Ingenuity Systems, Redwood City, CA), and these proteins were classified by molecular function annotation. Then the proteins with angiogenesis function were selected to generate the protein interaction network using the “Build➔Connect” process in the “Path Designer” module in IPA software. 2.7. Western blotting analysis Fourteen days after alkali burns, mice were sacrificed and their corneas were enucleated for western blotting examinations. Normal corneas served as control group. Corneas were placed into RIPA lysis buffer (Beyotime, Shanghai, China) containing protease inhibitor and phosphatase inhibitors. Protein concentrations were determined by BCA protein assay kit (Beyotime, Shanghai, China). Lysate samples were electrophoretically separated on 15% SDS-PAGE, transferred onto PVDF membrane, blocked with 5% nonfat milk for 1 h and incubated in primary antibody overnight at 4 °C. Antibodies to the following proteins were purchased from Abcam (Cambridge, MA, USA): PEDF (ab180711, ~ 46 kDa, with 1:500 dilution), Lcn-2 (ab63929, ~ 23 kDa, with 1:250 dilution), αB-crystallin (ab13496, ~21 kDa, with 1:1000 dilution). Membranes were washed and incubated with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (1:3000 dilution; ZSGB-BIO, Beijing, China), then detected by chemiluminescence western blotting detection reagents (Pierce ECL and SuperSignal, Thermo Fisher Scientific, Rockford, IL, USA). Each gel was loaded with
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molecular weight markers including proteins molecular weight from 10 to 250 kDa (Bio\\Rad). Each target protein was normalized to β-actin, and relative values were expressed as the protein of interest divided by the loading control. The results were expressed as the mean ± SD. Student's t-test was used for comparisons. p b 0.05 was used as a criterion for statistical significance. 3. Results 3.1. Alkali burn-induced corneal neovascularization altered the corneal proteome Corneal alkali burn is a common approach to model corneal injury and recovery [10]. Following the injury, the area affected by CNV increased over time, progressing most quickly at day 7 and reaching maximal length at day 14 (Fig. 1A). The histological changes following alkali burn included edema of the whole cornea, heavy infiltration and neovessels in corneal stroma (Fig. 1B). To explore the potential pathogenesis of CNV and novel targets for further investigation, we compared the proteomes between the normal corneal tissues and the neovascularized corneal tissues from mice 14 days following alkali burns using label-free quantitative proteomic analysis. In our labelfree quantitative proteomics analysis, the mass tolerance for peptide precursor ions and product ions was set at 6 and 20 ppm, respectively, and the false discovery rates at both peptide and protein levels were controlled to be b1%. Using these strict filtration criteria, a total of 16,126 unique peptides were identified and assigned to 2124 proteins. Moreover, the variation of the retention times across different LC − MS/MS runs were b 1 min, and the variations in global peptide intensities were statistically insignificant, demonstrating the high degree of consistency and reproducibility of this platform. As shown in Supplementary Table 1, the number of MS/MS scans, percentage of identified MS/MS scans, unique peptide sequences and protein groups had relative standard deviations lower than 10% across the 10 LC-MS/MS runs. Therefore, the identification results indicated that the data sets were suitable for comparative label-free analysis. Moreover, the results from the different experiments were also suitable for relative quantitation of proteins. As shown in Fig. 2A, a box plot analysis was applied to compare the Log2LFQ intensity distribution of the 10 individual samples. The mean of Log2LFQ intensity of each individual sample was nearly identical, which indicated that the results of LFQ analysis had no biases toward different samples. As shown in Fig. 2B, the correlation coefficient between two LC-MS/MS runs was higher than 0.79, which demonstrated that relative label-free quantitation was highly reproducible between each sample inside each cohort or between the LC-MS/MS runs from different cohorts. The system variation of our label-free proteomic
Fig. 1. Alkali burn-induced corneal neovascularization. (A) Front view and side view of corneas with or without corneal neovascularization under slit lamp. (B) Hematoxylin-eosin staining of corneas with or without corneal neovascularization. The scale bar represents 20 μm.
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Fig. 2. Box plots of the protein LFQ intensity (log 2) for each sample (A) and scatter plots and correlation between two samples (B). Hierarchical clustering analysis (C) of the 2124 proteins identified by label-free quantitative proteomics from the chemical-burned group (n = 5 mice) and normal group (n = 5 mice), and N: normal corneal tissues; C: chemical-burned corneal tissues.
analysis was well controlled in our experiment procedures, so the resulting data was convincing and can be used for further analysis. Hierarchical clustering analysis (HCA) of LFQ intensity for the 2124 identified proteins revealed clear separation of the chemical-burned group from the control group (Fig. 2C). 3.2. Analysis of 290 significant changed proteins The details for the 1682 quantified proteins were provided in Supplementary Table 2. The protein-level volcano plots of log 2 ratio and the p value between the normal group and chemical-burned group (N and C) were shown in Fig. 3A, and they were very similar to the normal distribution. Among these proteins, 290 proteins (p b 0.05 and fold change N 1.5) were significantly changed with 111 proteins up-regulated and 179 proteins down-regulated in the CNV model. GeneOntology (GO) was utilized to predict the cellular components annotation of these changed proteins using DAVID Bioinformatics
Resources (david.abcc.ncifcrf.gov). As shown in Fig. 3B, most changed proteins were located in cytoplasm, and about 11% (33 proteins) were located in mitochondrion. In addition, 11% (32 proteins) changed proteins were involved in cytoskeleton. The DAVID functional annotation tool was used to map up-regulated or down-regulated proteins to KEGG pathways with enrichment pvalue b 0.05 (Table 1). The up-regulated proteins were mainly involved in focal adhesion, ECM-receptor interaction, glycolysis/gluconeogenesis and oxidative phosphorylation pathways. Glycolysis pathway played an important role in angiogenesis [14]. Glucose, glutamine and alanine transformations sustain nucleotide and fatty acid synthesis. In contrast, hypoxic cells slow down their proliferation, enhance glycolysis to produce ATP and reject lactate which is recycled as fuel innormoxic cells. Thus, glucose is spared for biosynthesis and/or for hypoxic cell function. Emerging studies suggested that, in addition to growth factors, vascular metabolism also regulated angiogenesis and was a viable target for manipulating the microvasculature. Although there were 6 proteins
Fig. 3. (A) Volcano plot of the 1682 quantified proteins from the corneal tissues (the logarithmic ratio of protein LFQ intensities in the C/N tissues were plotted against negative logarithmic p values of the t-test performed from five replicates). (B) Cellular component annotation by Gene Ontology of the 290 significantly changed proteins. N: normal corneal tissues; C: chemical-burned corneal tissues.
M. Shen et al. / Biochimica et Biophysica Acta 1864 (2016) 787–793 Table 1 Enriched KEGG pathways from the significantly changed 290 proteins. Enriched pathways
Enriched p-Value proteins
Enriched pathways of up-regulated proteins in neovascularized corneal tissue mmu04510: focal adhesion 8 7.80E-04 mmu04512: ECM-receptor interaction 6 4.40E-04 mmu05012: Parkinson's disease 6 3.60E-03 mmu00010: glycolysis/gluconeogenesis 5 1.90E-03 mmu03320: PPAR signaling pathway 5 3.20E-03 mmu04540: gap junction 5 4.40E-03 mmu00190: oxidative phosphorylation 5 1.80E-02 mmu04260: cardiac muscle contraction 4 2.30E-02 Enriched pathways of down-regulated proteins in neovascularized corneal tissue mmu04810: regulation of actin cytoskeleton 10 1.40E-03 mmu04510: focal adhesion 9 3.10E-03 mmu00980: metabolism of xenobiotics by cytochrome P450 7 1.60E-04 mmu00982: drug metabolism 7 3.20E-04 mmu05412: arrhythmogenic right ventricular 7 3.20E-04 cardiomyopathy (ARVC) mmu04512: ECM-receptor interaction 6 3.70E-03 mmu04530: tight junction 6 2.70E-02 mmu00010: glycolysis/gluconeogenesis 5 1.00E-02 mmu05414: dilated cardiomyopathy 5 2.80E-02 mmu00350: tyrosine metabolism 4 1.30E-02 mmu00480: glutathione metabolism 4 2.70E-02 mmu00670: one carbon pool by folate 3 1.70E-02 mmu00360: phenylalanine metabolism 3 3.10E-02 mmu00340: histidine metabolism 3 3.90E-02
enriched in Parkinson's disease, these enriched proteins were mainly involved in oxidative phosphorylation. The down-regulated proteins were mainly involved in regulation of actin cytoskeleton and focal adhesion pathways. It indicated that endothelial cells could generate mechanical force in their cytoskeleton and exert it on their extracellular matrix adhesions feed back to modulate cell sensitivity to soluble angiogenic factors, and thereby control vascular development. Our study showed that glycolysis/gluconeogenesis pathway, regulation of actin cytoskeleton and focal adhesion pathways were the main pathways involved in CNV. 3.3. Angiogenesis-related protein analysis and verification by Western blotting Chemical burns resulted in progressive ocular surface inflammation. Even with appropriate management, they still may lead to corneal opacification, neovascularization and visual loss [15]. All of significantly changed angiogenesis-related proteins were listed in Table 2 (p b 0.05, fold change N 1.5). These 35 significantly changed proteins were further analyzed by protein-protein interaction network analysis using Ingenuity Pathway Analysis Software. As shown in Fig. 4A, 26 proteins were enriched and connected to each other in the protein-protein interaction network. Serine protease HTRA1 (ratio C/N: 40.08, p-value: 2.76E-6), which may regulate retinal angiogenesis, was up-regulated in CNV model with N 40-fold increase. The inflammation related proteins, S100 A8 (ratio (C/N): 9.3, p-value: 2.63E-3), S100 A9 (ratio (C/N): 16.4, p-value: 4.40E-4) and S100 A4 (ratio (C/N): 26.77, p-value: 1.68E-3) were also increased in CNV model respectively. The upregulation of αB-crystallin (ratio (C/N): 8.06, p-value: 2.15E-2) and Lcn-2 (ratio (C/N): 2.84, p-value: 4.27E-2) in CNV model was shown in our dataset. Of which, 9 proteins, including CD44 (ratio (C/N): 0.62, p-value: 2.5E-2), G6PD (ratio (C/N): 0.57, p-value: 7.44E-4), FBLN1 (ratio (C/N): 0.63, p-value: 4.59E-3), PAK1 (ratio (C/N): 0.37, p-value: 2.99E-3), SERPINB5 (ratio (C/N): 0.30, p-value: 3.84E-5), SERPINF1(PEDF) (ratio (C/N): 0.56, p-value = 6.39E-3), THBS4 (ratio (C/N): 0.32, p-value: 3.15E-3), TGFBI (ratio (C/N): 0.34, p-value: 6.95E-5) and TGM2 (ratio (C/N): 0.46, p-value: 1.84E-3), were significantly down-regulated in CNV model group. These proteins were further filtered by literature search on cornea-related research field. PEDF
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Table 2 List of the 35 significantly changed angiogenesis-related proteins. IPI number
Gene symbol
IPI00128040 IPI00124096 IPI00222556 IPI00129240 IPI00230768 IPI00403938 IPI00138274 IPI00320420 IPI00470178 IPI00884460 IPI00131223 IPI00308852 IPI00266899 IPI00115941
HTRA1 S100 A4 S100 A9 VTN S100 A8 TNC CRYAB CLU CSRP2 PRKCA THBS2 DAB2 FKBP1A LCN2
Protein name
Serine protease HTRA1 Protein S100-A4 Protein S100-A9 Vitronectin Protein S100-A8 Isoform 1 of Tenascin Alpha-crystallin B chain Clusterin Cysteine and glycine-rich protein 2 Protein kinase C alpha type Thrombospondin-2 Isoform p96 of Disabled homolog 2 Peptidyl-prolyl cis-trans isomerase Neutrophil gelatinase-associated lipocalin IPI00118413 THBS1 Thrombospondin-1 IPI00975193 FN1 Fibronectin 1 IPI00108041 STIM1 Stromal interaction molecule 1 IPI00227299 VIM Vimentin IPI00121209 APOA1 Apolipoprotein A-I IPI00223446 LAMA4 Laminin subunit alpha-4 IPI00322748 FBLN1 Isoform D of Fibulin-1 IPI00410802 CD44 CD44 antigen isoform a precursor IPI00318595 ERAP1 Endoplasmic reticulum aminopeptidase 1 IPI00606550 Ighg2b Isoform 1 of Ig gamma-2B chain C region IPI00228385 G6PD Glucose-6-phosphate 1-dehydrogenase IPI00323035 FBLN5 Fibulin-5 IPI00331088 SERPINF1 Pigment epithelium-derived factor IPI00853896 Tmsb4x Isoform Long of Thymosin beta-4 IPI00229475 JUP Junction plakoglobin IPI00126861 TGM2 Protein-glutamine gamma-glutamyltransferase 2 IPI00132993 PAK1 Serine/threonine-protein kinase PAK 1 IPI00122528 TGFBI Transforming growth factor-beta-induced protein ig-h3 IPI00130445 THBS4 Thrombospondin-4 IPI00229963 SERPINB5 Serpin B5 IPI00121378 ALCAM CD166 antigen
Ratio p-Value (C/N) 40.08 26.77 16.37 15.06 9.31 8.75 8.06 5.49 4.64 4.51 4.45 4.23 3.12 2.84
2.76E-06 1.68E-03 4.40E-04 3.67E-02 2.63E-03 4.21E-08 2.15E-02 1.94E-06 2.15E-03 3.81E-02 1.22E-03 1.32E-02 2.64E-02 4.27E-02
2.48 2.42 2.35 1.83 1.55 1.51 0.63 0.62 0.62
1.08E-03 2.12E-03 2.19E-02 6.99E-03 4.73E-02 7.88E-03 4.59E-03 2.50E-02 4.00E-02
0.61 1.82E-02 0.57 7.44E-04 0.56 0.56 0.52 0.47 0.46
9.93E-04 6.39E-03 4.78E-03 1.97E-05 1.84E-03
0.37 2.99E-03 0.34 6.95E-05 0.32 3.15E-03 0.30 3.84E-05 0.08 1.65E-04
N: normal corneal tissues; C: chemical-burned corneal tissues.
played an important role in anti-angiogenesis and in the physiology and morphology of the transparent lens [16]. Yang et al. reported that decreased expression of PEDF in primary human lens epithelial cells resulted in an increase of αB-crystallin expression, and αB-crystallin is a critical protein for maintaining lens clarity and potential role in angiogenesis [16]. The overexpression of Lcn-2 could up-regulate mesenchymal markers, including vimentin and fibronectin, and significantly increase cell motility and invasiveness [17]. In addition, Lcn-2 was found to be associated with epidermal growth factor-induced epithelio-mesenchymal transition (EMT) and angiogenesis process [17,18]. Thus, we selected these three angiogenesis-related factors (PEDF, αB-crystallin and Lcn-2) from by label-free quantitative proteomic results (Fig. 4B), for further verification by Western blotting analysis. The protein expression of Lcn-2 and αB-crystallin was detected to statistically increase in alkali-burned corneal tissues, while the PEDF protein expression was found to statistically decrease in chemical burned corneal tissues (Fig. 4C). 4. Discussion In this study, we induced corneal neovessels successfully by alkali burns, which is a common animal model for CNV [10]. CNV progressed over time and reached maximal length at day 14. Label-free quantitative proteomic approach was employed to analyze corneal tissue, resulting
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Fig. 4. (A) Angiogenesis-related protein interaction network was generated by Ingenuity Pathways Analysis. (B) Box plot for the LFQ intensity of αB-crystallin, Lcn2 and Serpinf1 (PEDF) in label-free quantitative proteomic analysis. (C) Western blotting of αB-crystallin, Lcn 2, and Serpinf1 (PEDF) in normal corneas and corneas with corneal neovascularization. β-actin was used as a loading control. Data are presented as a ratio of the proteins toβ-actin. (*p b 0.05 vs. normal corneas; n = 3). N: normal corneal tissues; C: chemical-burned corneal tissues.
in 2124 proteins identified and 1682 proteins quantified. Of these quantified proteins, 290 were differentially expressed in CNV model, and these significant changed proteins may be related to the pathogenesis of CNV. In our study, angiogenesis-related factors were analyzed. PEDF has been found to be the most potent endogenous inhibitor of angiogenesis [19]. It has been shown that PEDF can enhance development and survival of photoreceptors, protect ocular neurons against cell death, and inhibit the growth of ocular blood vessels [20]. PEDF can inhibit neovascularization in cornea and neutralizing antibodies to PEDF can stimulate the invasion of new vessels into the cornea [19]. However, the expression of PEDF in ischemic retina is controversial. It was reported that up-regulation of VEGF in combination with down-regulation of PEDF modulated the formation of ocular new vessels in ischemic retinal diseases [21–23], while others found that VEGF and PEDF factors were both increased [24–26]. αB-crystallin, a member of the super-family of small heat shock proteins, is well known as a molecular chaperone. In recent years, it has been proved that αB-crystallin is associated with tumor angiogenesis, metastasis formation and poor prognosis [27–30]. Data from previous studies implicated that αB-crystallin can modulate VEGF expression by stimulating VEGF secretion [30,31], protecting VEGF for proteolytic degradation and maintaining intracrine VEGF signaling [29]. αBcrystallin can also increase vascular survival during tube morphogenesis [27]. Furthermore, recent observations suggested the importance of αBcrystallin in retinal disorders. It has been found that αB-crystallin was expressed in proliferative diabetic retinopathy membranes [32] and in the retinal pigment epithelium cells of patients with early and advanced age-related macular degeneration [33]. In addition, αB-crystallin has been described as a new regulator of leukocyte recruitment [34]. Interestingly, it has been reported that αB-crystallin was also expressed in mammalian corneas and cultured corneal cells [35]. In this study, we found that αB-crystallin was changed significantly in normal and neovascularized corneal tissues through label-free proteomics test and was verified by western blotting. According to the above, we suggested that αB-crystallin was involved with CNV. However, it has been proposed that the development of corneal cellular haze during woundhealing resembles the development of oxidative stress-induced opacity in the crystalline lens [36]. Thus, we wondered whether αB-crystallin was related with corneal repair. Further experiments need to be performed to explore the dynamic changes of αB-crystallin during the pathophysiological processes of CNV, and to confirm the relationship between αB-crystallin and VEGF.
Neutrophil gelatinase-B associated lipocalin (Lcn-2or NGAL) is a 25kD protein of the lipocalin superfamily. Lcn-2 is siderophore-binding antimicrobial protein that is up-regulated in epithelial tissues during inflammation and seems to play an important role in this process [37].Lcn-2 is up-regulated in a number of human cancers. Lcn-2 could regulate hypoxia-inducible factor 1α (HIF-1α) through extracellular signal-regulated kinase (Erk) and induce VEGF through HIF-1α and promote angiogenesis [38]. In addition, the over-expression of Lcn-2 protein could bind to MMP-9, inhibit MMP-9 degradation and increase MMP-9 activity. In turn, MMP-9 activity promotes cancer progression by degrading the basement membranes and extracellular matrix, liberating VEGF, and thus enabling angiogenesis, invasion and metastasis. Lcn-2 may also play a role in tear film. The aqueous humor Lcn-2 levels increased in eyes with idiopathic acute anterior uveitis [39]. Microarray and protein analysis of human pterygium showed that Lcn-2 increased in the pterygium basal epithelial cells. Due to its collocation with MMPs including MMP-9, Lcn-2 may augment the MMPs driven dissolution of Bowman's layer and invasion by limbal basal epithelial cells [40]. In our study, Lcn-2 was up-regulated in alkali-burned corneal tissues, which was in accordance with mass spectrometry-based proteomics results. Mechanisms and mediators of pathologic angiogenesis are thought to differ from physiological angiogenesis, since the latter does not usually carry an inflammatory component [41]. The increment of Lcn-2 may attribute to inflammatory response of cornea and then contribute to alkali burn-induced CNV. 5. Conclusion In summary, we compared the cornea protein expression from 14 days after alkali burned mice corneas and normal ones, resulting in 2124 proteins identified and 1682 proteins quantified. A total of 290 proteins were changed significantly, from which three angiogenesisrelated proteins were selected for validation. Although the effects of these factors during the pathophysiological process of CNV are not fully understood, investigation of molecules relevant in vascular development will be useful in the ophthalmic clinic to specifically target angiogenesis, and treat or prevent corneal neovascularization. Dynamic changes of these proteins during the process of CNV will be analyzed in further research to understand the detailed information during angiogenesis process. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.bbapap.2016.04.001.
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Conflict of interest statement The authors have declared no conflict of interest. Transparency document The Transparency document associated with this article can be found, in online version. Acknowledgements This study was supported by The National Natural Science Foundation of China (grant No.21375138, to H.Z.; grant No. 81470637, to F.Y.; grant No. 31300681, to Y.T.), “one hundred talent program” of Chinese Academy of Sciences (to H.Z.), National Science and Technology Major Project of the Ministry of Science and Technology of China (grant No. 2013ZX09507001, to H.Z.) References [1] J.H. Chang, E.E. Gabison, T. Kato, D.T. Azar, Corneal neovascularization, Curr. Opin. Ophthalmol. 12 (2001) 242–249. [2] W. Stevenson, S.F. Cheng, M.H. Dastjerdi, G. Ferrari, R. Dana, Corneal neovascularization and the utility of topical VEGF inhibition: ranibizumab (Lucentis) vs bevacizumab (Avastin), Ocul. Surf. 10 (2012) 67–83. [3] T.F. Dyrlund, E.T. Poulsen, C. Scavenius, C.L. Nikolajsen, I.B. Thogersen, H. Vorum, J.J. Enghild, Human cornea proteome: identification and quantitation of the proteins of the three main layers including epithelium, stroma, and endothelium, J. Proteome Res. 11 (2012) 4231–4239. [4] R. Joseph, O.P. Srivastava, R.R. Pfister, Differential epithelial and stromal protein profiles in keratoconus and normal human corneas, Exp. Eye Res. 92 (2011) 282–298. [5] K. Nielsen, H. Vorum, P. Fagerholm, K. Birkenkamp-Demtroder, B. Honore, N. Ehlers, T.F. Orntoft, Proteome profiling of corneal epithelium and identification of marker proteins for keratoconus, a pilot study, Exp. Eye Res. 82 (2006) 201–209. [6] E.T. Poulsen, T.F. Dyrlund, K. Runager, C. Scavenius, T.P. Krogager, P. Hojrup, I.B. Thogersen, K.W. Sanggaard, H. Vorum, J. Hjortdal, J.J. Enghild, Proteomics of Fuchs' endothelial corneal dystrophy support that the extracellular matrix of Descemet's membrane is disordered, J. Proteome Res. 13 (2014) 4659–4667. [7] O.P. Srivastava, D. Chandrasekaran, R.R. Pfister, Molecular changes in selected epithelial proteins in human keratoconus corneas compared to normal corneas, Mol. Vis. 12 (2006) 1615–1625. [8] T. Parikh, N. Eisner, P. Venugopalan, Q. Yang, B.L. Lam, S.K. Bhattacharya, Proteomic analyses of corneal tissue subjected to alkali exposure, Invest. Ophthalmol. Vis. Sci. 52 (2011) 1819–1831. [9] C.L. Wu, H.C. Chou, J.M. Li, Y.W. Chen, J.H. Chen, Y.H. Chen, H.L. Chan, Hyaluronic acid-dependent protection against alkali-burned human corneal cells, Electrophoresis 34 (2013) 388–396. [10] M. Yoshizuka, Y. Katsume, S. Fujimoto, Y. Ogoh, Dynamic changes of the corneal epithelium and stroma after alkali burning, Kurume Med. J. 28 (1981) 23–27. [11] H. Zhou, F. Wang, Y. Wang, Z. Ning, W. Hou, T.G. Wright, M. Sundaram, S. Zhong, Z. Yao, D. Figeys, Improved recovery and identification of membrane proteins from rat hepatic cells using a centrifugal proteomic reactor, Mol. Cell. Proteomics 10 (2011) (O111 008425). [12] Y. Tao, L. Fang, Y. Yang, H. Jiang, H. Yang, H. Zhang, H. Zhou, Quantitative proteomic analysis reveals the neuroprotective effects of huperzine A for amyloid beta treated neuroblastoma N2a cells, Proteomics 13 (2013) 1314–1324. [13] W. Huang da, B.T. Sherman, R.A. Lempicki, Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources, Nat. Protoc. 4 (2009) 44–57. [14] K. De Bock, M. Georgiadou, S. Schoors, A. Kuchnio, B.W. Wong, A.R. Cantelmo, A. Quaegebeur, B. Ghesquiere, S. Cauwenberghs, G. Eelen, L.K. Phng, I. Betz, B. Tembuyser, K. Brepoels, J. Welti, I. Geudens, I. Segura, B. Cruys, F. Bifari, I. Decimo, R. Blanco, S. Wyns, J. Vangindertael, S. Rocha, R.T. Collins, S. Munck, D. Daelemans, H. Imamura, R. Devlieger, M. Rider, P.P. Van Veldhoven, F. Schuit, R. Bartrons, J. Hofkens, P. Fraisl, S. Telang, R.J. Deberardinis, L. Schoonjans, S. Vinckier, J. Chesney, H. Gerhardt, M. Dewerchin, P. Carmeliet, Role of PFKFB3-driven glycolysis in vessel sprouting, Cell 154 (2013) 651–663. [15] L.F. Martin, E.M. Rocha, S.B. Garcia, J.S. Paula, Topical Brazilian propolis improves corneal wound healing and inflammation in rats following alkali burns, BMC Complement. Alternat. Med. 13 (2013) 337. [16] J. Yang, L. Luo, X. Liu, M.I. Rosenblatt, B. Qu, Y. Liu, Y. Liu, Down regulation of the PEDF gene in human lens epithelium cells changed the expression of proteins vimentin and alphaB-crystallin, Mol. Vis. 16 (2010) 105–112. [17] J. Yang, D.R. Bielenberg, S.J. Rodig, R. Doiron, M.C. Clifton, A.L. Kung, R.K. Strong, D. Zurakowski, M.A. Moses, Lipocalin 2 promotes breast cancer progression, Proc. Natl. Acad. Sci. U. S. A. 106 (2009) 3913–3918.
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