vitreous proteomic analysis of proliferative vitreoretinopathy

RESEARCH ARTICLE

Vitreous proteomic analysis of proliferative

vitreoretinopathy

Jing Yu1*, Feng Liu2*, Shu-Jian Cui2*, Yan Liu1, Zheng-Yu Song1, Hui Cao1,Feng-E Chen1, Wei-Jun Wang1, Tao Sun1 and Fang Wang1

1 The First People Hospital affiliated to Shanghai Jiaotong University, Shanghai, P. R. China2 Chinese National Human Genome Center at Shanghai, Shanghai, P. R. China

Proliferative vitreoretinopathy (PVR) is the most common cause of anatomic failure in retinaldetachment surgery. To understand the molecular mechanisms, vitreous proteomes of patientswith PVR were investigated by two-dimensional-nano-liquid chromatography coupled with tan-dem mass spectrometry. Vitreous samples of moderate PVR (grade B), and severe PVR (grade Cor D) were aspirated during pars plana vitrectomy before infusion. In the current study, 129, 97and 137 proteins were identified in vitreous of normal control, moderate and severe PVR,respectively. In PVR vitreous samples, complement components, serine proteinase inhibitors,and extracellular proteins were up-regulated or appeared, while normal cytoskeleton and me-tabolism proteins were down-regulated or disappeared. It was noteworthy that the proteinsinvolved in transcription and translation regulation increased in vitreous with PVR. Among 102PVR-specific proteins, kininogen 1 was specifically detected in both vitreous and the corre-sponding serum. Therefore, it can be concluded that PVR is a complicated pathology processwith great amount of proteins involved in metabolism dysfunction, immune reactions, andcytoskeleton remolding. Kininogen 1 may be a candidate biomarker of PVR. Further investiga-tions of these special proteins will provide additional targets for treatment or prevention of ocularproliferative diseases.

Received: August 27, 2007Revised: March 25, 2008Accepted: May 12, 2008

Keywords:

Biomarker / Liquid chromatography / Proliferative vitreoretinopathy / Vitreous

Proteomics 2008, 8, 3667–3678 3667

1 Introduction

The term of proliferative vitreoretinopathy (PVR) is a pro-posed designation for the clinical condition previouslyknown variously as massive vitreous retraction, massive pre-retinal retraction, or massive periretinal proliferation. This is

an abnormality in which rhegmatogenous retinal detach-ment (RRD) is complicated by proliferation of membraneson both surfaces of the detached retina and on the posteriorsurface of the detached vitreous gel [1]. These membranescause subsequent tractional retinal detachment, resulting inserious vision problems. PVR occurs in 5 to 10% of RRD [2]and is the most common cause of anatomic failure in RRDsurgery. Although the RRD surgical success rates haveimproved significantly by vitrectomy combined with C3F8 gasor silicone tamponade and the adjunctive medical agents,such as daunorubicin [3], a combination of 5-fluorouracil andlow-molecular weight heparin [4] used to prevent PVR, it isstill a challenge in the clinical experience. The pathogenesisof PVR has not been fully understood until now. PVR may beviewed as a maladapted wound-healing phenomenon inwhich contractile periretinal cellular membranes can keep

Correspondence: Professor Fang Wang, Department of Ophthal-mology, the First People Hospital affiliated to Shanghai JiaotongUniversity, Shanghai, P. R. ChinaE-mail: [email protected]: 186-21-3301-1075

Abbreviations: bFGF, basic fibroblast growth factor; CK, cytoskel-eton; EC, extracellular; HMWK, high-molecular-weight kinino-gen; MH, macular holes; PDGF, platelet-derived growth factor;PDR, proliferative diabetic retinopathy; PVR, proliferative vitreo-retinopathy; RRD, rhegmatogenous retinal detachment; SC, spec-tral counts; Serpin, serine proteinase inhibitor; TGF, transforminggrowth factor; TNF, tumor necrosis factor; VN, vitronectin * These authors contributed equally to this work.

DOI 10.1002/pmic.200700824

© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

3668 J. Yu et al. Proteomics 2008, 8, 3667–3678

breaks open, create new ones, or distort retinal topographywith visually detrimental sequelae [5]. According to previousreports, some cells such as retinal pigment epithelial, glialcell, macrophage and fibroblast [6], as well as various factors,including transforming growth factor-beta 2 (TGF-beta2) [7],basic fibroblast growth factor (bFGF) [7, 8], platelet-derivedgrowth factor (PDGF) [8], tumor necrosis factor (TNF) [9], areknown to be involved during the destructive processes ofendogenous ocular tissue. Changes in the expression levelsof these proteins have been described by the measurement ofthese substances by ELISA in aqueous, vitreous humor andsubretinal fluid. In these studies, however, substances formeasurement were targeted in advance, and the targets werelimited because of the small amount of available samplematerial. Therefore, the interrelationship between variouscandidates for controlling disease processes has not yet beenexamined.

To facilitate the research in the pathogenesis of PVR andto find new clues for developing new therapeutic agents, thevitreous humor database of the proteins expressed in eyesneeds to be obtained. Traditionally, 2-DE has been the pri-mary technique for separation of complex protein mixtures.Due primarily to the limited dynamic range of this tech-nique, only 38 vitreous proteins were reportedly identified inthe vitreous humor of proliferative diabetic retinopathy(PDR) [10]. Recent advances in LC-MS/MS have greatlyimproved the dynamic range and sensitivity for analysis ofcomplex protein mixtures [11, 12]. Large-scale proteomeprofiling has been verified for different organisms, as well asmammalian tissues and cell lines by using multi-dimen-sional LC-MS/MS [11–13]. By adopting this technique, itwould be possible to scrutinize whole protein profiles ofvitreous humor in various destructive processes of oculartissue. Few studies have evaluated human vitreous humorsamples from RRD eyes with PVR by this method. In thisreport, we presented the protein profiles of human vitreoushumor from RRD eyes with moderate or severe PVR(according to the classification of retinal detachment withPVR of the retina society terminology committee, 1983) [1]and the normal donor eyes without ocular diseases as a con-trol. We found that some cytoskeleton (CK) proteins andproteins involved in metabolism were down-regulated, whilecomplement components, serine proteinase inhibitor (ser-pin), and extracellular (EC) proteins were up-regulated orappeared in PVR vitreous, contributing greatly to under-standing the process of PVR and novel therapeutics for thisdisease.

2 Materials and methods

2.1 Sample preparation

Twenty-four PVR patients with RRD (n = 24, 8 eyes of gradeB, C or D) were enrolled in the study. The patients with ocu-lar trauma, age-related macular degeneration, diabetes mel-

litus, history of ocular surgery and other systemic diseaseswere excluded. The research followed the tenets of Declara-tion of Helsinki for the use of human subjects. Informedconsents were obtained from all patients after verbal andwritten explanation of the nature and possible consequencesof the study. The ethics committee of the Shanghai JiaotongUniversity approved the research protocol. The severe PVR(grade C or D) [14] and moderate PVR (grade B) undilutedvitreous humor samples (0.3 to 1.0 mL volume) wereobtained during pars plana vitrectomy under visual controlby aspirating liquefied vitreous from the center of the vi-treous cavity with a syringe before the vitrectomy infusion.The corresponding serum samples were obtained beforesurgery. The control group from the normal human eyeswithout any known ocular diseases (n = 8), donated for cor-neal transplant in accordance with the Standardized Rulesfor Development and Applications of Organ Transplants,were obtained from the Eye Bank of Shanghai in China. Thenormal vitreous samples (0.8 to 1.0 mL volume) were allaspirated with a syringe at pars plana. The normal serumsamples were obtained from the 20 healthy volunteers with-out any known ocular and systemic diseases. Harvested vi-treous humor samples were collected in Eppendorf tubes,placed immediately on ice, centrifuged for 15 min at 12 000rpm to separate the cell contents, and stored at 2807C untiluse. The protein concentrations for the samples were meas-ured using a BCA protein assay kit (Pierce, Rockford, IL).

2.2 Vitreous sample processing and tryptic digestion

To avoid the individual difference, the vitreous samples inthe same group were mixed at same volume before theexperiment. In control (n = 8) and moderate PVR (n = 8)group, 100 mL of each sample was mixed. Since the proteinconcentrations of severe PVR (n = 16) were higher thanthose in control and moderate PVR samples, 25 mL of eachsample was mixed. Cold acetone (including 10% TCA and20 mM DTT) was added into the vitreous samples (800 mLof moderate PVR and control, 400 mL of severe PVR) with aratio of 5:1 v/v and the mixtures were stored at 2207Covernight. The mixtures were centrifuged for 10 min at12 000 rpm and the pellets were washed by 1 mL acetonethree times. The supernatant was removed and the pelletswere dried at room temperature. The pellets were re-sus-pended in Tris base buffer (50 mM Tris/pH 8.3, 5 mMEDTA, 50 mM DTT, and 100 mg/mL PMSF, 2 M Urea)under sonication. The proteins in the solution were digest-ed into peptides using sequencing grade trypsin (Roche,Mannheim, Germany) overnight at 377C with a 1:50 w/wtrypsin-to-protein ratio.

2.3 Proteomic analyses

The following procedures were used to determine the pro-tein profiles of vitreous samples according to the reference[15], with minor modification. Briefly, the protein mixtures


Proteomics 2008, 8, 3667–3678 Biomedicine 3669

from the samples were digested and then fractionated intoten subgroups by strong cation exchange (SCX) chromatog-raphy. The peptide mixtures of each SCX fraction were thenloaded onto a RP trap column (C18, 5 mm, 300 Å, 300 mmid65 mm, LC Packings) for desalting at a flow rate of 20 mL/min. The trap column was sequentially connected in-linewith an analytical 75 mm6150 mm C18 column (LC Pack-ings) and the peptide mixtures were eluted into QSTAR pul-ser i coupled with a Protana NanoES ESI source at a flow rateof 200 nL/min. Agilent 1100 capillary LC system (AgilentTechnologies) was used to deliver mobile phases A (0.5%acetic acid in water) and B (0.5% acetic acid in ACN) at alinear gradient from 5% B to 50% B within 60 min, alongwith a gradient from 50% B to 90% B within 30 min and then90% B for 15 min. A spray voltage of 2500 V was applied to a10-mm id PicoTip nanospray emitter (New Objective) con-nected at the end of the analytical column through a stainlessunion joint (Valco Instrument) to give a steady spray. TheMS/MS spectra were recorded with information-dependentacquisition and duty-cycle enhancement. The three mostintense ions with 2 to 4 charges in each survey scan werefragmented with rolling collision energy.

2.4 Database search and analysis

The MS/MS spectra were searched against the human pro-tein database (downloaded on 6 February 2007 from ftp://ftp.ncbi.nlm.nih.gov/refseq/H_sapiens/mRNA_Prot/)using MASCOT (http://www.matrixscience.com) as reported[16, 17]. The search parameters were as follows: MS/MSspectra with mass tolerance of 0.5 Da; modifications ofmethylation and oxidation were permitted. The MASCOTsearching results were combined and further filtered as fol-lows: (i) short matched peptides (five amino acids) wereremoved; (ii) peptide assignments that were not the first rankin the matching list were removed; (iii) peptides with MAS-COT score ,25 were removed. The relative abundance of in-dividual protein was assessed by spectral counting, in whichwe counted how many times the unlabeled version of a pro-tein was identified by the fragmentation spectra of its pep-tides. Spectral counts (SC) correlate with protein abundance[18]. To analyze the difference of abundance among the pro-teomes, proteins were divided into three groups according toSC: high-abundance group (SC �10), medium-abundancegroup (SC ranged from 5 to 9), and low-abundance group(SC ,5) [13].

2.5 Western blotting

To confirm 2-D-nano-LC-MS/MS findings of proteins specialto vitreous samples, we performed Western blotting analysisfor some of these proteins, i.e. kininogen 1, vitronectin (VN)precursor. Ten microliters of vitreous humor samples fromthe subjects with control and PVR B, 5 mL of vitreous humorsample from the subjects with PVR C and D, 1 mL of serumsample from the subjects with PVR B, C and D and normal

volunteers were analyzed. The samples were electrophoresedon 10% SDS-PAGE gel, and then electrophoretically trans-ferred to NC transfer membrane (Hybond-C; AmershamBiosciences UK limited, Arlington Heights, IL) at 30 V for0.5 h. Membranes were blocked for 2 h at room temperaturewith blocking buffer and incubated for 12 h at 47C with pri-mary antibodies (anti-kininogen 1 (mouse, R&D Systems,USA, 1:200); anti-VN (mouse, CHEMICON International,CA, 1:200). The membrane was washed four times for 5 minwith PBS containing 0.1% Tween-20 and followed by incu-bation with the secondary antibody for an additional 30 min.The membrane was then washed several times and scannedby Odyssey infrared imaging system (LI-COR, Lincoln, NE)at a 700 to 800 channel wavelength.

2.6 Statistical analysis

Student’s t-tests, correlation analysis and chi-square testswere performed in the current study (SPSS 11.5, Chicago,IL). A p value of 0.05 or less was considered statistically sig-nificant.

3 Results

3.1 Average vitreous protein concentration

Average vitreous protein concentrations in PVR samples(grade B, C, D) were significantly higher than in controlsamples (t = 6.186, 4.343, 6.417, p = 0.000, 0.003, 0.000)(Fig. 1). Furthermore, the average vitreous humor proteinconcentration of PVR samples had a positive correlation withthe severe grade of PVR (Pearson correlation r = 0.740, p =0.000). It suggested that some proteins newly produced orsome proteins from serum entered into the vitreous in thePVR procedure. Therefore, the vitreous protein concentra-tion itself could be regarded as a biomarker, which couldroughly reflect the severity of PVR.

Figure 1. Average protein concentration in control and PVRsamples. Average vitreous protein concentrations in PVRsamples were significantly higher than in control samples(**p,0.01).



3.2 Preliminary proteome characteristics of control

and PVR vitreous

The integrated 255 unique proteins derived from 2723 pep-tides were unambiguously identified by 2-D-nano-LC-MS/MS in control and PVR vitreous samples (Supporting Infor-mation Table 1). The details of peptide information areshown in Supporting Information Table 2. There were 129,97 and 137 proteins identified in control, moderate PVR andsevere PVR samples, respectively (Fig. 2A, Table 1). Only 35proteins were shared among control, moderate and severePVR vitreous samples. Beside the 35 overlapping proteins,there were another 13 vitreous proteins shared betweencontrol and moderate PVR, while only one protein was com-mon in control and severe PVR vitreous. On the contrary, themoderate and severe PVR shared more common proteins (24proteins). It suggested that when PVR process got worse, thenormal vitreous proteins got less or disappeared while

abnormal proteins increased simultaneously. In PVR vi-treous samples, the numbers of proteins with two or moreidentified peptides in moderate (52.6%) and severe PVR(38.7%) were less than in control samples (73.6%) were. Theproteins with a molecular weight (MW) of less than 60 kDawere detected in the majority (87.6%) in control vitreous.Significantly more than those detected in moderate (64.9%)and severe PVR (55.6%) vitreous samples (Fisher’s exact, p =0.006, 0.000). By contrast, 67.6% (69/102) of the PVR-spe-cific proteins were more than 60 kDa. To further character-ize the molecular features of PVR vitreous humor, the sub-cellular locations and functions categories of identifiedvitreous proteins were investigated based on gene ontology(GO: http://www.geneontology.org/) (Figs. 2B and C). WhenPVR developed, the secreted proteins (EC proteins) weresignificantly increased in moderate and severe PVR (Fisher’sexact, p = 0.000, 0.000). In addition, membrane and nucleusproteins were also up-regulated slightly but signifi-

Figure 2. Integrated analyses of proteome data from control, moderate and PVR vitreous. (A) The diagram shows the proteins identifiedfrom the control, moderate PVR and severe PVR samples. The numbers of proteins identified in the three samples are shown in the circles.(B) The predicted subcellular locations of vitreous proteins identified. The number of proteins that located at EC (extracellular), Mem(membrane), Nuc (nucleus) in PVR vitreous samples were significantly increased comparing with control samples (*p,0.05, **p,0.01). CK(cytoskeleton), Cyt (cytoplasm), IC (intracellular), NA (no annotation). (C) Function categorizations of vitreous proteins identified. Proteinsinvolved in the protein synthesis, including transcription or translation regulation significantly increased in PVR vitreous samples(*p ,0.05). (D) The distribution of high-, medium- and low-abundance vitreous proteins. The low-abundance proteins in severe PVRsamples obviously surpassed those in control samples (* p,0.05).



Table 1. Vitreous proteins identified by 2-D-nano-LC-MS/MS in control, moderate and severe PVR samplesa)

GIb) Protein name Summarized MASCOT scorec) SCd) Location Function

C M S SN C M S SN

Vitreous proteins shared among control, moderate and severe PVR samples4557871 Transferrin 7278.6 8315.9 7780.4 15560.7 195 227 232 464 EC Transport4507725 Transthyretin 758.4 1076.0 1894.0 3788.0 23 31 59 118 EC Transport4502027 Albumin precursor 735.5 765.7 1950.7 3901.4 21 23 51 102 EC Antioxidant4502005 Alpha-2-HS-glycoprotein 357.4 804.7 1484.7 2969.4 7 20 32 64 EC Antioxidant4506453 Retinol-binding protein 3 precursor 1806.2 2019.7 1200.3 2400.5 47 53 31 62 EC Retinol binding42716297 Clusterin 1422.1 1565.5 1269.3 2538.7 35 40 35 70 EC Apoptosis39725934 Serpin F, pigmentary epithelium derived factor

(PEDF)713.9 556.3 1094.8 2189.6 26 19 34 68 EC Antioxidant

50363217 Serpin A, antitrypsin 226.3 1005.7 959.4 1918.8 7 32 32 64 EC Antioxidant21071030 Alpha 1B-glycoprotein 71.6 691.8 813.9 1627.8 3 19 20 40 EC NA11321561 Hemopexin 56.6 426.3 349.6 699.2 2 17 8 16 EC Transport32171249 Prostaglandin H2 D-isomerase 107.7 150.6 366.5 732.9 4 3 8 16 EC Transport115298678 Complement component 3 precursor 213.0 224.8 277.6 555.1 1 7 10 20 EC Antioxidant73858568 Complement component 1 Inhibitor precursor 27.2 120.7 258.6 517.3 1 3 7 14 EC Antioxidant4503107 Cystatin C precursor 187.6 155.7 230.7 461.4 6 5 8 16 NA Antioxidant4557485 Ceruloplasmin precursor 100.8 256.4 223.0 446.1 4 10 7 14 EC Transport7657419 Opticin precursor 328.4 202.7 36.5 73.0 10 7 1 2 EC CK organization9257232 Orosomucoid 1 precursor 84.4 291.7 71.7 143.3 3 7 2 4 EC NA4501883 Actin 197.7 45.4 44.2 88.4 5 1 1 2 CK CK organization4503143 Cathepsin D preproprotein 89.9 70.6 110.4 220.8 4 3 5 10 EC Antioxidant4502261 Serpin C1, antithrombin 28.6 166.5 104.3 208.6 1 5 4 8 EC Antioxidant113413194 Prostate, ovary, testis expressed protein 73.4 45.4 44.2 88.4 2 1 1 2 NA NA4759166 Secreted phosphoprotein 1 isoform b 28.9 30.4 61.8 123.6 1 1 2 4 EC Cytokine activity

Vitreous proteins shared between control and moderate PVR samples4507729 Tubulin, beta 1002.9 54.8 _ _ 25 3 _ _ CK CK organization33286418 Pyruvate kinase 3 954.4 63.4 _ _ 24 1 _ _ Cyt Binding4503571 Enolase 1 792.0 95.7 _ _ 33 5 _ _ Cyt Glucose metablism5803011 Enolase 2 485.6 161.2 _ _ 13 4 _ _ Cyt Glucose metablism7669492 Glyceraldehyde-3-phosphate dehydrogenase 667.5 56.2 _ _ 20 4 _ _ Cyt Glucose metablism4503377 Dihydropyrimidinase-like 2 406.7 29.9 _ _ 11 1 _ _ NA Signal transduction66346689 Dickkopf homolog 3 precursor 43.0 161.2 _ _ 5 4 _ _ EC Signal transduction21536286 Brain creatine kinase 79.3 27.4 _ _ 2 1 _ _ Cyt Metablism5453678 Epididymal secretory protein E1 precursor 27.0 52.2 _ _ 1 1 _ _ NA Metablism

Special vitreous proteins shared between moderate and severe PVR samples4504893 Kininogen 1 _ 240.8 307.1 614.1 _ 7 8 16 EC Binding50345296 Complement component 4B preproprotein _ 161.1 359.9 719.9 _ 5 11 22 EC Antioxidant67190748 Complement component 4A preproprotein _ 182.4 373.7 747.4 _ 5 12 24 EC Antioxidant50659080 Serpin A, antitrypsin _ 254.1 268.7 537.3 _ 5 6 12 EC Inflammation38044288 Gelsolin _ 257.9 52.4 104.8 _ 4 1 2 CK CK organization32483410 Vitamin D-binding protein precursor _ 194.8 149.9 299.8 _ 4 3 6 EC Transport4557321 Apolipoprotein A-I preproprotein _ 113.6 213.7 427.4 _ 3 6 12 EC Metablism4503635 Coagulation factor II precursor _ 172.1 56.2 112.4 _ 5 1 2 EC Apoptosis4504489 Histidine-rich glycoprotein precursor _ 34.2 147.4 294.7 _ 1 4 8 EC Metablism11321593 Insulin-like growth factor binding protein 6 _ 55.0 133.6 267.1 _ 1 3 6 EC Cell proliferation67782358 Complement factor B preproprotein _ 55.5 118.9 237.7 _ 1 4 8 EC Complement4504781 Inter-alpha (globulin) inhibitor H1 _ 30.6 108.5 217.0 _ 1 3 6 EC Metablism119392081 Complement factor I _ 30.6 108.5 217.0 _ 1 3 6 EC Complement4502337 Alpha-2-glycoprotein 1, zinc _ 76.2 83.9 167.8 _ 1 2 4 EC Cell proliferation6631090 Interphotoreceptor matrix proteoglycan 1 _ 32.0 81.7 163.4 _ 1 2 4 EC CK organization16418467 Leucine-rich alpha-2-glycoprotein 1 _ 68.6 78.0 156.0 _ 2 2 4 EC Transferase7657490 Clusterin-like 1 (retinol) _ 44.5 68.5 137.0 _ 1 1 2 NA Cell death4502149 Apolipoprotein A-II preproprotein _ 41.3 61.1 122.2 _ 1 2 4 EC Lipid transport88853069 Vitronectin precursor _ 86.3 45.1 90.2 _ 2 1 2 EC Cell proliferation



Table 1. Continued


C M S SN C M S SN

57242793 Interphotoreceptor matrix proteoglycan 2 _ 40.6 43.0 86.0 _ 1 1 2 EC CK organization4502511 Complement component 9 _ 53.7 28.6 57.3 _ 1 1 2 Mem NA50083295 Papilin _ 25.3 27.8 55.6 _ 1 1 2 EC Cell proliferation

Vitreous proteins shared between control and severe PVR samples5729770 Tripeptidyl-peptidase I preproprotein 25.1 _ 49.1 98.3 1 _ 1 2 Cyt Lipid Metablism

Special vitreous proteins in severe PVR samples4826762 Haptoglobin _ _ 555.9 1111.7 _ _ 20 40 EC Metablism21361845 Peptidoglycan recognition protein L precursor _ _ 107.7 215.4 _ _ 2 4 IC Metablism113418914 Unc4.1 homeobox _ _ 94.3 188.6 _ _ 1 2 NA NA33946299 PRP39 pre-mRNA processing factor _ _ 70.0 140.0 _ _ 1 2 IC Protein synthesis4557327 Apolipoprotein H precursor _ _ 69.5 139.0 _ _ 2 4 EC Transport4557287 Angiotensinogen preproprotein _ _ 64.3 128.6 _ _ 2 4 EC Signal transduction57164942 Colonic and hepatic tumor over-expressed

protein_ _ 62.9 125.8 _ _ 2 4 Nuc Protein synthesis

8393956 Serpin B13, ovalbumin _ _ 62.9 125.8 _ _ 2 4 NA Protein metablism28076869 Serpin B4, ovalbumin _ _ 62.4 124.8 _ _ 2 4 Cyt Protein metablism118582275 Superoxide dismutase 3 _ _ 59.5 118.9 _ _ 2 4 EC Superoxide metablism70778918 Inter-alpha globulin inhibitor H2 polypeptide _ _ 57.7 115.3 _ _ 2 4 EC Metablism55743122 Retinol-binding protein 4 precursor _ _ 56.2 112.4 _ _ 2 4 EC Transport68161548 MORC family CW-type zinc finger 1 _ _ 50.7 101.3 _ _ 2 4 Nuc Cell proliferation5031863 Galectin 3 binding protein _ _ 47.2 94.3 _ _ 1 2 EC Binding22538461 Nuclear receptor co-repressor 1 _ _ 46.9 93.7 _ _ 1 2 Nuc Chromatin modification4504347 Alpha globin _ _ 42.0 84.0 _ _ 1 2 IC Transport4557018 Chitinase 3-like 1 _ _ 34.6 69.1 _ _ 1 2 EC Catalytic activity71061468 Centromere protein _ _ 31.4 62.9 _ _ 1 2 Nuc Cell cycle5031787 Mitochondrial ribosomal protein S31 _ _ 31.4 62.9 _ _ 1 2 Cyt Protein synthesis4507593 Tumor necrosis factor superfamily, member 10 _ _ 31.4 62.9 _ _ 1 2 EC Apoptosis118026942 Zinc finger protein Cezanne _ _ 31.4 62.9 _ _ 1 2 Cyt Signal transduction6006001 Plasma glutathione peroxidase 3 precursor _ _ 30.5 61.1 _ _ 1 2 EC Protein metablism118442839 Complement factor H-related 1 _ _ 30.3 60.6 _ _ 1 2 EC Complement4505379 Nuclear factor -like 1 _ _ 29.7 59.4 _ _ 1 2 Nuc Transcription113422499 Hypothetical protein _ _ 29.4 58.7 _ _ 1 2 NA NA110349788 Absent, small, or homeotic 1 _ _ 28.6 57.2 _ _ 1 2 Nuc Transcription113205091 BAI1-associated protein 2-like 2 _ _ 28.6 57.2 _ _ 1 2 NA NA8923771 ELG protein _ _ 28.6 57.2 _ _ 1 2 NA NA31563507 GRIP and coiled-coil domain-containing 2 _ _ 28.6 57.2 _ _ 1 2 IC Signal transduction118498337 HECT domain containing 1 _ _ 28.6 57.2 _ _ 1 2 IC Ubiquitin cycle63054866 Leucine rich repeat containing 16 _ _ 28.6 57.2 _ _ 1 2 NA Transferase55749678 Hypothetical protein LOC22981 _ _ 28.6 57.2 _ _ 1 2 NA Binding4505221 Matrix metalloproteinase 8 preproprotein _ _ 28.6 57.2 _ _ 1 2 EC Glucose metablism20336209 Transcriptional regulator ATRX _ _ 28.6 57.2 _ _ 1 2 Nuc Protein synthesis4557417 CD14 antigen precursor _ _ 28.5 57.0 _ _ 1 2 Mem Apoptosis13899247 Retbindin _ _ 28.5 57.0 _ _ 1 2 NA NA7706190 Spectrin, beta, non-erythrocytic 5 _ _ 28.5 57.0 _ _ 1 2 CK CK organization66932947 Alpha-2-macroglobulin precursor _ _ 28.2 56.5 _ _ 1 2 EC Transport88974389 Exocyst complex component _ _ 27.8 55.6 _ _ 1 2 NA NA110349713 Titin _ _ 27.4 54.8 _ _ 1 2 Mem Glucose metablism71067341 Cerebral cavernous malformation 2 _ _ 26.7 53.4 _ _ 1 2 Cyt Signal transduction116256485 Chromosome 6 open reading frame 10 _ _ 26.7 53.4 _ _ 1 2 Mem NA31377468 Dedicator of cytokinesis 2 _ _ 26.7 53.4 _ _ 1 2 CK CK organization4503515 eukaryotic translation initiation factor 3 _ _ 26.7 53.4 _ _ 1 2 Cyt Translation11545801 GALNAC-T11 _ _ 26.7 53.4 _ _ 1 2 Mem Glucose metablism46195767 Membrane-spanning 4-domains, A 10 _ _ 26.7 53.4 _ _ 1 2 Mem Signal transduction60685229 Myotubularin related protein 15 _ _ 26.7 53.4 _ _ 1 2 NA DNA repair



Table 1. Continued


C M S SN C M S SN

4758956 Peripheral benzodiazepine receptor-associatedprotein 1

_ _ 26.7 53.4 _ _ 1 2 Cyt Receptor binding

113423509 SET domain containing 1A _ _ 26.7 53.4 _ _ 1 2 NA NA21071070 Transmembrane channel-like 1 _ _ 26.7 53.4 _ _ 1 2 Mem NA78000163 Sorbin and SH3 domain containing 1 _ _ 26.5 52.9 _ _ 1 2 Mem Signal transduction40538730 Calmodulin regulated spectrin-associated

protein 1_ _ 26.4 52.8 _ _ 1 2 NA NA

20149629 DEAD (Asp-Glu-Ala-Asp) box polypeptide 47 _ _ 26.0 52.1 _ _ 1 2 Cyt Protein synthesis10835242 Protein kinase, cGMP-dependent, type I _ _ 25.5 51.0 _ _ 1 2 Cyt CK organization73858566 Heparin cofactor II precursor _ _ 25.2 50.3 _ _ 1 2 EC Blood coagulation

Special vitreous proteins in moderate PVR samples29788768 Tubulin, beta 1098.38 25 Cyt CK organization57242755 Calsyntenin 1 _ 210.35 _ _ _ 4 _ _ Mem Binding4502147 Amyloid beta A4 precursor-like protein 2 _ 189.9 _ _ _ 6 _ _ Mem Binding40548389 Dickkopf homolog 3 precursor 131.98 4 EC Signal transduction4504349 Beta globin _ 106.0 _ _ _ 3 _ _ IC Transport86788015 EGF-containing fibulin-like extracellular matrix

protein1_ 41.9 _ _ _ 1 _ _ EC Visual perception

50845384 ADAM metallopeptidase 1 preproprotein _ 35.9 _ _ _ 1 _ _ EC Signal transduction89057120 Complement C3 precursor _ 30.4 _ _ _ 1 _ _ NA NA4502067 Alpha-1-microglobulin/ bikunin precursor _ 29.7 _ _ _ 1 _ _ EC Inflammation18450369 Protein tyrosine phosphatase A _ 27.3 _ _ _ 1 _ _ Mem Metablism4505047 Lumican precursor _ 27.0 _ _ _ 1 _ _ EC CK organization41406055 Amyloid beta A4 protein precursor _ 26.2 _ _ _ 1 _ _ EC Apoptosis113428622 Fc fragment of IgG binding protein _ 25.3 _ _ _ 1 _ _ NA NA89994744 Zinc finger protein 236 _ 25.1 _ _ _ 1 _ _ IC Transcription

Special vitreous proteins in control samples21361322 Tubulin, beta 430.1 _ _ _ 11 _ _ _ CK CK organization4507645 Triosephosphate isomerase 1 356.5 _ _ _ 8 _ _ _ NA Metablism16554592 Enolase 3 218.9 _ _ _ 6 _ _ _ IC Glucose metablism4507149 Superoxide dismutase 1, soluble 218.7 _ _ _ 6 _ _ _ Cyt Superoxide metablism113408757 Triose-phosphate isomerase 212.2 _ _ _ 4 _ _ _ NA NA58743306 Alpha-tubulin isotype H2-alpha 171.7 _ _ _ 5 _ _ _ Micro Signal transduction32455264 Peroxiredoxin 171.3 _ _ _ 4 _ _ _ NA Cell proliferation14389309 Tubulin, alpha 161.5 _ _ _ 3 _ _ _ Micro Binding31543380 DJ-1 protein 140.8 _ _ _ 3 _ _ _ Cyt Signal transduction4885063 Fructose-bisphosphate aldolase C 133.4 _ _ _ 4 _ _ _ NA Glucose metablism55956788 Nucleolin 123.7 _ _ _ 3 _ _ _ Nuc Binding24234686 Heat shock 70kDa protein 8 119.1 _ _ _ 3 _ _ _ IC Protein folding46409270 Hypothetical protein LOC112714 113.1 _ _ _ 3 _ _ _ EC Cell motility4505763 Phosphoglycerate kinase 1 113.0 _ _ _ 3 _ _ _ NA Glucose metablism4503051 Collapsin response mediator protein 1 105.0 _ _ _ 3 _ _ _ NA Nucleotide metablism5453549 Thioredoxin peroxidase 95.5 _ _ _ 2 _ _ _ NA NA4557325 Apolipoprotein E precursor 83.7 _ _ _ 2 _ _ _ NA NA4507521 Transketolase 72.3 _ _ _ 2 _ _ _ NA Metablism4557395 Carbonic anhydrase II 67.0 _ _ _ 2 _ _ _ Cyt Glucose metablism62414289 Vimentin 60.3 _ _ _ 2 _ _ _ Cyt Cell motility34577110 Aldolase A 60.2 _ _ _ 2 _ _ _ NA Glucose metablism4885413 Histidine triad nucleotide binding protein 1 56.1 _ _ _ 1 _ _ _ CK Signal transduction116008178 S-arrestin 54.6 _ _ _ 2 _ _ _ Cyt Transcription19923206 Glutamine synthetase 54.0 _ _ _ 2 _ _ _ NA Glutamine synthesis4557032 Lactate dehydrogenase B 42.2 _ _ _ 1 _ _ _ Cyt Glucose metablism4503063 Crystallin 38.0 _ _ _ 1 _ _ _ NA Lens structure18201905 Glucose phosphate isomerase 35.1 _ _ _ 1 _ _ _ EC Glucose metablism



Table 1. Continued


C M S SN C M S SN

5902018 Brain-specific protein p25 alpha 33.5 _ _ _ 1 _ _ _ NA NA7706369 Brain protein 44-like 30.5 _ _ _ 1 _ _ _ NA NA83700235 Eukaryotic translation initiation factor 4A 30.1 _ _ _ 1 _ _ _ NA NA11056044 Pyrophosphatase 1 29.5 _ _ _ 1 _ _ _ Cyt Metablism4505753 Phosphoglycerate mutase 27.8 _ _ _ 1 _ _ _ Cyt Glucose metablism4504067 Aspartate aminotransferase 1 27.2 _ _ _ 1 _ _ _ Cyt Protein metablism74271826 Glutamine synthetase 27.0 _ _ _ 1 _ _ _ NA NA18375673 Regulator of nonsense transcripts 1 26.9 _ _ _ 1 _ _ _ Cyt Cell cycle55749932 Desmin 25.8 _ _ _ 1 _ _ _ Nuc CK organization14249342 Internexin neuronal intermediate filament

protein alpha25.8 _ _ _ 1 _ _ _ Nuc CK organization

4885513 Neurofilament 3 25.8 _ _ _ 1 _ _ _ Nuc CK organization52317140 Olfactory receptor 51, A 7 25.4 _ _ _ 1 _ _ _ NA NA14249316 Hypothetical protein LOC84800 25.2 _ _ _ 1 _ _ _ NA CK organization

a) C, control; M, moderate PVR; S, severe PVR; SN , Normalization of severe PVR; IC, intracellular; Cyt, cytoplasm; Mem, membrane; Nuc,nucleus; Micro, microtubule; NA, No annotation.

b) The GI numbers of representative protein sequences from NCBI (www.ncbi.nlm.nih.gov). The details of the proteome can be seen in theSupporting Information Table 1.

c) The scores are summed MASCOT scores of peptides of the certain protein.d) The number of spectral counts of vitreous proteins identified.

cantly (Fisher’s exact, p = 0.000, 0.044). The proteins inmoderate and severe PVR vitreous humor involved in pro-tein synthesis, including transcription or translation regula-tion were up-regulated compared with control proteome(Fisher’s exact, p = 0.006, 0.000). These data suggested thatproteins, especially the secreted proteins with high MW,were synthesized increasing in PVR process. However,nearly one-third or more proteins in control vitreous werewithout a clear subcellular location and functional catego-rization, but had a clear gene resource. The low-abundanceproteins in severe PVR samples were enriched obviouslycompared with those in control samples (Fisher’s exact, p =0.013) (Fig. 2D). However, the common cytokines in PVR,such as TGF, bFGF, PDGF, could not be detected in the PVRsamples except TNF. Perhaps some low-abundance proteinsin PVR samples were removed from the protein list due tothe higher cut-off value of MASCOT scores (25 or more) inthe current study.

3.3 Comparison of PVR vitreous proteome with

control vitreous proteome

To compare PVR proteome with control and moderate PVRproteome, the data should be normalized. Since the volumeof severe PVR samples was half of the control and moderatePVR samples, the summarized scores and SC of severe PVRsamples should be doubled when they were compared withcontrol and moderate PVR proteome. In this case, significantup-regulation was defined as SC ratio �3 and SC distance

�5; significant down-regulation was defined as SC ratio�0.33 and SC distance was �25. SC ratio = (PVR SC)/(con-trol SC); SC distance = (PVR SC) – (control SC). Twelve pro-teins, majority of which were serum high-abundance pro-teins such as transferrin, albumin precursor, alpha2-HS-gly-coprotein, alpha1B-glycoprotein, serpins family andcomplement components were significantly up-regulated inmoderate and severe PVR vitreous comparing with controlsamples (Fig. 3A). Meanwhile, 11 proteins, including tubu-lin (three isoforms), pyruvate kinase 3 (three isoforms), eno-lase (two isoforms), and glyceraldehyde-3-phosphate dehy-drogenase (GAPDH), were significantly down-regulated inmoderate and severe PVR vitreous comparing with controlsamples (Fig. 3B). Nineteen important enzymes involved inglycolysis process, including pyruvate kinases and enolases,were detected in control proteome. These enzymes were sig-nificantly decreased in moderate PVR, even disappeared insevere PVR. This indicated that glycolysis process wasaffected obviously when PVR happened.

It was interesting to find that many CK proteins identi-fied in control samples, such as tubulin and actin familymembers, were down-regulated dramatically in moderatePVR samples while remained undetectable in severe PVRvitreous humor. Meanwhile, matrix metalloproteinase 8 pre-proprotein, which related to CK grading [19], could bedetected in PVR vitreous simultaneously. These data mightindicate that CK was remodeled during PVR process.

It was noteworthy that some groups of proteins signifi-cantly changed in PVR proteome. The members of serpin



Figure 3. Comparison of PVR and control proteome. C, control;M, moderate PVR; S, severe PVR. (A) Twelve proteins were sig-nificantly up-regulated in moderate or severe PVR comparingwith control samples. (B) Eleven proteins were significantlydown-regulated in moderate or severe PVR comparing with con-trol samples. (Significant up-regulation was defined as SC ratio�3 and SC distance �5; significant down-regulation was definedas SC ratio �0.33 and SC distance was �25. SC ratio = (PVR SC)/(control SC); SC distance = (PVR SC) – (control SC).

family, such as antitrypsin, were significantly up-regulated.Furthermore, other members, such as serpin A3, B4 and B13were identified only in PVR samples. Other common pro-teins detected in the PVR vitreous humor included comple-ment components C3, C4A, C4B, and C9, as well as comple-ment factors B, H, and I. Enolases, the relative high abun-dance proteins in control vitreous, were significant down-regulated in moderate PVR and were undetectable in severePVR vitreous.

3.4 The verification of proteins identified in PVR

samples by Western blotting analysis

The images of MS/MS spectra of kininogen 1 and VN iden-tified by LC-MS/MS are shown in Figs. 4A and B. Both pro-teins were specifically detected in PVR vitreous samples butwere not detected in control samples (Figs. 4C and E). Kini-nogen 1 could be detected in the corresponding serum ofPVR but was undetectable in the normal serum (Fig. 4D).VN could be detected in the corresponding PVR serum aswell as in the normal serum (Fig. 4F). Furthermore, both

proteins expressed higher in vitreous with PVR C and D thanin vitreous with PVR B. It was noteworthy that kininogen 1could be detected in all PVR vitreous samples (24/24, 100%)and the corresponding serum samples. VN was detected in21/24 vitreous samples with PVR (three samples with PVR Bwere undetected) and 24/24 in the corresponding serumsamples. Therefore, kininogen 1 may be one of the candidateserum biomarkers for evaluation of the PVR severity grade.

4 Discussion

In the present study, we performed a systematic comparisonof proteomic with vitreous surveys for characterizing thePVR and normal vitreous proteome. Integration of the data-sets from control, moderate, and severe PVR proteomes, willresult in a comprehensive understanding of protein func-tions of a given biofluid. There were 255 distinct proteinsidentified by 2-D-nano-LC-MS/MS in the integrated controland PVR proteome, which was significantly more than theprevious reports of vitreous proteome separated and identi-fied by 2-DE combined with MS [10, 20, 21]. It implied that2-D-nano-LC-MS/MS was a better optional to obtain richproteins information from the biofliud, especially in smallamount samples. However, the protein identifications wererarely shared, i.e. only 35 proteins were in both PVR andcontrol vitreous, while 24 proteins were shared in moderateand severe PVR vitreous. Furthermore, nearly 40% of theintegrated proteome were specific proteins in PVR. It sug-gested that PVR was a complicated process with a greatamount of proteins newly produced. It was very interestingthat the number of proteins identified in moderate PVR wassmaller than in the control samples, although the averageprotein concentration of moderate PVR was higher than incontrol. Eighty proteins (62%) in control were undetected ineither moderate or severe PVR. This suggested that the nor-mal vitreous proteins were reduced at the onset of PVR. Atthe early stage of PVR, the complement components andserpins significantly increased, while the normal CK, such astubulins was down-regulated. When PVR got severe, theproteins involved in cell proliferation increased significantly,while the normal CK proteins decreased or disappeared. Themajority of these increased proteins were involved in proteinsynthesis, including cell cycle, signal transduction, tran-scription or translation regulation. These outcomes indi-cated a significant difference in the constitution of the vi-treous proteome between PVR and control samples. Thecurrent study also provides proof of the destruction of blood-retina barriers in the PVR samples [22], in which manycommon serum proteins, such as hemopexin, apolipopro-tein A-I, A-II, H and complement components significantlyincreased.

Although PVR and PDR are kinds of ischemic diseaseshaving different causes and clinical characteristics, retinalmembranes from both conditions share the features offibroplasia, excessive matrix protein deposition, and cellular



Figure 4. Proteins were identified by MS/MS and Western blotting. (A) The MS/MS spectrum of peptide IGEIKEETTSHLR, was from kini-nogen 1; (B) The MS/MS spectrum of peptide LIRDVWGIEGPIDAAFTR, was from VN precursor. C,F were the outcomes of Western blot-ting. V (vitreous), S (serum), B (PVR B), C (PVR C), D (PVR D). The long and short arrows in (C) and (D) show the two versions of kininogen 1of high and low MW, respectively. Kininogen 1(C) and VN (E) were detected in mixture vitreous humor of PVR B, C and D but were notdetected in control vitreous. Kininogen 1(D) was detected in mixture serum of PVR B, C and D but was not detected in normal serum; VN (F)was detected in mixture serum samples of PVR B, C and D as well as in normal serum.

infiltration. Yamane and coauthors [10] successfully analyzedand identified 38 proteins in the PDR proteome of vitreoushumor using 2-DE combined with MS. Among them, pig-mentary epithelium-derived factor (PEDF), prostaglandin-D2 synthase, and interphotoreceptor retinol-binding proteinwere special proteins in vitreous humor that could be detect-ed in PDR and macular hole (MH) samples (control group),but were not detected in the corresponding serum samples.In our results, three proteins were detected in both controland PVR vitreous samples, supporting their outcomes. Incontrast, the neuron-specific enolase existed in PVR samplesas well as in normal vitreous with high abundance in ourstudy. This suggested that it was not a newly produced pro-tein in PVR. Although MH is a non-proliferative disease,which could be set as a control group of PDR or PVR, it is stilla certain kind of retinal disease of disease. The fact thatvitreous proteins in MH samples were reduced or that 2-DEcombined with MS was not sensitive enough to detect theproteins might contribute to this phenomenon. In the cur-rent study, enolases, including both non-neuron-specific and

neuron-specific enolases, were significantly down-regulatedin moderate PVR and disappeared in severe PVR vitreous.This indicated that enolases existed in normal eyes anddecreased with PVR development. Enolases are the essentialglycolytic enzymes involved in the glucose metabolism.Other proteins involved in glycolysis metabolism, such aspyruvate kinase, were also significantly down-regulated inPVR vitreous.

Several studies documented that both complement com-ponents and immunoglobulins (Igs) could be detected in theepiretinal membranes of PVR [23, 24]. Complement systemis a principal constituent of humoral immune reactions andis involved in opsonization, inflammation, lysis, andimmune complex clearance. In our results, the high-abun-dance proteins such as complement C3, C4A and C4B, wereup-regulated significantly. In addition, complement factorsB, H, and I could also be detected in the PVR vitreous humor,supporting Grisanti’ report [23]. Furthermore, retinalarrestin detected in PVR vitreous is a kind of autoantigen,which can activate positive T cell proliferative responses [25].



However, Igs were undetected in the PVR or control vitreoussamples, which agreed with the reports that T lymphocytes,but not B lymphocytes or Igs, were found in the epiretinalmembrane [26].

In the current study, CK remodeling in PVR processrevealed that normal CK proteins were down-regulated ordisappeared while secreted proteins significantly increasedin PVR vitreous. Previous reports documented that theextracellular matrix components, fibronectin, laminin, andvitronectin (VN) were the major components of the epir-etinal and subretinal membranes of PVR, which weredetected in vitreous [27, 28]. However, in our study, only VNcould be detected in both moderate and severe PVR vitreous.Furthermore, VN existed in normal serum as well as in PVRserum, suggesting the serum-origin of VN in vitreous.

Among the special proteins of PVR proteome, whichcould be detected in the corresponding serum samples withPVR, kininogen 1 was one of the relative high-abundanceproteins. In our study, both high-molecular-weight kinino-gen (HMWK, 120 kDa) and low-molecular weight kinino-gen (LMWK, 47 kDa) could be detected by the Westernblotting, although only LMWK could be identified by the2-D-nano-LC-MS/MS, due possibly to the technology lim-itation of the 2-D-nano-LC-MS/MS. HMWK is an abundantsingle-chain protein with six domains. It is cleaved by kal-likrein, which releases bradykinin (BK) domain 4, to gen-erate 2-chain kininogen (HKa) [29]. HKa domains 3 and 5may contribute to the pathogenesis of inflammatory dis-eases by releasing interleukin-1beta from human mono-cytes using intracellular signaling pathways initiated bybeta2 integrins and gC1q receptor (gC1qR) [30]. gC1qR mayparticipate in tissue remodeling and inflammation to thepericellular environment to modulate local protease activityand regulate HMWK activation [31]. Mechanistically, tissuekallikrein/kinin leads to an increase in nitric oxide levelsand Akt activation, and reduces reactive oxygen species for-mation, TGF-beta1 expression, and nuclear factor-kappaBactivation [32]. Kallikreins, which activate HMWK to HKa,can be regarded as anti-inflammatory and anti-oxidativeagents in protecting the brain against ischemic stroke-induced injuries [33]. It is worthy to note that several hu-man kallikreins can be the biomarkers in serum to aid inthe diagnosis and monitoring some caners, such as ovarian,prostate and breast cancer [34, 35]. Previous researches havedocumented that kallikrein-kinin system (KKS) plays animportant role in chronic inflammatory diseases such asrheumatoid arthritis [36], Crohn’s disease [37], athero-sclerosis [38], proliferative granuloma [39], Alzheimer’s dis-ease [40]. Decreasing of HKa and BK would inhibit the localinflammatory process in the joints and initiate systemicinflammation [36, 41, 42]. As PVR is a chronic inflamma-tion process in essence, KKS may also play an essential rolein PVR pathology. Therefore, kininogen, HKa and kallik-reins may be appropriate targets for further drug discoveryin the therapy of PVR. In addition, it is noteworthy thatkininogen 1 maybe one of the candidate serum biomarkers

of PVR to evaluate PVR severity grade. Further research isneeded to validate this hypothesis and develop new ther-apeutic agents for prevention and control of PVR.

This work was supported by Shanghai Educational Com-mittee Key Laboratory Foundation Project (2006BZ068).

The authors have declared no conflict of interest.

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