exosomal proteins in the aqueous humor as novel biomarkers in patients with neovascular age-related...

Exosomal Proteins in the Aqueous Humor as Novel Biomarkers inPatients with Neovascular Age-related Macular DegenerationGum-Yong Kang,†,○ Joo Young Bang,†,○ Ae Jin Choi,‡ Jeehyun Yoon,‡ Won-Chul Lee,§

Soyoung Choi,∥,⊥ Soojin Yoon,# Hyung Chan Kim,‡,▽ Je-Hyun Baek,† Hyung Soon Park,†

Hyunjung Jade Lim,∥,⊥ and Hyewon Chung*,‡,⊥,▽

†Diatech Korea Co., Ltd., Young-Shin Boulevard, 57-5, Munjeong-dong, Songpa-gu, Seoul 138-826, Korea‡Department of Ophthalmology, Konkuk University School of Medicine, 120 Neungdong-ro, Gwangjin-gu, Seoul 143-701, Korea§Department of Biomedical Sciences, Seoul National University Graduate School, Seoul 151-742, Korea∥Department of Biomedical Science & Technology, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 143-701, Korea⊥Institute of Biomedical Science and Technology, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 143-701, Korea#Department of Molecular Biotechnology, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 143-701, Korea▽Department of Ophthalmology, Konkuk University Medical Center, 120-1 Neungdong-ro, Gwangjin-gu, Seoul 143-729, Korea

*S Supporting Information

ABSTRACT: Age-related macular degeneration (AMD) describes theprogressive degeneration of the retinal pigment epithelium (RPE), retina, andchoriocapillaris and is the leading cause of blindness in people over 50. Themolecular mechanisms underlying this multifactorial disease remain largelyunknown. To uncover novel secretory biomarkers related to the pathogenesis ofAMD, we adopted an integrated approach to compare the proteins identified inthe conditioned medium (CM) of cultured RPE cells and the exosomes derivedfrom CM and from the aqueous humor (AH) of AMD patients by LC−ESI−MS/MS. Finally, LC−MRM was performed on the AH from patients andcontrols, which revealed that cathepsin D, cytokeratin 8, and four other proteinsincreased in the AH of AMD patients. The present study has identifiedpotential biomarkers and therapeutic targets for AMD treatment, such asproteins related to the autophagy−lysosomal pathway and epithelial−mesenchymal transition, and demonstrated a novel and effective approach to identifying AMD-associated proteins that mightbe secreted by RPE in vivo in the form of exosomes. The proteomics-based characterization of this multifactorial disease couldhelp to match a particular marker to particular target-based therapy in AMD patients with various phenotypes.

KEYWORDS: age-related macular degeneration, retinal pigment epithelium, exosome, aqueous humor, proteomics, biomarker,cathepsin D, epithelial−mesenchymal transition

■ INTRODUCTION

Neovascular age-related macular degeneration (AMD), which ischaracterized by choroidal neovascularization (CNV), is theleading cause of blindness in people over 50 years of age.Neovascular AMD is an advanced form of AMD and causessevere vision loss in 80−90% of AMD patients.1 Despiteextensive basic and clinical research, including studies of AMDrisk genotypes,2−5 the causes of this disease remain elusive. Theunderlying cellular pathologies suggested to date includeoxidative stress, hypoxia, chronic inflammation, and theaccumulation of lipofuscin, which is derived from incompletelydigested photoreceptor outer segments in the retinal pigmentepithelium (RPE) and extracellular deposits such as drusen.6

Functional abnormalities or the degeneration of the RPE arebelieved to be the initiators and major pathologies of AMDalong with the accumulation of drusen, which subsequentlyleads to photoreceptor damage in the neural retina and a loss of

vision.7 The RPE is located between the photoreceptors of theretina and the choroid and is exposed to an oxidativeenvironment as a result of its high oxygen tension (70−90mmHg), high metabolic rate, and the accumulation oflipofuscin. Moreover, continuous exposure to light causesRPE cells to consume a large amount of oxygen to completethe complex processes in the visual cycle, nutrient transport,and phagocytosis of photoreceptor outer segments. In neo-vascular AMD, the prevention of CNV development andgrowth by the RPE or by other mechanisms seems to be largelyunsuccessful.8 Additionally, the decrease in many effectiveantioxidant enzymes in RPE is reportedly associated with thedevelopment of dry AMD, which is the more prevalent form ofAMD.9,10 Thus, it is important to understand the molecular and

Received: July 19, 2013Published: January 8, 2014

Article

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© 2014 American Chemical Society 581 dx.doi.org/10.1021/pr400751k | J. Proteome Res. 2014, 13, 581−595

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proteomic changes that occur in the RPE during diseaseprogression because the failure of or decrease in RPEadaptations to aging or stress may be the major mechanismof the pathogenesis of dry AMD and CNV. Yuan andassociates11 studied the quantitative proteomics of the macularregion of the Bruch membrane/choroid complex from thecadavers of donors with various stages of AMD. These authorsfound many proteins, including those considered to be secretedfrom AMD tissues, that were elevated or reduced comparedwith their levels in normal donors. These authors suggestedthat galectin-3, α-defensins, and other proteins might be used aspotential AMD biomarkers. Despite the interest in the RPE,little information about the molecular response to AMD isavailable because RPE samples cannot be taken from livepatients for proteomic analysis.Several growth factors have been implicated in the

pathogenesis of CNV; among these, vascular endothelialgrowth factor (VEGF) is known to be the most potent inducerof CNV. Ranibizumab (Lucentis, Novartis, Switzerland), arecombinant, humanized monoclonal antibody against VEGF-A, has been approved by the FDA for the treatment of CNVpatients with AMD. Intravitreal injection of ranibizumabreduces vascular leakage and angiogenesis, the main therapeuticmodalities for CNV. It has been suggested that the VEGF levelin the aqueous humor (AH) may reflect the VEGF level in thevitreous fluid.12 The AH, the fluid that fills the anterior segmentof the eye, supplies nutrients to the avascular tissues of the eyeand removes metabolic waste. This fluid is producedcontinuously by the ciliary processes and is drained at theanterior chamber angle via the trabecular meshwork, with aturnover rate of between 30 min and 2 h.13 Because AH isderived through the filtration of plasma in the capillary networkof the ciliary processes by an active transport mechanism, thecomposition of the fluid at the time of production is similar tothat of the plasma. However, the AH also contains manyproteins that have been secreted from ocular cells; thus, thewhole protein profile in the AH is distinct from that of theplasma.14 The movement of the lens, which is located betweenthe vitreous humor and AH, during accommodation may resultin the mixing of vitreous and AH fluid.15 The proteomicprofiling and alteration of the AH from patients with severalocular diseases, such as acute corneal rejection,16 cancer-associated retinopathy,17 proliferative diabetic retinopathy,18

and primary open angle glaucoma,19 have been reported.Recently, a whole-proteome analysis of the AH to identifybiomarkers for ocular disease was described by Escoffier andassociates. These authors used liquid chromatography (LC)-based separation directly coupled to mass spectrometry(MS).14 Although several studies20,21 have reported changesin the expression of several growth factors and inflammatorycytokines in the AH of patients with neovascular AMD beforeand during anti-VEGF therapy, other studies have found nosignificant difference in the expression of VEGF between AMDpatients and control subjects.22 Thus, it is possible that proteinsor cytokines other than VEGF will serve as better biomarkers ofAMD.Recently, Wang and associates described a novel mechanism

of drusen formation.23 They found that the intracellularproteome profile of drusen is markedly similar to that ofexosomes and suggested that drusen formation is initiated byintracellular proteins of the RPE that become extracellular viaexosomal release. Exosomes are endosome-derived micro-vesicles with a diameter of 30−100 nm. They are released

from most cell types through the fusion of multivesicular bodieswith the plasma membrane.24,25 Exosomes have been reportedin many biological fluids in vivo, including blood, urine, saliva,amniotic fluid, malignant ascites, pleural effusion, bronchoal-veolar lavage fluid, synovial fluid, and breast milk.26−36 Manycells have also been reported to release exosomes into culturemedium in vitro.36 Exosomes contain membrane proteins,intracellular proteins, RNA, DNA, and microRNAs24,36,37 andhave been suggested to have potential diagnostic andtherapeutic applications.36,37 The reported functions ofexosomes include the regulation of programmed cell death,angiogenesis, inflammation, coagulation, and the interactionbetween tumor cells and their environment.25,36 However, noinformation is available on the exosomes in ocular fluids ortissues from patients in vivo, although exosomes have beenidentified in ocular samples from donated eyes and cell linesused to study glaucoma.38,39

ARPE-19 cells, a spontaneously arising human RPE cell linewith normal karyology, express the RPE-specific markersCRALBP and RPE6540 and have structural and functionalproperties characteristic of RPE cells in vivo.40−42 Additionally,oxidatively stressed ARPE-19 cells have been used widely instudies investigating the pathogenesis of AMD.43,44 Wespeculated that proteins secreted from the RPE, retina, andCNV, possibly in the form of secretory vesicles such asexosomes, could be identified in the AH of patients. Thus, inthe present study, we adopted an integrated approach toidentify proteins possibly secreted from the RPE in the AH ofpatients with AMD and to gain insight into the pathogenicmechanism in the RPE during the course of the disease in vivo.We profiled the whole proteome of the conditioned medium(CM) from ARPE-19 cells and compared them with theexosomes derived from the CM of ARPE-19 cells and theexosomes from the AH of AMD patients. We isolated andcharacterized the exosomes in the AH of AMD patients for thefirst time and profiled their whole proteomes. We detectedvarious proteins in the CM from ARPE-19 cells that haveimplications for the status of the RPE and the disease andfurther quantified six proteins that were found in either theexosomes derived from the CM of ARPE-19 cells or theexosomes from the AH of AMD patients. Six proteins selectedusing this novel comparative approach were analyzed further byliquid chromatography multiple reaction monitoring (LC−MRM) for verification as potential biomarkers. The aim of thepresent study was to unravel novel molecular aspects of AMDand to identify new biomarkers associated with this disease.

■ MATERIALS AND METHODS

ARPE-19 Cell Cultures and the Secretome of the CellCulture Supernatant (ARPE-19 CM)

Human retinal pigment epithelial ARPE-19 cells were culturedin DMEM/F-12 (Gibco) supplemented with 10% fetal bovineserum (FBS, Gibco) and 1% penicillin-streptomycin (Gibco).Approximately 5 × 106 cells were plated in each 100 mmculture dish and maintained at 37 °C in a 5% CO2 incubator toallow proliferation. When cell confluence reached ∼90%, thecells were washed three times in PBS and then treated with 400μM paraquat (Sigma) at 37 °C for 24 h under serum-freeconditions. At the same time, total cell lysates were preparedfrom the cells that produced the CM; these lysates were usedlater in parallel with exosomes for Western blot analyses. Atotal of 100 mL of CM was collected and centrifuged at 480g

Journal of Proteome Research Article

dx.doi.org/10.1021/pr400751k | J. Proteome Res. 2014, 13, 581−595582

for 10 min and then at 1900g for 10 min to remove dead cellsand cell debris. The CM was concentrated to ∼1 mL using anAmicon Ultracel-10K molecular weight cutoff centrifugal filterdevice (Millipore) for secretome analysis by liquid chromatog-raphy−electrospray ionization tandem mass spectrometry(LC−ESI−MS/MS).

Subjects and AH Sample Collection

AH samples were collected at the Department of Ophthalmol-ogy, Konkuk University Medical Center, Seoul, Korea. FromSeptember 1, 2011 to July 31, 2013, a total of 26 patients withuntreated neovascular AMD and 18 patients undergoingcataract surgery (controls) were enrolled in this study. The26 sets of patient samples analyzed consisted of samples frompatients before treatment (intravitreal injection of ranibizumab)and samples taken from patients at 1 month after the firsttreatment for a total of 52 AH samples. The 26 patients were alltreatment-naıve; that is, they had not received any type oftreatment for neovascular AMD prior to their inclusion in thestudy. Patients with other ophthalmic diseases (e.g., glaucoma,uveitis, or progressive retinal disease), uncontrolled systemicdiseases (e.g., uncontrolled diabetes mellitus or arthritis), orwho had undergone laser or intraocular surgery were excluded.The control subjects underwent routine senile cataract surgeryfor visual rehabilitation. AH samples from patients undergoingcataract surgery were used as a control rather than samplesfrom normal eyes for ethical reasons. We matched the ages ofthe patients with those of the control subjects, and the extent ofthe cataracts in each individual corresponded to the patient’sage. The control subjects did not have any eye disease otherthan cataracts. The clinical data from the patients and controlsare summarized in Table 1. Control samples were obtainedimmediately before cataract surgery. Samples from neovascularAMD patients were obtained before performing the firstintravitreal injection of 0.5 mg ranibizumab and 1 month afterthe injection (before performing the second intravitrealinjection of 0.5 mg ranibizumab). Nine sets of samples (27samples) were used for the preparation of exosomes from AH(sample set 1 in Table 1), and the subsequent whole-proteinprofiling was performed by LC−ESI−MS/MS analysis. Becausethe number of exosomes per AH sample was small, rather thanprofiling each set of samples individually, we performedproteomic profiling of nine pooled samples each for thecontrols and for the AMD patients both before and aftertreatment. Three sets of samples (nine samples) were preparedfor the Western blot analysis of several proteins (sample set 2in Table 1). Finally, 14 sets of patient samples (28 samples)

and 6 samples from control subjects were analyzed by LC−MRM (sample set 3 in Table 1).For the electron microscopic examination and Western blot

analysis of exosomes from AH, 190 AH samples were collectedfrom patients who had neovascular AMD but did not meet thecriteria for the experiments described above. These sampleswere also collected prior to intravitreal anti-VEGF injection.However, these 190 patients had already received some type oftreatment for their disease, such as intravitreal injection ofranibizumab or bevacizumab; some patients had other ocular oruncontrolled systemic diseases such as glaucoma or uncon-trolled diabetes, and some patients had previously received lasertreatments or other intraocular surgeries. The patients rangedin age from 52 to 92 years (average, 73.8 ± 9.3 years) andincluded 101 men and 89 women.All sample collections and intravitreal injections were

performed using standard sterile procedures, and AH sampleswere obtained by anterior chamber paracentesis using a 30gauge needle. No complications were encountered afterparacentesis of the anterior chamber. Samples of the AH(100−150 μL) in safe-lock microcentrifuge tubes (1.5 mL)were immediately frozen at −80 °C and stored until analysis.The study followed the guidelines of the Declaration ofHelsinki, and informed written consent was obtained from allpatients and control subjects. The procedure for AH collectionwas approved by the Institutional Review Board of KonkukUniversity Medical Center, Seoul, Korea.

Isolation and Morphologic and BiochemicalCharacterization of Exosomes from the CM of ARPE-19 CellCulture and the AH of AMD Patients

Exosomes were isolated from the CM of ARPE-19 cell culture(ARPE-19 Exosomes) and the AH from AMD patients andcontrols (AH Exosomes) using ExoQuick Exosome Precip-itation Solution (System Bioscience, SBI) according to themanufacturer’s protocol. In brief, after centrifuging at 3000g for15 min to remove cells and cell debris, ExoQuick reagent wasadded to the supernatant and mixed well. Then, the mixturewas stored overnight at 4 °C. Subsequently, the ExoQuick/sample mixture was centrifuged at 1500g for 30 min. Aftercentrifugation, the exosomes appeared as a faint yellow-whitepellet at the bottom of the tube. The supernatant was aspirated,and all traces of fluid were removed after the residual ExoQuicksolution was spun down by centrifugation at 1500g for 5 min.The exosome pellet was resuspended and subsequently used fortransmission electron microscopy (TEM), Western blotanalysis, and LC−ESI−MS/MS.

Table 1. Summary of the Demographic Characteristics of Age-related Macular Degeneration (AMD) Patients and ControlSubjects

sample set 1: profiling of exosomal proteinsin AHa sample set 2: WBb of AH sample set 3: LC−MRM of AH

AMDc AMD AMD

property befored aftere control before after control before after control

no. of AH samples 9 9 9 3 3 3 14 14 6age (mean ± SD, years) 69.8 ± 6.1 70.6 ± 4.0 74.7 ± 7.5 71.0 ± 6.6 69.9 ± 7.4 67.3 ± 6.8sex (men:women) 5:4 5:4 2:1 2:1 9:5 5:1diabetes mellitus (no.) 2 2 1 1 3 1hypertension (no.) 5 3 1 1 4 2

aAH: aqueous humor. bWB: Western blot analysis. cAMD: age-related macular degeneration. dBefore: before treatment with ranibizumab. eAfter:one month after treatment with ranibizumab.



For TEM, exosomes were directly adsorbed onto Formvar-carbon-coated 400 mesh copper EM grids (PELCO, TEDPELLA) and dried for 20 min at RT. The specimens werenegatively stained with freshly prepared 2.0% aqueous uranylacetate (Fluka), dried, and then photographed using a JEM-1100 transmission electron microscope at an accelerationvoltage of 80 kV (JEOL, Japan). Exosomes were defined asrelatively homogeneously sized (approximately 50−150 nm indiameter) round membranous vesicles.45

For Western blot analysis, exosome pellets from the CM andfrom the AH of AMD patients were resuspended in PBScontaining protease inhibitor cocktail (Roche). The proteinconcentration of the suspension was determined by a modifiedBradford Assay (Bio-Rad Laboratories). Cell lysates andexosome sample preparations containing 10−15 μg proteinwere loaded per well. The membrane was blocked with 5%nonfat dried milk for 1 h and incubated overnight at 4 °C withthe following antibodies: anti-CD63 (Santa Cruz), anti-Hsp70(BD Sciences), anti-Tsg101 (Abcam), anticathepsin D (SantaCruz), anti-RPE65 (Abcam), antiglutamine synthetase(Abcam), anti-Thy-1 (Santa Cruz), or anticytokeratin 8(Abcam). Horseradish peroxidase-conjugated goat antirabbitor antimouse IgG (Cell Signaling) secondary antibodies wereused. A chemiluminescence substrate (ECL Prime, Amersham)was used to visualize the immunoreactive proteins.For immunocytochemistry, ARPE-19 cells were fixed with

4% paraformaldehyde for 1 h at room temperature and thenpermeabilized with 0.2% Triton X-100 for 10 min. Afterblocking with 2% bovine serum albumin, the fixed cells wereincubated overnight at 4 °C with anti-CD63 (1/1000)antibody. The cultures were then treated with a fluorescence-conjugated secondary antibody (Alexa Fluor 555 antirabbitIgG; 1:1000; Invitrogen) for 2 h at room temperature. Fornegative controls, cultures were treated with the secondaryantibody only. The mounted slides were observed under aconfocal microscope (FV-1000 Spectral, Olympus) at anexcitation wavelength of 568 nm (×800).Mice

Mice were maintained in accordance with the policies of theKonkuk University Institutional Animal Care and UseCommittee (IACUC). Mice were housed in a controlledbarrier facility in the Laboratory Animal Research Center inKonkuk University. All animals were handled in compliancewith the ARVO Statement of the Use of Animals inOphthalmic and Visual Research. Eight-week-old C57BL/6mice were killed with CO2. Eyes were enucleated, and theretinas were carefully pushed out to isolate RPE cells. RPE cellswere lysed in RIPA buffer (Thermo) with phosphatase inhibitor(Thermo) and phenylmethylsulfonyl fluoride (PMSF, Sigma).Supernatants were obtained by centrifugation at 15 000 rpm for10 min and used for Western blot analysis of RPE65.

Rat Muller Cell Cultures

Primary Muller cell cultures were generated as previouslydescribed.46 In brief, one week old Sprague−Dawley rats werekilled with CO2 and the eyes were enucleated into DMEM(Gibco) supplemented with 1% penicillin-streptomycin(Gibco) and incubated overnight at 37 °C with 5% CO2.The retinas were carefully pushed out from the eyecups anddissociated into single cells in DMEM with 10% FBS (Gibco)and 1% penicillin-streptomycin. The cells were seeded into 100mm culture dishes and placed in an incubator with 5% CO2 at37 °C. After 3 days, the medium was exchanged with fresh

medium; the medium was exchanged every 2 or 3 daysthereafter. When the cell confluence reached 80−90%, the cellswere detached with 0.25% trypsin/EDTA (Gibco), and equalamounts of cells were placed into two or three culture dishes.The medium was exchanged every 2 or 3 days. Cells frompassage 1 were used in all experiments after phenotypiccharacterization by Western blot analysis and immunofluor-escence using a known marker (glutamine synthetase, GS) thatis typical of Muller cells. Muller cell-derived exosomes wereobtained in the same manner as described above. CM fromcontrol cultures and the cultures exposed to 50 μM paraquatfor 24 h were used for exosome isolation and subsequent LC−ESI−MS/MS analysis.

Tryptic Digestion for ARPE-19 CM, ARPE-19 Exosomes, andAH Exosomes

The proteins separated by SDS-PAGE were excised from thegel and the gel pieces containing protein were destained with50% acetonitrile (ACN) containing 50 mM NH4HCO3 andvortexed until GelCode Blue stain reagent (Thermo Scientific,Rockford, IL) was completely removed. These gel pieces werethen dehydrated in 100% ACN and vacuum-dried for 20 min inspeedVac. For the digestion, gel pieces were reduced using 10mM DTT in 50 mM NH4HCO3 for 45 min at 56 °C, followedby alkylation by 55 mM iodoacetamide in 50 mM NH4HCO3for 30 min in dark. Finally, each gel piece was treated with 12.5ng/μL sequencing-grade-modified trypsin (Promega, Madison,WI) in 50 mM NH4HCO3 buffer (pH 7.8) at 37 °C forovernight. Following digestion, tryptic peptides were extractedwith 5% formic acid in 50% ACN solution at room temperaturefor 20 min. The supernatants were collected and dried bySpeedVac. The samples were desalted using C18 ZipTips(Millipore, MA) before LC−ESI−MS/MS analysis.

LC−ESI−MS/MS Analysis, Database Search, and WesternBlot Verification of Biomarker Candidates in the AH

Tryptic peptides were loaded onto a fused silica microcapillarycolumn (12 cm × 75 μm) packed with C18 resin (5 μm, 200Å). Nano-LC (EksigentnanoLC Ultra 2D, EksigentTechnolo-gies, Dublin, CA) separation was conducted under a lineargradient from 3 to 40% solvent B (0.1% formic acid in 100%ACN) with a flow rate of 250 nL/min for 60 min. The columnwas directly connected to an LTQ linear ion-trap massspectrometer (Thermo Fisher Scientific, San Jose, CA)equipped with a nanoelectrospray ion source. The electrosprayvoltage was 0.95 kV, and the threshold for switching from MSto MS/MS was 500. The normalized collision energy for MS/MS was 35% of the main radio frequency (RF) amplitude, andthe duration of activation was 30 ms. The spectra were acquiredin data-dependent scan mode. Each full MS scan was followedby MS/MS scans of the five most intense peaks. The repeatpeak count for dynamic exclusion was 1, and the repeatduration was 30 s. The dynamic exclusion duration was 180 s,and the width of exclusion mass was ±1.5 Da. The list size ofdynamic exclusion was 50.The LC−ESI−MS/MS spectra were analyzed using the

BioWorks Software (version Rev. 3.3.1 SP1, Thermo FisherScientific, San Jose, CA) with the SEQUEST search engine,which searches the National Center for BiotechnologyInformation (http://www.ncbi.nlm.nih.gov/) nonredundanthuman protein database (version: July 20, 2011; included 70112 proteins). The search conditions were as follows: trypsinenzyme specificity, no more than two missed cleavages, peptidetolerance of ±2 amu, a mass error of ±1 amu on fragment ions,



http://www.ncbi.nlm.nih.gov/

and fixed modifications of carbamidomethylation on cysteine(+57 Da) and oxidation of methionine (+16 Da) residues. Thedelta CN was 0.1; the Xcorr values were 1.8 (+1 charge state),2.3 (+2), and 3.5 (+3); and the consensus score was 10.13 forthe SEQUEST criteria. We analyzed the samples in triplicateand selected proteins that were identified in at least tworeplicate analyses.Before performing LC−MRM, we performed a verification of

several candidate proteins by Western blot analysis using theAH of AMD patients and control subjects. For cathepsin D,cytokeratin 8, and cytokeratin 14 (anticytokeratin 14 antibody,Novus Biologicals), three patient samples, and their age- andsex-matched control subjects were assayed by Western blotting.

LC−MRM

For the LC−MRM experiment, six target proteins weredigested by trypsin in silico using MRMPilot 2.1 software(AB SCIEX, Foster City, CA). The software was used to selectthe best peptides (no modification, no methionine, no cysteineresidues, two tryptic ends, and no missed cleavage sites) andtransitions (higher m/z value than the precursor m/z) for LC−MRM experiments. We then performed an NCBI Protein Blastquery to determine whether these peptides were unique. AHsamples (each 10 μg) from 14 patients and 6 control subjectswere dissolved in 6 M urea and 50 mM ammonium bicarbonate(pH 7.8) in HPLC-grade water. Denatured AH proteins werereduced with 5 mM DTT for 2 h, followed by 1 h of 5 mMiodoacetamide treatment in the dark for alkylation. AlkylatedAH samples were digested in solution with sequencing grademodified trypsin (Promega, Madison, WI) overnight at 37 °C.Formic acid was then added to the sample to stop the digestion.The MRM mode was used on a QTRAP 5500 hybrid triplequadrupole/linear ion trap mass spectrometer (AB SCIEX)equipped with a nanospray ionization source for the

quantitative analysis of specific peptides of a protein of interest.A given MRM Q1/Q3 ion value (precursor/fragment ion pair)was monitored to select a specifically targeted peptidecorresponding to each candidate protein. The MRM scan wasperformed in a positive mode with ion spray voltages in the1800−2100 V range. The MRM mode settings were as follows:curtain gas and spray gas were set at 10 and 20 psi, respectively,and the collision gas was set to unit resolution. Thedeclustering potential (DP) was set to 100 V. The massresolution was set to unit using an advanced MS parameter. Forthe correct LC−MRM, monitoring of the selected peptide byenhanced product ion (EPI) scan was performed withthreshold switching of 100 counts and the selection of rollingcollision energy. In positive mode, a product of 30, scan range100−1000 Da, and two scans were used. In the advanced MStab, the quadrupole resolution was set to low, the scan speedwas 10 000 amu/s, and a dynamic fill time was selected.

■ RESULTS

Overall Strategy for Experimental Procedures

The present study was carried out in three stages. In the firststage, we profiled the whole secretome, that is, the CM, fromcontrol and oxidatively stressed ARPE-19 cells (ARPE-19 CM)by LC−ESI−MS/MS. In stage 2, we characterized theexosomal proteins in the CM of control and oxidativelystressed ARPE-19 cells (ARPE-19 Exosomes) and in the AH ofcontrols and AMD patients (AH Exosomes) and profiled theirwhole proteomes using LC−ESI−MS/MS. In stage 3, wecombined the data from stage 1 with those generated in stage 2and selected proteins for LC−MRM. We determined thetransitions for the LC−MRM runs and performed LC−MRManalysis of the six proteins that were found in either of two

Figure 1. Ultrastructural and biochemical characterization of the exosomes isolated from the conditioned medium (CM) of ARPE-19 cell culture(ARPE-19 Exosomes) and the AH (AH Exosomes). (A) Representative negative staining electron micrograph of exosomes released from the CM ofARPE-19 cells exposed to 400 μM paraquat for 24 h (left) and the AH of AMD patients (right) (bar, 100 nm). (B) Western blot analysis ofexosomes using antibodies recognizing the known exosomal marker CD63. The same amounts (15 μg) of cell lysates (from the ARPE-19 cells thatproduced the CM), exosomes from the CM, and exosomes from the AH of AMD patients were loaded on the same gel (con: control culture; para:culture exposed to 400 μM paraquat for 24 h). The expression of CD63 was increased in the exosomes from the AH of AMD patients and CMexposed to 400 μM paraquat for 24 h compared with CM from control cultures. (C) Fluorescent confocal photomicrographs of ARPE-19 cellsimmunostained for CD63. Compared with controls, ARPE-19 cells exposed to 400 μM paraquat for 24 h showed an increased globular pattern ofCD63 staining (scale bar, 10 μm). For nuclear counterstaining, TO-PRO-3 (blue) was used. (D) Western blot analysis for further validation ofexosomal proteins using antibodies against Hsp70 and Tsg101.



proteomic profile comparisons (ARPE-19 CM vs ARPE-19Exosomes or AH Exosomes).

Proteomic Analyses of the ARPE-19 CM

Paraquat was added to ARPE-19 cells to mimic the heightenedoxidative stress of the cellular environment in neovascularAMD. We identified a total of 701 proteins in the ARPE-19CM (Supplemental Table 1 in the Supporting Information).We hypothesized that some of these proteins might be found inpatients’ AH because they are secreted from the RPE or thatthe changes in their expression in AMD patients are partiallydue to the secretory activity of the RPE.

Isolation and Characterization of ARPE-19 Exosomes andAH Exosomes

For further verification of the proteins possibly secreted by theRPE of AMD patients and to narrow down the list of proteinsthat could be most relevant to our study, we analyzed thesecretory vesicles in CM and AH. Among the microvesicles thatcells secrete to the extracellular spaces, we chose to focus onexosomes because many previous studies have reported theirbiological significance in body fluids including as vehicles forexternalization of important intracellular proteins.28,29,34,36

Exosomes have not been found in patients’ AH to date.Treatment with 400 μM paraquat for 24 h did not induce cell

death or apoptosis in ARPE-19 cells, as determined by FACSanalysis, whereas concentrations higher than 500 μM werecytotoxic (data not shown). This result confirms that theharvested exosomes and exosomal release of proteins were notproduced as a consequence of cell death.The exosome pellets from CM of ARPE-19 cell culture and

the AH of controls and AMD patients were obtained withExoQuick Exosome Precipitation Solution according to themanufacturer’s protocol, as described in the Materials andMethods. TEM revealed that the ARPE-19 Exosomes (from theCM of ARPE-19 cells exposed to paraquat) and the AHExosomes (from the AH of AMD patients) appeared ashomogeneous round-shaped membrane vesicles with diametersof 50−100 nm (Figure 1A). To further characterize theexosomes, we used Western blot analysis to examine whethercommon exosomal marker proteins were present in the purifiedexosome pellet. The most widely used markers includetetraspanins (CD9, CD63, CD81, CD82) and Hsp70, andWestern blot analysis is widely used for rapid confirmation ofexosome presence.36 Equivalent amounts of proteins from theAH Exosomes, ARPE-19 Exosomes, and the total cell lysates ofARPE-19 cells were loaded on the same gel. We detected CD63in the ARPE-19 Exosomes and the AH Exosomes (Figure 1B).The AH contained a large number of exosomes, which was

Figure 2. (A) Venn diagram of the identified whole proteins from the CM of ARPE-19 cell culture (ARPE-19 CM), ARPE-19 Exosomes, and AHExosomes. (B) Western blot analysis of exosomes using antibodies against Cathepsin D. Cathepsin D was increased in cell lysates exposed tooxidative stress compared with controls as well as in the exosomes isolated from the AH of AMD patients and in the CM from oxidatively stressedARPE-19 cells compared with exosomes from control CM. (C−E) Distribution and classification of proteins from the ARPE-19 CM, ARPE-19Exosomes, and AH Exosomes. The cellular distribution (C), molecular function (D), and biological process (E) profiles of the identified proteins areshown.



Table2.Listof

Com

mon

ProteinsIdentified

byLC

−ESI−MS/MSAnalysisandExistingin

AllThree

Profiles:Con

dition

edMedium

ofARPE-19CellC

ultures(A

RPE-19CM),

Exosomes

Isolated

from

theCM

ofARPE-19CellCultures(A

RPE-19Exosomes),andExosomes

Isolated

from

theAH

(AH

Exosomes)(N

o.1-23)a

no.

accession

entrez

reference

MW

scoreb

coverage

peptide

commonc /targetd

1119395750

3848

keratin

,typeIIcytoskeletal1

65998.94

350.32

49.8

327

common

2119395754

3852

keratin


62340.01

180.24

25.3

44common

3126012571

3339

basementmem

brane-specificheparansulfate

proteoglycan

core

proteinprecursor

468533.1

1070.33

39542

common

447132557

2335

fibronectin

isoform

1preproprotein

272157.3

770.36

42.6

780

common

566932947

2alpha-2-macroglobulin

precursor

163188.3

90.24

6.8

38common

64502027

213

serum

albumin

preproprotein

69321.63

680.37

813574

common

7115298678

718

complem

entC3precursor

187029.3

260.26

22.5

77common

8189083782

2934

gelsolin

isoform

c81890.13

60.24

11.1

15common

9110611235

80781

collagenalpha-1(XVIII)

chainisoform

1precursor

153671.8

210.38

19.3

130

common

10324021745

vitamin

D-binding

proteinisoform

3precursor

55041.09

90.22

25.6

17common

11156523970

197

alpha-2-HS-glycoprotein

39315.71

40.18

1221

common

1242740907

1191

clusterin

precursor

52461.05

70.31

20.3

35common

13291045230

7273

titin

isoform

novex-2

3011590

30.14

0.2

3common

145031863

3959

galectin-3-binding

protein

65289.4

180.33

41.4

219

common

154502261

462

antithrom

bin-IIIprecursor

52568.98

30.22

813

common

164503143

1509

cathepsinD

preproprotein

44523.66

30.13

11.2

4common/target

1715147337

51366

E3ubiquitin

-protein

ligaseUBR5

309156.7

20.14

0.8

2common

184557321

335

apolipoprotein

A-Ipreproprotein

30758.94

50.2

22.8

12common

19324021738

amyloidbeta

A4proteinisoform

hprecursor

84986.14

10.19

1.7

3common

2034734068

2192

fibulin-1

isoform

Aprecursor

61538.3

50.23

13.4

13common

21226246554

643677

hypotheticalproteinLO

C643677

801429.1

10.15

0.3

2common

224505881

5340

plasminogen

isoform

1precursor

90510.23

20.22

36

common

23153266841

350

beta-2-glycoprotein1precursor

38272.67

30.16

116

common

2412667788

4627

myosin-9

226390.6

610.28

30.4

133

target

254504919

3856

keratin


53671.19

290.32

48168

target

26213688375

59actin

,aortic

smooth

muscle

41981.82

50.28

18.6

53target

27194248072

3303

heat

shock70

kDaprotein1A

/1B

70009.18

30.23

7.3

15target

2815431310

3861

keratin

,typeIcytoskeletal14

51589.5

40.24

11.9

9target

aIn

additio

n,thetableincludes

targetproteins

forLC

−MRM

(nos.16,24−28).bScore:generatedby

Bioworks

(3.3.1)proteinconsensusscores.cCom

mon:C

ommon

inallthree

profiles(ARPE

-19CM,

ARPE

-19Exosom

es,and

AH

Exosom

es).dTarget:Targetproteins

forLC

−MRM

that

arepresentin

ARPE

-19CM

andeither

ARPE

-19Exosomes

orAH

Exosomes.



reflected by the marked CD63 content found in this fraction byWestern blot analysis. Increased expression of globular CD63was also detected in oxidatively stressed ARPE-19 cellscompared with control cells (Figure 1C). We noted that theoverall number of exosomes increased in the CM of paraquat-treated ARPE-19 cells compared with the control cells (datanot shown). We also confirmed the expression of Hsp70 andtumor susceptibility gene 101 (Tsg101), which is a componentof ESCRT (endosomal sorting complexes required fortransport) in ARPE-19 Exosomes (Figure 1D). Thus, wehypothesized that the proteins in the AH of AMD patientsmight be secreted from the RPE via exosomes.RPE cells are mainly responsible for the pathogenesis of

AMD. However, the possibility that the exosomes in the AH ofAMD patients were mainly derived from other ocular cells, suchas Muller cells or ganglion cells, could not be excluded. Thus,we performed Western blot analysis of the AH Exosomes andeach cell type (as positive controls) using RPE-, Muller cell-,and retinal ganglion cell-specific markers. Bands on Westernblots specific for glutamine synthetase (GS, for Muller cells) orThy-1 (for retinal ganglion cells) were not detected in the AHExosomes compared with each positive control (SupplementalFigure 1A in the Supporting Information). Western blotanalysis of an RPE-specific marker protein, RPE65, wasperformed for AH Exosomes from AMD patients and RPEcells freshly obtained from adult C57BL/6 mice (positivecontrol). We detected a single clear band of approximately 60and 50 kDa for RPE cells freshly obtained from adult C57BL/6mice and for AH Exosomes, respectively (Supplemental Figure1B in the Supporting Information). There has been no previousstudy regarding RPE65 in the AH, and thus we were not certainwhether RPE65 in the AH of AMD patients was modified ortruncated into fragments during the disease course. In theprevious study,47 RPE65 was ubiquitinated or truncated intofragments (45 and 20 kDa) under oxidative stress. There is noguarantee that cell-specific markers such as RPE65 for RPEcells, GS for Muller cells, or Thy-1 for retinal ganglion cellscould be detected in exosomes derived from RPE cells, Mullercells, or retinal ganglion cells because of detection limits orbecause cell-specific markers might not always be incorporatedinto the exosomes. Taken together, these results indicate thatexosomes in the AH of AMD patients contained exosomesfrom the RPE of AMD patients, although other cells such asMuller or retinal ganglion cells might secrete exosomes into theAH of AMD patients.

Proteomic Analysis of ARPE-19 CM versus ARPE-19Exosomes or AH Exosomes

To further explore the possibility that some of the proteins inthe AH were secreted via exosomes, the exosome proteomeobtained from ARPE-19 Exosomes and AH Exosomes wasanalyzed using LC−ESI−MS/MS. In total, we identified 575and 171 proteins that were detected in triplicate experiments inARPE-19 Exosomes and AH Exosomes, respectively (Figure 2Aand Supplemental Tables 2 and 3 in the SupportingInformation). These proteins included members of the annexinfamily (annexin A1, A2, A3, A4, A5), the heat shock proteinfamily (Hsp70 and 90 alpha), cytoskeletal proteins (cytokeratin1, 5, 7, 8, 18, and 19), chaperone proteins, members of theubiquitin−proteasome pathway, proteases and protease inhib-itors, coagulation and complement cascades, proteins involvedin transport and metabolism, signaling molecules, and house-keeping proteins (e.g., glyceraldehyde 3-phosphate dehydro-

genase, GAPDH). Among these proteins, cathepsin D wasconfirmed as upregulated in AH Exosomes from AMD patientsand ARPE-19 Exosomes compared with the exosomes in theCM of the control culture by Western blot analysis (Figure2B). The elevated level of cathepsin D may reflect a cellularadaptive response by the autophagy−lysosomal pathway inAMD patients to resist oxidative stress.The 25 proteins that are most often identified in exosomes

(ExoCarta, http://www.exocarta.org) were also found in theexosomes in this study. We also identified new proteins thathad not been previously described in exosomes by examiningAH Exosomes (Supplemental Table 4 in the SupportingInformation). In addition to cathepsin D and Hsp70, whichwere found in ARPE-19 Exosomes or AH Exosomes byWestern blot analysis (Figure 1D, 2B), actin (aortic smoothmuscle), myosin-9, cytokeratin 8, and cytokeratin 14 werefound in ARPE-19 Exosomes or AH Exosomes by proteomicanalysis (Table 2). The molecular function, biological process,cellular component, and pathway annotations of these proteinswere classified using PANTHER (http://www.pantherdb.org/). As shown in Figure 2C−E, the majority of these proteins areinvolved in metabolic processes, immune system processes,response to stimulus, or developmental processes. The proteinsare associated with various types of activities, mainly bindingactivity, catalytic activity, structural molecular activity, andenzyme regulator activity.We further investigated the possibility that other ocular cells

such as Muller cells contributed the exosomes in the AH ofAMD patients, although a Muller cell-specific marker, GS, wasnot identified in the AH Exosomes (Supplemental Figure 1A inthe Supporting Information). After characterization of theMuller cell cultures (Supplemental Figure 2A in the SupportingInformation), we profiled the entire proteome of Muller cellexosomes obtained from the CM of Muller cell cultures(Supplemental Table 6 in the Supporting Information). A totalof 116 and 106 proteins were identified in the Muller cellexosomes (control) and Muller cell exosomes exposed to 50μM paraquat for 24 h, respectively. The ARPE-19 Exosomescontained 37 proteins that were detected in the AH Exosomes(Supplemental Table 5 in the Supporting Information),whereas 18 proteins were present in both AH Exosomes andMuller cell exosomes.We also performed a Western blot analysis of cathepsin D

and cytokeratin 8 in ARPE-19 Exosomes, AH Exosomes, andexosomes from Muller cells in addition to cell lysates fromARPE-19 cells. As shown in Supplemental Figure 2B in theSupporting Information, neither cathepsin D nor cytokeratin 8was detected in exosomes from Muller cells exposed to 50 μMparaquat for 24 h, in contrast with the strong expressiondetected in exosomes from ARPE-19 cells exposed to paraquatas well as exosomes from the AH of AMD patients.Collectively, the above results suggest that RPE is potentiallythe major source of the exosomes in the AH of AMD patients.

Selection of the Six Target Proteins for LC−MRM

A large volume of pooled AH sample is required for exosomepreparation, limiting the clinical utility of AH components asAMD biomarkers. We further investigated the diagnosticefficacy of target proteins in AH using individual AHspecimens. In the whole-proteome profiling of ARPE-19 CM,candidate proteins were identified by comparison with datafrom ARPE-19 Exosomes or AH Exosomes (Table 2). A totalof 701 identified proteins were searched against previously



http://www.exocarta.org

http://www.pantherdb.org/

published studies of proteins or genes identified in AH, RPEcell culture media, or proteins found in donor eyes withAMD11,48−50 to determine their relevance to AMD or AMD-related conditions such as oxidative stress. Target proteins forLC−MRM were selected based on two criteria: (1) the proteinmust be present in the ARPE-19 CM profile as well as in eitherthe ARPE-19 Exosomes or AH Exosomes profile (Table 2 and3) and (2) the peptides of a target protein must be frequentlyobserved in MS scans because these proteins are easilyobserved in LC−MRM assays. On the basis of these criteria,six candidate proteins considered to be potentially originatedfrom the RPE of AMD patients and that might be present inthe AH of AMD patients were selected and used in aquantitative LC−MRM assay to measure the levels of theseproteins in the AH samples from AMD patients. The followingproteins were selected: actin (aortic smooth muscle), myosin-9,Hsp70, cathepsin D, and cytokeratin 8 and 14 (Table 3).Western Blot Analysis for Biomarker Candidates in the AH

Before performing LC−MRM of six target proteins in the AHfrom individual patients, we performed verification of severalproteins by Western blot analysis. Cathepsin D, cytokeratin 8,and cytokeratin 14 were increased in the AH of three patientsbefore treatment compared with their matched controls (Figure3). The levels of protein expression decreased, were unchanged,or increased slightly after treatment.

Biomarker Verification Using LC−MRM from Individual AHSamples

A total of six candidate proteins were subjected to LC−MRMassays. It is critical to select unique tryptic peptides for targetproteins with good MS signals. We used MRMPilot 2.1software (AB SCIEX, Foster City, CA) to select multiple trypticpeptides for the given target proteins. The MRMPilot analysisprovided good candidates for target peptides (no modification,no methionine, no cysteine residue, two tryptic ends, and nomissed cleavage sites) and transitions (higher m/z value thanthe precursor m/z) for MRM analyses. We verified theuniqueness of these peptides and chose appropriate peptides

using an NCBI Protein Blast search. Finally, we chose threepeptides per protein and three transitions per peptide andperformed an MRM analysis. However, several transitionsshowing good signals were finally chosen for targetquantification. The MRM transitions were optimized for 9peptides and 14 transitions of 6 proteins, as shown in Table 4.The selected peptides were examined by LC−MRM experi-ments using a QTRAP 5500 triple quadrupole/linear ion trapmass spectrometer (AB SCIEX, Foster City, CA). As aninternal standard, we utilized a beta-galactosidase digest (100fmol). The expression levels of all six proteins were elevated inthe AH of AMD patients compared with the average value ofthe controls (Figure 4A). All target proteins were increased bymore than approximately 1.5-fold compared with the controlgroups, as determined by t-test analysis. Further evaluation ofthese proteins as biomarkers was conducted using receiveroperating characteristic (ROC) curve analysis, which is widelyused in case-control studies. A ROC analysis was performedwith six proteins (one representative peptide of each protein).The ROC curves showed that these proteins can be used todiscriminate AMD patients from control subjects. The mostnotable indicator protein was cytokeratin 8 with an AUC of0.929 (Figure 4B).

■ DISCUSSION

The first whole-proteome analysis of AH proteins was reportedin 2008; this analysis was performed mostly by 2DE using frogeyes.15 Recently, Chowdhury and associates identified 676proteins in the AH of patients undergoing cataract surgery.49

Izzotti and associates analyzed the expression of 1264 proteinsusing glass antibody−microarrays and detected remarkablechanges in the AH proteins of glaucomatous patients.19

Another recent investigation reported the proteomic profilingof the AH of patients with neovascular AMD and thequantification of several proteins.51 In the present investigation,we focused on identifying novel proteins possibly secreted fromthe RPE to elucidate the mechanism of AMD and its responseto the current standard treatment as well as to identify potentialbiomarkers of the disease. We identified exosomes in the AH ofneovascular AMD patients for the first time and quantified thechanges in the expression of six target proteins by LC−MRM.We isolated exosomes from the AH of AMD patients and

control subjects using ExoQuick52,53 and used LC−ESI−MS/MS and Western blot analysis to identify their proteincomposition. The number of microvesicles released fromneural cells is reported to be low compared with the numbersreleased by other cells such as endothelial cells, stem cells,tumor cells, or platelets.54 In addition, AH has a relatively lowprotein content. Because RPE-specific exosomes might bediluted in the AH, a simple and efficient method should be usedto obtain exosomes from this fluid. The SBI ExoQuick exosomeprecipitation reagent used in this study effectively isolatedexosomes and their proteins for further LC−ESI−MS/MS and

Table 3. Selection of Six Target Proteins That Are Common to the Investigated Sample Sets for LC−MRM

protein name ARPE-19 CM ARPE-19 Exosomes AH Exosomes

actin, aortic smooth muscle O Omyosin-9 O OHsp70 O Ocathepsin D O O Ocytokeratin 8 O Ocytokeratin 14 O O

Figure 3. Western blot analysis of cathepsin D, cytokeratin 8, andcytokeratin 14 in the AH of three patients before (‘P’) and aftertreatment (‘T’) compared with their matched controls (‘C’). Thelevels of protein expression in the AH of three patients beforetreatment were increased compared with controls. The levels ofprotein expression decreased, were unchanged, or increased slightlyafter treatment.



Western blot analyses.52,53 Our results showed that theexosomal preparation from the AH contained previouslyreported exosomal marker proteins, supporting the validity ofthis method. We also identified endosomal proteins, annexins,heat shock proteins, cytoskeletal proteins, complements,signaling mediators, and migration- and adhesion-relatedproteins in the AH Exosomes. Among these, Hsp70, a well-known exosomal marker, was increased in untreated patientsand decreased after anti-VEGF treatment, as shown by LC−MRM analysis. Heat shock proteins have also been reported tobe present in glaucomatous AH.55 Hsp70, which acts as amolecular chaperone, is typically undetectable under normalconditions but highly induced in cells that are experiencingstress.56,57 Strong Hsp70 immunoreactivity has also beendetected in the muscles, endothelial cell layers, andinflammatory infiltrates of carotid plaques.58 Collectively,these results suggest that the induction of heat shock proteinsis one mechanism that protects against the accumulation ofmisfolded proteins, which might occur during the course ofAMD or could also be an indicator of vascular damage in theCNV. Thus, Hsp70 should be further investigated as a potentialbiomarker of cellular stress and targeted for therapeutics tostimulate endogenous adaptive and protective mechanisms toameliorate the disease process.

RPE-Secreted Proteins and Exosomes: The BiologicalSignificance of Cathepsin D in AMD

We quantified six proteins by LC−MRM in the AH from 14AMD patients and 6 control subjects. These proteins wereselected as potential candidate biomarkers of AMD or aspotential contributors to the pathogenic mechanism of AMD;they are most likely secreted by the RPE, the progressivedegeneration of which is believed to be the initiating event ofAMD. Understanding the adaptation or damage response of theRPE in the context of AMD could improve both diagnosis andtherapy of this complex disease.Yuan and associates11 reported that secreted proteins

accounted for a large proportion of the proteins found to beelevated (∼44% secreted) or decreased (∼38% secreted) inAMD tissues from cadaveric donors. A comparison of theprotein lists reporting the differential expression of secretedproteins in the RPE cells of AMD and control donors48 and thesecretome of RPE cell cultures in this study showed that manyproteins, including actin (aortic smooth muscle), myosin-9,galectin 3-binding protein, lysozyme, metalloproteinase inhib-itor, pigment epithelium-derived factor (PEDF), vitamin D-

binding protein, complement factors C3, annexin A1,cytokeratin 14, and cathepsin D, could be considered secretoryproteins from the RPE of AMD patients. Many well-knownexosomal proteins were also found in the above list of possiblesecreted proteins. Although it remains to be establishedwhether the exosomes in AH are secreted by the RPE, wefound that the ARPE-19 Exosomes contain 37 proteins thatwere also detected in the AH Exosomes (Supplemental Table 5in the Supporting Information). Thus, we speculate that RPEsecretes many proteins in exosomal vesicles. The exocyticactivity of the RPE has functional significance in thepathogenesis of AMD because this mechanism is implicatedin the formation of drusen, extracellular deposits thataccumulate between the RPE and choroid, which is considereda risk factor for developing AMD. Moreover, this findingsupports the feasibility of using ARPE-19 cell cultures for futurestudies of exosome release from RPE cells, an importantdevelopment given the difficulty of obtaining large amounts ofAH from patients or mice in vivo or CM from primary humanRPE cell culture. The results from ARPE-19 cell cultures couldbe extrapolated and tested further by in vivo studies. Like othertissues and cells, exosomes of RPE cells might have promisingroles as diagnostic and therapeutic targets and in furtherresearch to elucidate the mechanism of AMD pathogenesis. Forexample, the proteins that are exported in exosomes might beresponsible for cellular resistance to cell death in AMD.We found that the number of exosomes released from the

ARPE-19 cells markedly increased when these cells wereexposed to oxidative stress, which mimics a disease conditionand is known to be associated with an increased risk of AMD.We were able to quantify the relative amount of cathepsin D,which accumulates in cells with autophagosome−lysosomefusion and the activation of autophagy59 and is known asprincipal lysosomal protease in the RPE,60 in the AH of AMDpatients and controls by LC−MRM and in exosomes isolatedfrom the AH of AMD patients and controls by LC−ESI−MS/MS. The levels of cathepsin D in exosomes from the AH andCM as well as from the AH of three AMD patients were alsomeasured by Western blot analysis. We suggest that autophagicactivity increased as a survival mechanism in response to theoxidative conditions in AMD patients and that the upregulationof cathepsin D activity is required for the proteolytic activityneeded for the breakdown of toxic materials sequestered by theautophagosomes in RPE.7 The LC−MRM analysis of cathepsinD showed that although the average level in patients was higher

Table 4. LC−MRM Transition Chart for the Identification of Putative Protein Biomarkers

no. accession # protein name peptide sequence fragment ion Q1 Q3 dwell CE

1 213688375 actin, aortic smooth muscle QEYDEAGPSIVHR 2/y9 750.86 965.52 50 38QEYDEAGPSIVHR 2/y8 750.86 836.47 38

2 12667788 myosin-9 SGFEPASLK 2/y6 468.25 644.36 50 26LVWVPSDK 2/y6 472.27 731.37 26

3 194248072 heat shock protein 70 LLQDFFNGR 2/y6 555.29 755.35 50 294 4503143 cathepsin D QPGITFIAAK 2/y9 523.31 375.24 50 28

QPGITFIAAK 2/y6 523.31 650.39 50 28VGFAEAAR 2/y5 410.72 517.27 50 23VGFAEAAR 2/y6 410.72 664.34 50 23VGFAEAAR 2/y7 410.72 721.36 50 23

5 4504919 cytokeratin 8 WSLLQQQK 2/y5 515.79 644.37 50 28YEELQSLAGK 2/y7 569.29 716.43 50 30

6 15431310 cytokeratin 14 DAEEWFFTK 2/y8 586.77 1057.5 50 31DAEEWFFTK 2/y6 586.77 857.42 50 31



than that in the controls, its level was decreased in somepatients. This finding might indicate variability in personaladaptive response, highlighting the potential efficacy ofpersonalized therapy in which the status of autophagic activityis identified for each individual. High concentrations ofautophagy-related proteins in the AH may be a part of thedefense of the RPE against AMD; that is, the RPE producesthese proteins locally to minimize disease activity.61 Thus, the

pharmacological manipulation of autophagy or related signalingpathways may be attractive therapeutic strategies for AMD. AsFader and associates suggested,62 the induction of autophagymay decrease the secretion of exosomes to the extracellularspace as multivesicular bodies are diverted to be fused withautophagosomes and subsequently degraded intracellularly bythe lysosome. Wang and associates described increasedautophagy and increased exocytotic activity in aged RPE and

Figure 4. (A) LC−MRM analysis of six selected proteins in AH sets (x axis, C: control, P: patient before treatment with ranibizumab, T: patient 1month after treatment with ranibizumab; y axis, mean value: corrected value of peak area with internal standard). The relative abundances of sixproteins were elevated in the AH samples from patients before and 1 month after treatment compared with those from the control group asdetermined by t-test analysis. (The respective fold changes of ‘P’ and ‘T’ each relative to ‘C’ are: actin, aortic smooth muscle at 3.24 and 2.80;myosin-9 at 1.73 and 1.54; heat shock protein 70 at 1.41 and 1.23; cathepsin D at 1.62 and 1.47; cytokeratin 8 at 2.09 and 1.75; and cytokeratin 14 at2.10 and 1.53). (B) ROC curves of six selected proteins. A representative peptide from each protein was used in the analyses. The area under thecurve (AUC) at a 95% confidence level is shown.



the presence of autophagy and exosome markers in drusen ofdonated eyes with AMD.23 Increased exocytic activity,including the formation of endosomes and multivesicularbodies and the release of exosomes from the cell, wouldpromote cell health by expelling damaged, toxic macro-molecules, or undigested intracellular proteins into theextracellular space.63 However, the increased formation ofdrusen with increasing exocytic activity would aggravate theinterference with the exchange of metabolites and wasteproducts between the choriocapillaris and RPE and furthercompromise the function of the stressed RPE. Thus, increasingautophagy activity in a discretely controlled manner maydecrease the formation of drusen in patients with AMD andprevent the progression of dry AMD and the development ofneovascular AMD. Recognition of the functional status of theautophagy-lysosomal pathway and exocytic activity would helpto treat AMD patients and improve therapeutic outcomes.

Cytokeratins and the Epithelial Mesenchymal Transition

The presence and increased levels of cytokeratin 8 and 14 inthe AH of AMD patients have not been previously reported,although a comparative proteomic analysis of the expression ofseveral cytokeratins, including cytokeratin 14 in patients withSLE64 and cytokeratins 1, 9, and 10 in human saliva,65 has beenreported. Cytokeratin 8 and 14 expression is increased in AMDpatients versus controls, as shown by LC−MRM and Westernblot analysis (Figures 3 and 4). Kongara and associates66

indicated that abnormal keratin accumulation in mammarytumors may be a histologic marker of defective autophagystatus and oxidative stress, and it may indicate more aggressivedisease. As these authors suggested, and as previouslymentioned, defects in autophagy may be associated with amore aggressive course of AMD. In contrast, Lau andassociates67 have suggested that the upregulation of cytokeratin8 may be responsible for apoptotic resistance. Cytokeratin hasbeen extensively studied in many tissues, including the liver andvarious tumors.67−69 However, the study of cytokeratin 8 inRPE has been limited, although it has been identified as an RPEepithelial marker.61 Whether cytokeratin upregulation inneovascular AMD patients is an endogenous adaptive responseassociated with a favorable clinical course and good treatmentoutcomes or an indicator of more aggressive disease and pooroutcomes should be determined in future studies with largersample sizes.In human CNVM, many RPE cells represented trans-

differentiated RPE.70 This finding suggests that during thecourse of AMD, RPE cells are de- or trans-differentiated, whichmight induce the deterioration of RPE function as well as AMDprogression. Prolonged treatment with VEGF-A and VEGF-Bwas reported to induce typical epithelial−mesenchymaltransition (EMT) phenotypes in a human pancreatic cellline.71 We speculate that EMT of RPE cells may also affect thesensitivity of AMD to treatment. There have been no reportsregarding EMT as a possible mechanism for resistance to anti-VEGF treatment in AMD. However, EMT has beenincreasingly reported to cause resistance to drugs, includinganti-VEGF drugs, and to increase metastasis in variouscancers.72 We expect that this study will lead to the validationof this EMT-related biomarker as a predictor of AMDdevelopment, progression, and responsiveness to anti-VEGFtreatments. It is possible that normal epithelial cells from theAMD may undergo EMT during the process of neovascularAMD development. Specific plasma or serum cytokeratin

markers are routinely used in prognostic and monitoring assaysfor several types of malignancies.68 Our detection ofcytokeratins in the AH of AMD patients suggests that theseproteins may be very interesting potential biomarkers that,along with clinical data such as those provided by opticalcoherence tomography of macula, might be used to monitorpatients with neovascular AMD.

■ CONCLUSIONS

The prevalence of advanced AMD in the United States isprojected to increase by 50%, to ∼3 million individuals, by theyear 2020, largely due to the rapid growth of the elderlypopulation. However, there are no therapies available to repairretinal damage in advanced neovascular AMD. In addition,there is no treatment that effectively prevents dry AMD and theprogression from dry to neovascular AMD. We have shownthat proteins secreted from the RPE in vivo can be obtainedfrom the AH of patients with neovascular AMD and providedthe first evidence that exosomes are present in the AH of thesepatients. We adopted an integrated approach to compare theAH with the CM of a well-known RPE cell culture system invitro to discover and select potential candidate proteins forfurther validation. We believe that comparing the proteomes ofthe AH of neovascular AMD patients with those of controlsubjects is a powerful strategy for directly identifying themechanisms responsible for the AMD process in vivo. Sixproteins were found in the AH Exosomes or in the ARPE-19Exosomes or both; thus, the secretion of these proteins mayinvolve exosomal exocytosis, similar to that involved in drusenformation. Despite the large quantitative and qualitativevariability of the protein contents in individual humansamples,24 LC−MRM analysis of samples from 14 patientsshowed that these proteins were increased in patients comparedwith control subjects and decreased after treatment. Becausethe current treatment with ranibizumab is not the ideal therapyfor neovascular AMD, the expression of AMD-related proteinsin patients may decrease, remain unchanged, or increase furtherafter treatment depending on the biological behavior of thedisease or its response to anti-VEGF therapy, which will likelyvary among patients and proteins. Whether the increased ordecreased abundance of these proteins in AMD reflects theconsequences or the causes of AMD remains to be determined,but identifying such proteins may enhance our understandingof the biological pathways involved in this complex disease.Our study has identified several potential biomarkers and

therapeutic target proteins in AMD, such as molecularchaperone proteins (heat shock proteins) and proteins relatedto the autophagy-lysosomal pathway, and EMT. Furtherresearch into the biology of disease progression and themechanistic study of disease control will be driven by thesefindings in the AH and its exosomes. The differential expressionof these candidate proteins in both the AH and plasma will alsobe further verified in AMD with various disease courses versuscontrols to confirm their utility as molecular diagnostic markersand for personalized medicine. The proteomics-based charac-terization of this multifactorial disease may thus help to match aparticular marker to particular target-based therapy in AMDpatients with various phenotypes. Incorporating the analysis ofbiomarkers, including the EMT markers identified in this study,in randomized clinical trials of anti-VEGF therapy in neo-vascular AMD patients, may also provide biomarkers predictiveof response and resistance to anti-VEGF therapies in AMD.



■ ASSOCIATED CONTENT*S Supporting Information

Protein lists. Western blot analysis of glutamine synthetase,Thy-1 and RPE65. Characterization of rat Muller cells cultures.Western blot analysis of cathepsin D and cytokeratin 8 inARPE-19 Exosomes, AH Exosomes, and exosomes from Mullercells as well as ARPE-19 cell lysates. This material is availablefree of charge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author

*E-mail: [email protected]. Phone: 82-2-2030-7657. Fax:82-2-2030-5273.Author Contributions○G.-Y.K. and J.Y.B. contributed equally to this work.Notes

The authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThis research was supported by the National ResearchFoundation of Korea (NRF) funded by the Ministry ofScience, ICT & Future Planning (2012M3A9B2028333 and2012R1A1A11012171).

■ ABBREVIATIONSAMD, age-related macular degeneration; CNV, choroidalneovascularization; RPE, retinal pigment epithelium; VEGF,vascular endothelial growth factor; AH, aqueous humor; LC−ESI−MS/MS, liquid chromatography-electrospray ionizationtandem mass spectrometry; LC−MRM, liquid chromatographymultiple reaction monitoring; CM, conditioned medium;Hsp70, heat shock protein 70; Hspβ-1, heat shock protein β-1; Tsg101, tumor susceptibility gene 101; ESCRT, endosomalsorting complexes required for transport; GAPDH, glycer-aldehyde 3-phosphate dehydrogenase; PEDF, pigment epithe-lium-derived factor; EMT, epithelial-mesenchymal transition

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exosomal proteins in the aqueous humor as novel biomarkers in patients with neovascular age-related...

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