Exosomal Proteins in the Aqueous Humor as Novel Biomarkers in

Article pubs.acs.org/jpr

Exosomal Proteins in the Aqueous Humor as Novel Biomarkers in Patients with Neovascular Age-related Macular Degeneration Gum-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 the progressive degeneration of the retinal pigment epithelium (RPE), retina, and choriocapillaris and is the leading cause of blindness in people over 50. The molecular mechanisms underlying this multifactorial disease remain largely unknown. To uncover novel secretory biomarkers related to the pathogenesis of AMD, we adopted an integrated approach to compare the proteins identified in the conditioned medium (CM) of cultured RPE cells and the exosomes derived from 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 and controls, which revealed that cathepsin D, cytokeratin 8, and four other proteins increased in the AH of AMD patients. The present study has identified potential biomarkers and therapeutic targets for AMD treatment, such as proteins related to the autophagy−lysosomal pathway and epithelial− mesenchymal transition, and demonstrated a novel and effective approach to identifying AMD-associated proteins that might be secreted by RPE in vivo in the form of exosomes. The proteomics-based characterization of this multifactorial disease could help 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

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vision.7 The RPE is located between the photoreceptors of the retina and the choroid and is exposed to an oxidative environment as a result of its high oxygen tension (70−90 mmHg), high metabolic rate, and the accumulation of lipofuscin. Moreover, continuous exposure to light causes RPE cells to consume a large amount of oxygen to complete the complex processes in the visual cycle, nutrient transport, and phagocytosis of photoreceptor outer segments. In neovascular AMD, the prevention of CNV development and growth by the RPE or by other mechanisms seems to be largely unsuccessful.8 Additionally, the decrease in many effective antioxidant enzymes in RPE is reportedly associated with the development of dry AMD, which is the more prevalent form of AMD.9,10 Thus, it is important to understand the molecular and

INTRODUCTION Neovascular age-related macular degeneration (AMD), which is characterized by choroidal neovascularization (CNV), is the leading cause of blindness in people over 50 years of age. Neovascular AMD is an advanced form of AMD and causes severe vision loss in 80−90% of AMD patients.1 Despite extensive basic and clinical research, including studies of AMD risk genotypes,2−5 the causes of this disease remain elusive. The underlying cellular pathologies suggested to date include oxidative stress, hypoxia, chronic inflammation, and the accumulation of lipofuscin, which is derived from incompletely digested photoreceptor outer segments in the retinal pigment epithelium (RPE) and extracellular deposits such as drusen.6 Functional abnormalities or the degeneration of the RPE are believed to be the initiators and major pathologies of AMD along with the accumulation of drusen, which subsequently leads to photoreceptor damage in the neural retina and a loss of © 2014 American Chemical Society

Received: July 19, 2013 Published: January 8, 2014 581

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

Journal of Proteome Research

Article

from most cell types through the fusion of multivesicular bodies with the plasma membrane.24,25 Exosomes have been reported in many biological fluids in vivo, including blood, urine, saliva, amniotic fluid, malignant ascites, pleural effusion, bronchoalveolar lavage fluid, synovial fluid, and breast milk.26−36 Many cells have also been reported to release exosomes into culture medium in vitro.36 Exosomes contain membrane proteins, intracellular proteins, RNA, DNA, and microRNAs24,36,37 and have been suggested to have potential diagnostic and therapeutic applications.36,37 The reported functions of exosomes include the regulation of programmed cell death, angiogenesis, inflammation, coagulation, and the interaction between tumor cells and their environment.25,36 However, no information is available on the exosomes in ocular fluids or tissues from patients in vivo, although exosomes have been identified in ocular samples from donated eyes and cell lines used to study glaucoma.38,39 ARPE-19 cells, a spontaneously arising human RPE cell line with normal karyology, express the RPE-specific markers CRALBP and RPE6540 and have structural and functional properties characteristic of RPE cells in vivo.40−42 Additionally, oxidatively stressed ARPE-19 cells have been used widely in studies investigating the pathogenesis of AMD.43,44 We speculated that proteins secreted from the RPE, retina, and CNV, possibly in the form of secretory vesicles such as exosomes, could be identified in the AH of patients. Thus, in the present study, we adopted an integrated approach to identify proteins possibly secreted from the RPE in the AH of patients with AMD and to gain insight into the pathogenic mechanism 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 the exosomes derived from the CM of ARPE-19 cells and the exosomes from the AH of AMD patients. We isolated and characterized the exosomes in the AH of AMD patients for the first time and profiled their whole proteomes. We detected various proteins in the CM from ARPE-19 cells that have implications for the status of the RPE and the disease and further quantified six proteins that were found in either the exosomes derived from the CM of ARPE-19 cells or the exosomes from the AH of AMD patients. Six proteins selected using this novel comparative approach were analyzed further by liquid chromatography multiple reaction monitoring (LC− MRM) for verification as potential biomarkers. The aim of the present study was to unravel novel molecular aspects of AMD and to identify new biomarkers associated with this disease.

proteomic changes that occur in the RPE during disease progression because the failure of or decrease in RPE adaptations to aging or stress may be the major mechanism of the pathogenesis of dry AMD and CNV. Yuan and associates11 studied the quantitative proteomics of the macular region of the Bruch membrane/choroid complex from the cadavers of donors with various stages of AMD. These authors found many proteins, including those considered to be secreted from AMD tissues, that were elevated or reduced compared with their levels in normal donors. These authors suggested that galectin-3, α-defensins, and other proteins might be used as potential AMD biomarkers. Despite the interest in the RPE, little information about the molecular response to AMD is available because RPE samples cannot be taken from live patients for proteomic analysis. Several growth factors have been implicated in the pathogenesis of CNV; among these, vascular endothelial growth factor (VEGF) is known to be the most potent inducer of CNV. Ranibizumab (Lucentis, Novartis, Switzerland), a recombinant, humanized monoclonal antibody against VEGFA, has been approved by the FDA for the treatment of CNV patients with AMD. Intravitreal injection of ranibizumab reduces vascular leakage and angiogenesis, the main therapeutic modalities for CNV. It has been suggested that the VEGF level in the aqueous humor (AH) may reflect the VEGF level in the vitreous fluid.12 The AH, the fluid that fills the anterior segment of the eye, supplies nutrients to the avascular tissues of the eye and removes metabolic waste. This fluid is produced continuously by the ciliary processes and is drained at the anterior chamber angle via the trabecular meshwork, with a turnover rate of between 30 min and 2 h.13 Because AH is derived through the filtration of plasma in the capillary network of the ciliary processes by an active transport mechanism, the composition of the fluid at the time of production is similar to that of the plasma. However, the AH also contains many proteins that have been secreted from ocular cells; thus, the whole protein profile in the AH is distinct from that of the plasma.14 The movement of the lens, which is located between the vitreous humor and AH, during accommodation may result in the mixing of vitreous and AH fluid.15 The proteomic profiling and alteration of the AH from patients with several ocular diseases, such as acute corneal rejection,16 cancerassociated retinopathy,17 proliferative diabetic retinopathy,18 and primary open angle glaucoma,19 have been reported. Recently, a whole-proteome analysis of the AH to identify biomarkers for ocular disease was described by Escoffier and associates. These authors used liquid chromatography (LC)based separation directly coupled to mass spectrometry (MS).14 Although several studies20,21 have reported changes in the expression of several growth factors and inflammatory cytokines in the AH of patients with neovascular AMD before and during anti-VEGF therapy, other studies have found no significant difference in the expression of VEGF between AMD patients and control subjects.22 Thus, it is possible that proteins or cytokines other than VEGF will serve as better biomarkers of AMD. Recently, Wang and associates described a novel mechanism of drusen formation.23 They found that the intracellular proteome profile of drusen is markedly similar to that of exosomes and suggested that drusen formation is initiated by intracellular proteins of the RPE that become extracellular via exosomal release. Exosomes are endosome-derived microvesicles with a diameter of 30−100 nm. They are released

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MATERIALS AND METHODS

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

Human retinal pigment epithelial ARPE-19 cells were cultured in DMEM/F-12 (Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin (Gibco). Approximately 5 × 106 cells were plated in each 100 mm culture dish and maintained at 37 °C in a 5% CO2 incubator to allow proliferation. When cell confluence reached ∼90%, the cells were washed three times in PBS and then treated with 400 μM paraquat (Sigma) at 37 °C for 24 h under serum-free conditions. At the same time, total cell lysates were prepared from the cells that produced the CM; these lysates were used later in parallel with exosomes for Western blot analyses. A total of 100 mL of CM was collected and centrifuged at 480g 582



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Table 1. Summary of the Demographic Characteristics of Age-related Macular Degeneration (AMD) Patients and Control Subjects sample set 1: profiling of exosomal proteins in AHa

sample set 2: WBb of AH

c

AMD

AMD

property

befored

aftere

control

no. of AH samples age (mean ± SD, years) sex (men:women) diabetes mellitus (no.) hypertension (no.)

9

9

9 70.6 ± 4.0 5:4 2 3

69.8 ± 6.1 5:4 2 5

before

sample set 3: LC−MRM of AH AMD

after

3 3 74.7 ± 7.5 2:1 1 1

control 3 71.0 ± 6.6 2:1 1 1

before

after

14 14 69.9 ± 7.4 9:5 3 4

control 6 67.3 ± 6.8 5:1 1 2

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

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 collected from patients who had neovascular AMD but did not meet the criteria for the experiments described above. These samples were also collected prior to intravitreal anti-VEGF injection. However, these 190 patients had already received some type of treatment for their disease, such as intravitreal injection of ranibizumab or bevacizumab; some patients had other ocular or uncontrolled systemic diseases such as glaucoma or uncontrolled diabetes, and some patients had previously received laser treatments or other intraocular surgeries. The patients ranged in age from 52 to 92 years (average, 73.8 ± 9.3 years) and included 101 men and 89 women. All sample collections and intravitreal injections were performed using standard sterile procedures, and AH samples were obtained by anterior chamber paracentesis using a 30 gauge needle. No complications were encountered after paracentesis 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 of Helsinki, and informed written consent was obtained from all patients and control subjects. The procedure for AH collection was approved by the Institutional Review Board of Konkuk University Medical Center, Seoul, Korea.

for 10 min and then at 1900g for 10 min to remove dead cells and cell debris. The CM was concentrated to ∼1 mL using an Amicon Ultracel-10K molecular weight cutoff centrifugal filter device (Millipore) for secretome analysis by liquid chromatography−electrospray ionization tandem mass spectrometry (LC−ESI−MS/MS). Subjects and AH Sample Collection

AH samples were collected at the Department of Ophthalmology, Konkuk University Medical Center, Seoul, Korea. From September 1, 2011 to July 31, 2013, a total of 26 patients with untreated neovascular AMD and 18 patients undergoing cataract surgery (controls) were enrolled in this study. The 26 sets of patient samples analyzed consisted of samples from patients before treatment (intravitreal injection of ranibizumab) and samples taken from patients at 1 month after the first treatment for a total of 52 AH samples. The 26 patients were all treatment-naı̈ve; that is, they had not received any type of treatment for neovascular AMD prior to their inclusion in the study. Patients with other ophthalmic diseases (e.g., glaucoma, uveitis, or progressive retinal disease), uncontrolled systemic diseases (e.g., uncontrolled diabetes mellitus or arthritis), or who had undergone laser or intraocular surgery were excluded. The control subjects underwent routine senile cataract surgery for visual rehabilitation. AH samples from patients undergoing cataract surgery were used as a control rather than samples from normal eyes for ethical reasons. We matched the ages of the patients with those of the control subjects, and the extent of the cataracts in each individual corresponded to the patient’s age. The control subjects did not have any eye disease other than cataracts. The clinical data from the patients and controls are summarized in Table 1. Control samples were obtained immediately before cataract surgery. Samples from neovascular AMD patients were obtained before performing the first intravitreal injection of 0.5 mg ranibizumab and 1 month after the injection (before performing the second intravitreal injection of 0.5 mg ranibizumab). Nine sets of samples (27 samples) were used for the preparation of exosomes from AH (sample set 1 in Table 1), and the subsequent whole-protein profiling was performed by LC−ESI−MS/MS analysis. Because the number of exosomes per AH sample was small, rather than profiling each set of samples individually, we performed proteomic profiling of nine pooled samples each for the controls and for the AMD patients both before and after treatment. Three sets of samples (nine samples) were prepared for the Western blot analysis of several proteins (sample set 2 in Table 1). Finally, 14 sets of patient samples (28 samples)

Isolation and Morphologic and Biochemical Characterization of Exosomes from the CM of ARPE-19 Cell Culture 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 and controls (AH Exosomes) using ExoQuick Exosome Precipitation Solution (System Bioscience, SBI) according to the manufacturer’s protocol. In brief, after centrifuging at 3000g for 15 min to remove cells and cell debris, ExoQuick reagent was added to the supernatant and mixed well. Then, the mixture was stored overnight at 4 °C. Subsequently, the ExoQuick/ sample mixture was centrifuged at 1500g for 30 min. After centrifugation, the exosomes appeared as a faint yellow-white pellet at the bottom of the tube. The supernatant was aspirated, and all traces of fluid were removed after the residual ExoQuick solution was spun down by centrifugation at 1500g for 5 min. The exosome pellet was resuspended and subsequently used for transmission electron microscopy (TEM), Western blot analysis, and LC−ESI−MS/MS. 583



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medium; the medium was exchanged every 2 or 3 days thereafter. When the cell confluence reached 80−90%, the cells were detached with 0.25% trypsin/EDTA (Gibco), and equal amounts of cells were placed into two or three culture dishes. The medium was exchanged every 2 or 3 days. Cells from passage 1 were used in all experiments after phenotypic characterization by Western blot analysis and immunofluorescence using a known marker (glutamine synthetase, GS) that is typical of Müller cells. Müller cell-derived exosomes were obtained in the same manner as described above. CM from control cultures and the cultures exposed to 50 μM paraquat for 24 h were used for exosome isolation and subsequent LC− ESI−MS/MS analysis.

For TEM, exosomes were directly adsorbed onto Formvarcarbon-coated 400 mesh copper EM grids (PELCO, TED PELLA) and dried for 20 min at RT. The specimens were negatively stained with freshly prepared 2.0% aqueous uranyl acetate (Fluka), dried, and then photographed using a JEM1100 transmission electron microscope at an acceleration voltage of 80 kV (JEOL, Japan). Exosomes were defined as relatively homogeneously sized (approximately 50−150 nm in diameter) round membranous vesicles.45 For Western blot analysis, exosome pellets from the CM and from the AH of AMD patients were resuspended in PBS containing protease inhibitor cocktail (Roche). The protein concentration of the suspension was determined by a modified Bradford Assay (Bio-Rad Laboratories). Cell lysates and exosome sample preparations containing 10−15 μg protein were loaded per well. The membrane was blocked with 5% nonfat dried milk for 1 h and incubated overnight at 4 °C with the following antibodies: anti-CD63 (Santa Cruz), anti-Hsp70 (BD Sciences), anti-Tsg101 (Abcam), anticathepsin D (Santa Cruz), anti-RPE65 (Abcam), antiglutamine synthetase (Abcam), anti-Thy-1 (Santa Cruz), or anticytokeratin 8 (Abcam). Horseradish peroxidase-conjugated goat antirabbit or antimouse IgG (Cell Signaling) secondary antibodies were used. 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 then permeabilized with 0.2% Triton X-100 for 10 min. After blocking with 2% bovine serum albumin, the fixed cells were incubated overnight at 4 °C with anti-CD63 (1/1000) antibody. The cultures were then treated with a fluorescenceconjugated secondary antibody (Alexa Fluor 555 antirabbit IgG; 1:1000; Invitrogen) for 2 h at room temperature. For negative controls, cultures were treated with the secondary antibody only. The mounted slides were observed under a confocal microscope (FV-1000 Spectral, Olympus) at an excitation wavelength of 568 nm (×800).

Tryptic Digestion for ARPE-19 CM, ARPE-19 Exosomes, and AH Exosomes

The proteins separated by SDS-PAGE were excised from the gel and the gel pieces containing protein were destained with 50% acetonitrile (ACN) containing 50 mM NH4HCO3 and vortexed until GelCode Blue stain reagent (Thermo Scientific, Rockford, IL) was completely removed. These gel pieces were then dehydrated in 100% ACN and vacuum-dried for 20 min in speedVac. For the digestion, gel pieces were reduced using 10 mM DTT in 50 mM NH4HCO3 for 45 min at 56 °C, followed by alkylation by 55 mM iodoacetamide in 50 mM NH4HCO3 for 30 min in dark. Finally, each gel piece was treated with 12.5 ng/μL sequencing-grade-modified trypsin (Promega, Madison, WI) in 50 mM NH4HCO3 buffer (pH 7.8) at 37 °C for overnight. Following digestion, tryptic peptides were extracted with 5% formic acid in 50% ACN solution at room temperature for 20 min. The supernatants were collected and dried by SpeedVac. The samples were desalted using C18 ZipTips (Millipore, MA) before LC−ESI−MS/MS analysis. LC−ESI−MS/MS Analysis, Database Search, and Western Blot Verification of Biomarker Candidates in the AH

Tryptic peptides were loaded onto a fused silica microcapillary column (12 cm × 75 μm) packed with C18 resin (5 μm, 200 Å). Nano-LC (EksigentnanoLC Ultra 2D, EksigentTechnologies, Dublin, CA) separation was conducted under a linear gradient 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 column was directly connected to an LTQ linear ion-trap mass spectrometer (Thermo Fisher Scientific, San Jose, CA) equipped with a nanoelectrospray ion source. The electrospray voltage was 0.95 kV, and the threshold for switching from MS to MS/MS was 500. The normalized collision energy for MS/ MS was 35% of the main radio frequency (RF) amplitude, and the duration of activation was 30 ms. The spectra were acquired in data-dependent scan mode. Each full MS scan was followed by MS/MS scans of the five most intense peaks. The repeat peak count for dynamic exclusion was 1, and the repeat duration was 30 s. The dynamic exclusion duration was 180 s, and the width of exclusion mass was ±1.5 Da. The list size of dynamic exclusion was 50. The LC−ESI−MS/MS spectra were analyzed using the BioWorks Software (version Rev. 3.3.1 SP1, Thermo Fisher Scientific, San Jose, CA) with the SEQUEST search engine, which searches the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/) nonredundant human protein database (version: July 20, 2011; included 70 112 proteins). The search conditions were as follows: trypsin enzyme specificity, no more than two missed cleavages, peptide tolerance of ±2 amu, a mass error of ±1 amu on fragment ions,

Mice

Mice were maintained in accordance with the policies of the Konkuk University Institutional Animal Care and Use Committee (IACUC). Mice were housed in a controlled barrier facility in the Laboratory Animal Research Center in Konkuk University. All animals were handled in compliance with the ARVO Statement of the Use of Animals in Ophthalmic and Visual Research. Eight-week-old C57BL/6 mice were killed with CO2. Eyes were enucleated, and the retinas were carefully pushed out to isolate RPE cells. RPE cells were lysed in RIPA buffer (Thermo) with phosphatase inhibitor (Thermo) and phenylmethylsulfonyl fluoride (PMSF, Sigma). Supernatants were obtained by centrifugation at 15 000 rpm for 10 min and used for Western blot analysis of RPE65. Rat Müller Cell Cultures

Primary Müller cell cultures were generated as previously described.46 In brief, one week old Sprague−Dawley rats were killed 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 and dissociated into single cells in DMEM with 10% FBS (Gibco) and 1% penicillin-streptomycin. The cells were seeded into 100 mm culture dishes and placed in an incubator with 5% CO2 at 37 °C. After 3 days, the medium was exchanged with fresh 584



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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 of ARPE-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 of exosomes using antibodies recognizing the known exosomal marker CD63. The same amounts (15 μg) of cell lysates (from the ARPE-19 cells that produced 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 CM exposed to 400 μM paraquat for 24 h compared with CM from control cultures. (C) Fluorescent confocal photomicrographs of ARPE-19 cells immunostained for CD63. Compared with controls, ARPE-19 cells exposed to 400 μM paraquat for 24 h showed an increased globular pattern of CD63 staining (scale bar, 10 μm). For nuclear counterstaining, TO-PRO-3 (blue) was used. (D) Western blot analysis for further validation of exosomal proteins using antibodies against Hsp70 and Tsg101.

and fixed modifications of carbamidomethylation on cysteine (+57 Da) and oxidation of methionine (+16 Da) residues. The delta 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 for the SEQUEST criteria. We analyzed the samples in triplicate and selected proteins that were identified in at least two replicate analyses. Before performing LC−MRM, we performed a verification of several candidate proteins by Western blot analysis using the AH 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- and sex-matched control subjects were assayed by Western blotting.

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 peptide corresponding to each candidate protein. The MRM scan was performed in a positive mode with ion spray voltages in the 1800−2100 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. The declustering potential (DP) was set to 100 V. The mass resolution was set to unit using an advanced MS parameter. For the correct LC−MRM, monitoring of the selected peptide by enhanced product ion (EPI) scan was performed with threshold switching of 100 counts and the selection of rolling collision energy. In positive mode, a product of 30, scan range 100−1000 Da, and two scans were used. In the advanced MS tab, the quadrupole resolution was set to low, the scan speed was 10 000 amu/s, and a dynamic fill time was selected.

LC−MRM

For the LC−MRM experiment, six target proteins were digested by trypsin in silico using MRMPilot 2.1 software (AB SCIEX, Foster City, CA). The software was used to select the best peptides (no modification, no methionine, no cysteine residues, two tryptic ends, and no missed cleavage sites) and transitions (higher m/z value than the precursor m/z) for LC− MRM experiments. We then performed an NCBI Protein Blast query to determine whether these peptides were unique. AH samples (each 10 μg) from 14 patients and 6 control subjects were dissolved in 6 M urea and 50 mM ammonium bicarbonate (pH 7.8) in HPLC-grade water. Denatured AH proteins were reduced with 5 mM DTT for 2 h, followed by 1 h of 5 mM iodoacetamide treatment in the dark for alkylation. Alkylated AH samples were digested in solution with sequencing grade modified 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 triple quadrupole/linear ion trap mass spectrometer (AB SCIEX) equipped with a nanospray ionization source for the

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RESULTS

Overall Strategy for Experimental Procedures

The present study was carried out in three stages. In the first stage, we profiled the whole secretome, that is, the CM, from control and oxidatively stressed ARPE-19 cells (ARPE-19 CM) by LC−ESI−MS/MS. In stage 2, we characterized the exosomal proteins in the CM of control and oxidatively stressed ARPE-19 cells (ARPE-19 Exosomes) and in the AH of controls and AMD patients (AH Exosomes) and profiled their whole proteomes using LC−ESI−MS/MS. In stage 3, we combined the data from stage 1 with those generated in stage 2 and selected proteins for LC−MRM. We determined the transitions for the LC−MRM runs and performed LC−MRM analysis of the six proteins that were found in either of two 585



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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 AH Exosomes. (B) Western blot analysis of exosomes using antibodies against Cathepsin D. Cathepsin D was increased in cell lysates exposed to oxidative stress compared with controls as well as in the exosomes isolated from the AH of AMD patients and in the CM from oxidatively stressed ARPE-19 cells compared with exosomes from control CM. (C−E) Distribution and classification of proteins from the ARPE-19 CM, ARPE-19 Exosomes, and AH Exosomes. The cellular distribution (C), molecular function (D), and biological process (E) profiles of the identified proteins are shown.

death or apoptosis in ARPE-19 cells, as determined by FACS analysis, whereas concentrations higher than 500 μM were cytotoxic (data not shown). This result confirms that the harvested exosomes and exosomal release of proteins were not produced 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 with ExoQuick Exosome Precipitation Solution according to the manufacturer’s protocol, as described in the Materials and Methods. TEM revealed that the ARPE-19 Exosomes (from the CM of ARPE-19 cells exposed to paraquat) and the AH Exosomes (from the AH of AMD patients) appeared as homogeneous round-shaped membrane vesicles with diameters of 50−100 nm (Figure 1A). To further characterize the exosomes, we used Western blot analysis to examine whether common exosomal marker proteins were present in the purified exosome pellet. The most widely used markers include tetraspanins (CD9, CD63, CD81, CD82) and Hsp70, and Western blot analysis is widely used for rapid confirmation of exosome presence.36 Equivalent amounts of proteins from the AH Exosomes, ARPE-19 Exosomes, and the total cell lysates of ARPE-19 cells were loaded on the same gel. We detected CD63 in the ARPE-19 Exosomes and the AH Exosomes (Figure 1B). The AH contained a large number of exosomes, which was

proteomic profile comparisons (ARPE-19 CM vs ARPE-19 Exosomes or AH Exosomes). Proteomic Analyses of the ARPE-19 CM

Paraquat was added to ARPE-19 cells to mimic the heightened oxidative stress of the cellular environment in neovascular AMD. We identified a total of 701 proteins in the ARPE-19 CM (Supplemental Table 1 in the Supporting Information). We hypothesized that some of these proteins might be found in patients’ AH because they are secreted from the RPE or that the changes in their expression in AMD patients are partially due to the secretory activity of the RPE. Isolation and Characterization of ARPE-19 Exosomes and AH Exosomes

For further verification of the proteins possibly secreted by the RPE of AMD patients and to narrow down the list of proteins that could be most relevant to our study, we analyzed the secretory vesicles in CM and AH. Among the microvesicles that cells secrete to the extracellular spaces, we chose to focus on exosomes because many previous studies have reported their biological significance in body fluids including as vehicles for externalization 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 586


587

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

2192 643677 5340 350 4627 3856 59 3303 3861

197 1191 7273 3959 462 1509 51366 335

3848 3852 3339 2335 2 213 718 2934 80781

entrez keratin, type II cytoskeletal 1 keratin, type II cytoskeletal 5 basement membrane-specific heparan sulfate proteoglycan core protein precursor fibronectin isoform 1 preproprotein alpha-2-macroglobulin precursor serum albumin preproprotein complement C3 precursor gelsolin isoform c collagen alpha-1(XVIII) chain isoform 1 precursor vitamin D-binding protein isoform 3 precursor alpha-2-HS-glycoprotein clusterin precursor titin isoform novex-2 galectin-3-binding protein antithrombin-III precursor cathepsin D preproprotein E3 ubiquitin-protein ligase UBR5 apolipoprotein A-I preproprotein amyloid beta A4 protein isoform h precursor fibulin-1 isoform A precursor hypothetical protein LOC643677 plasminogen isoform 1 precursor beta-2-glycoprotein 1 precursor myosin-9 keratin, type II cytoskeletal 8 actin, aortic smooth muscle heat shock 70 kDa protein 1A/1B keratin, type I cytoskeletal 14

reference 65998.94 62340.01 468533.1 272157.3 163188.3 69321.63 187029.3 81890.13 153671.8 55041.09 39315.71 52461.05 3011590 65289.4 52568.98 44523.66 309156.7 30758.94 84986.14 61538.3 801429.1 90510.23 38272.67 226390.6 53671.19 41981.82 70009.18 51589.5

MW 350.32 180.24 1070.33 770.36 90.24 680.37 260.26 60.24 210.38 90.22 40.18 70.31 30.14 180.33 30.22 30.13 20.14 50.2 10.19 50.23 10.15 20.22 30.16 610.28 290.32 50.28 30.23 40.24

scoreb 49.8 25.3 39 42.6 6.8 81 22.5 11.1 19.3 25.6 12 20.3 0.2 41.4 8 11.2 0.8 22.8 1.7 13.4 0.3 3 11 30.4 48 18.6 7.3 11.9

coverage 327 44 542 780 38 3574 77 15 130 17 21 35 3 219 13 4 2 12 3 13 2 6 6 133 168 53 15 9

peptide

common common common common common common common common common common common common common common common common/target common common common common common common common target target target target target

commonc/targetd

In addition, the table includes target proteins for LC−MRM (nos. 16, 24−28). bScore: generated by Bioworks (3.3.1) protein consensus scores. cCommon: Common in all three profiles (ARPE-19 CM, ARPE-19 Exosomes, and AH Exosomes). dTarget: Target proteins for LC−MRM that are present in ARPE-19 CM and either ARPE-19 Exosomes or AH Exosomes.

a

accession

no.

Table 2. List of Common Proteins Identified by LC−ESI−MS/MS Analysis and Existing in All Three Profiles: Conditioned Medium of ARPE-19 Cell Cultures (ARPE-19 CM), Exosomes Isolated from the CM of ARPE-19 Cell Cultures (ARPE-19 Exosomes), and Exosomes Isolated from the AH (AH Exosomes) (No. 1-23)a

Journal of Proteome Research Article



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reflected by the marked CD63 content found in this fraction by Western blot analysis. Increased expression of globular CD63 was also detected in oxidatively stressed ARPE-19 cells compared with control cells (Figure 1C). We noted that the overall number of exosomes increased in the CM of paraquattreated ARPE-19 cells compared with the control cells (data not shown). We also confirmed the expression of Hsp70 and tumor susceptibility gene 101 (Tsg101), which is a component of ESCRT (endosomal sorting complexes required for transport) in ARPE-19 Exosomes (Figure 1D). Thus, we hypothesized that the proteins in the AH of AMD patients might 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 of AMD patients were mainly derived from other ocular cells, such as Müller cells or ganglion cells, could not be excluded. Thus, we performed Western blot analysis of the AH Exosomes and each cell type (as positive controls) using RPE-, Müller cell-, and retinal ganglion cell-specific markers. Bands on Western blots specific for glutamine synthetase (GS, for Müller cells) or Thy-1 (for retinal ganglion cells) were not detected in the AH Exosomes compared with each positive control (Supplemental Figure 1A in the Supporting Information). Western blot analysis of an RPE-specific marker protein, RPE65, was performed for AH Exosomes from AMD patients and RPE cells freshly obtained from adult C57BL/6 mice (positive control). We detected a single clear band of approximately 60 and 50 kDa for RPE cells freshly obtained from adult C57BL/6 mice and for AH Exosomes, respectively (Supplemental Figure 1B in the Supporting Information). There has been no previous study regarding RPE65 in the AH, and thus we were not certain whether RPE65 in the AH of AMD patients was modified or truncated into fragments during the disease course. In the previous study,47 RPE65 was ubiquitinated or truncated into fragments (45 and 20 kDa) under oxidative stress. There is no guarantee that cell-specific markers such as RPE65 for RPE cells, GS for Müller cells, or Thy-1 for retinal ganglion cells could be detected in exosomes derived from RPE cells, Müller cells, or retinal ganglion cells because of detection limits or because cell-specific markers might not always be incorporated into the exosomes. Taken together, these results indicate that exosomes in the AH of AMD patients contained exosomes from the RPE of AMD patients, although other cells such as Müller or retinal ganglion cells might secrete exosomes into the AH of AMD patients.

genase, GAPDH). Among these proteins, cathepsin D was confirmed as upregulated in AH Exosomes from AMD patients and ARPE-19 Exosomes compared with the exosomes in the CM of the control culture by Western blot analysis (Figure 2B). The elevated level of cathepsin D may reflect a cellular adaptive response by the autophagy−lysosomal pathway in AMD patients to resist oxidative stress. The 25 proteins that are most often identified in exosomes (ExoCarta, http://www.exocarta.org) were also found in the exosomes in this study. We also identified new proteins that had not been previously described in exosomes by examining AH Exosomes (Supplemental Table 4 in the Supporting Information). In addition to cathepsin D and Hsp70, which were found in ARPE-19 Exosomes or AH Exosomes by Western blot analysis (Figure 1D, 2B), actin (aortic smooth muscle), myosin-9, cytokeratin 8, and cytokeratin 14 were found in ARPE-19 Exosomes or AH Exosomes by proteomic analysis (Table 2). The molecular function, biological process, cellular component, and pathway annotations of these proteins were classified using PANTHER (http://www.pantherdb.org/ ). As shown in Figure 2C−E, the majority of these proteins are involved in metabolic processes, immune system processes, response to stimulus, or developmental processes. The proteins are associated with various types of activities, mainly binding activity, catalytic activity, structural molecular activity, and enzyme regulator activity. We further investigated the possibility that other ocular cells such as Müller cells contributed the exosomes in the AH of AMD patients, although a Müller cell-specific marker, GS, was not identified in the AH Exosomes (Supplemental Figure 1A in the Supporting Information). After characterization of the Müller cell cultures (Supplemental Figure 2A in the Supporting Information), we profiled the entire proteome of Müller cell exosomes obtained from the CM of Müller cell cultures (Supplemental Table 6 in the Supporting Information). A total of 116 and 106 proteins were identified in the Müller cell exosomes (control) and Müller cell exosomes exposed to 50 μM paraquat for 24 h, respectively. The ARPE-19 Exosomes contained 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 and Müller cell exosomes. We also performed a Western blot analysis of cathepsin D and cytokeratin 8 in ARPE-19 Exosomes, AH Exosomes, and exosomes from Müller cells in addition to cell lysates from ARPE-19 cells. As shown in Supplemental Figure 2B in the Supporting Information, neither cathepsin D nor cytokeratin 8 was detected in exosomes from Müller cells exposed to 50 μM paraquat for 24 h, in contrast with the strong expression detected in exosomes from ARPE-19 cells exposed to paraquat as well as exosomes from the AH of AMD patients. Collectively, the above results suggest that RPE is potentially the major source of the exosomes in the AH of AMD patients.

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

To further explore the possibility that some of the proteins in the AH were secreted via exosomes, the exosome proteome obtained from ARPE-19 Exosomes and AH Exosomes was analyzed using LC−ESI−MS/MS. In total, we identified 575 and 171 proteins that were detected in triplicate experiments in ARPE-19 Exosomes and AH Exosomes, respectively (Figure 2A and Supplemental Tables 2 and 3 in the Supporting Information). These proteins included members of the annexin family (annexin A1, A2, A3, A4, A5), the heat shock protein family (Hsp70 and 90 alpha), cytoskeletal proteins (cytokeratin 1, 5, 7, 8, 18, and 19), chaperone proteins, members of the ubiquitin−proteasome pathway, proteases and protease inhibitors, coagulation and complement cascades, proteins involved in transport and metabolism, signaling molecules, and housekeeping proteins (e.g., glyceraldehyde 3-phosphate dehydro-

Selection of the Six Target Proteins for LC−MRM

A large volume of pooled AH sample is required for exosome preparation, limiting the clinical utility of AH components as AMD biomarkers. We further investigated the diagnostic efficacy of target proteins in AH using individual AH specimens. In the whole-proteome profiling of ARPE-19 CM, candidate proteins were identified by comparison with data from ARPE-19 Exosomes or AH Exosomes (Table 2). A total of 701 identified proteins were searched against previously 588



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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

actin, aortic smooth muscle myosin-9 Hsp70 cathepsin D cytokeratin 8 cytokeratin 14

O O O O O O

O O O O O

AH Exosomes

O O

using an NCBI Protein Blast search. Finally, we chose three peptides per protein and three transitions per peptide and performed an MRM analysis. However, several transitions showing good signals were finally chosen for target quantification. The MRM transitions were optimized for 9 peptides and 14 transitions of 6 proteins, as shown in Table 4. The selected peptides were examined by LC−MRM experiments using a QTRAP 5500 triple quadrupole/linear ion trap mass spectrometer (AB SCIEX, Foster City, CA). As an internal standard, we utilized a beta-galactosidase digest (100 fmol). The expression levels of all six proteins were elevated in the AH of AMD patients compared with the average value of the controls (Figure 4A). All target proteins were increased by more than approximately 1.5-fold compared with the control groups, as determined by t-test analysis. Further evaluation of these proteins as biomarkers was conducted using receiver operating characteristic (ROC) curve analysis, which is widely used in case-control studies. A ROC analysis was performed with six proteins (one representative peptide of each protein). The ROC curves showed that these proteins can be used to discriminate AMD patients from control subjects. The most notable indicator protein was cytokeratin 8 with an AUC of 0.929 (Figure 4B).

published studies of proteins or genes identified in AH, RPE cell culture media, or proteins found in donor eyes with AMD11,48−50 to determine their relevance to AMD or AMDrelated conditions such as oxidative stress. Target proteins for LC−MRM were selected based on two criteria: (1) the protein must be present in the ARPE-19 CM profile as well as in either the ARPE-19 Exosomes or AH Exosomes profile (Table 2 and 3) and (2) the peptides of a target protein must be frequently observed in MS scans because these proteins are easily observed in LC−MRM assays. On the basis of these criteria, six candidate proteins considered to be potentially originated from the RPE of AMD patients and that might be present in the AH of AMD patients were selected and used in a quantitative LC−MRM assay to measure the levels of these proteins in the AH samples from AMD patients. The following proteins 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 AH from individual patients, we performed verification of several proteins by Western blot analysis. Cathepsin D, cytokeratin 8, and cytokeratin 14 were increased in the AH of three patients before treatment compared with their matched controls (Figure 3). The levels of protein expression decreased, were unchanged, or increased slightly after treatment.

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DISCUSSION

The first whole-proteome analysis of AH proteins was reported in 2008; this analysis was performed mostly by 2DE using frog eyes.15 Recently, Chowdhury and associates identified 676 proteins in the AH of patients undergoing cataract surgery.49 Izzotti and associates analyzed the expression of 1264 proteins using glass antibody−microarrays and detected remarkable changes in the AH proteins of glaucomatous patients.19 Another recent investigation reported the proteomic profiling of the AH of patients with neovascular AMD and the quantification of several proteins.51 In the present investigation, we focused on identifying novel proteins possibly secreted from the RPE to elucidate the mechanism of AMD and its response to the current standard treatment as well as to identify potential biomarkers of the disease. We identified exosomes in the AH of neovascular AMD patients for the first time and quantified the changes 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 protein composition. The number of microvesicles released from neural cells is reported to be low compared with the numbers released by other cells such as endothelial cells, stem cells, tumor cells, or platelets.54 In addition, AH has a relatively low protein content. Because RPE-specific exosomes might be diluted in the AH, a simple and efficient method should be used to obtain exosomes from this fluid. The SBI ExoQuick exosome precipitation reagent used in this study effectively isolated exosomes and their proteins for further LC−ESI−MS/MS and

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

Biomarker Verification Using LC−MRM from Individual AH Samples

A total of six candidate proteins were subjected to LC−MRM assays. It is critical to select unique tryptic peptides for target proteins with good MS signals. We used MRMPilot 2.1 software (AB SCIEX, Foster City, CA) to select multiple tryptic peptides for the given target proteins. The MRMPilot analysis provided good candidates for target peptides (no modification, no methionine, no cysteine residue, two tryptic ends, and no missed cleavage sites) and transitions (higher m/z value than the precursor m/z) for MRM analyses. We verified the uniqueness of these peptides and chose appropriate peptides 589



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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

12667788

myosin-9

3 4

194248072 4503143

heat shock protein 70 cathepsin D

5

4504919

cytokeratin 8

6

15431310

cytokeratin 14

2/y9 2/y8 2/y6 2/y6 2/y6 2/y9 2/y6 2/y5 2/y6 2/y7 2/y5 2/y7 2/y8 2/y6

750.86 750.86 468.25 472.27 555.29 523.31 523.31 410.72 410.72 410.72 515.79 569.29 586.77 586.77

965.52 836.47 644.36 731.37 755.35 375.24 650.39 517.27 664.34 721.36 644.37 716.43 1057.5 857.42

50

2

QEYDEAGPSIVHR QEYDEAGPSIVHR SGFEPASLK LVWVPSDK LLQDFFNGR QPGITFIAAK QPGITFIAAK VGFAEAAR VGFAEAAR VGFAEAAR WSLLQQQK YEELQSLAGK DAEEWFFTK DAEEWFFTK

38 38 26 26 29 28 28 23 23 23 28 30 31 31

Western blot analyses.52,53 Our results showed that the exosomal preparation from the AH contained previously reported exosomal marker proteins, supporting the validity of this method. We also identified endosomal proteins, annexins, heat shock proteins, cytoskeletal proteins, complements, signaling mediators, and migration- and adhesion-related proteins in the AH Exosomes. Among these, Hsp70, a wellknown exosomal marker, was increased in untreated patients and decreased after anti-VEGF treatment, as shown by LC− MRM analysis. Heat shock proteins have also been reported to be present in glaucomatous AH.55 Hsp70, which acts as a molecular chaperone, is typically undetectable under normal conditions but highly induced in cells that are experiencing stress.56,57 Strong Hsp70 immunoreactivity has also been detected in the muscles, endothelial cell layers, and inflammatory infiltrates of carotid plaques.58 Collectively, these results suggest that the induction of heat shock proteins is one mechanism that protects against the accumulation of misfolded proteins, which might occur during the course of AMD or could also be an indicator of vascular damage in the CNV. Thus, Hsp70 should be further investigated as a potential biomarker of cellular stress and targeted for therapeutics to stimulate endogenous adaptive and protective mechanisms to ameliorate the disease process.

50 50 50 50 50 50 50 50 50 50 50

binding protein, complement factors C3, annexin A1, cytokeratin 14, and cathepsin D, could be considered secretory proteins from the RPE of AMD patients. Many well-known exosomal proteins were also found in the above list of possible secreted proteins. Although it remains to be established whether the exosomes in AH are secreted by the RPE, we found that the ARPE-19 Exosomes contain 37 proteins that were also detected in the AH Exosomes (Supplemental Table 5 in the Supporting Information). Thus, we speculate that RPE secretes many proteins in exosomal vesicles. The exocytic activity of the RPE has functional significance in the pathogenesis of AMD because this mechanism is implicated in the formation of drusen, extracellular deposits that accumulate between the RPE and choroid, which is considered a risk factor for developing AMD. Moreover, this finding supports the feasibility of using ARPE-19 cell cultures for future studies of exosome release from RPE cells, an important development given the difficulty of obtaining large amounts of AH from patients or mice in vivo or CM from primary human RPE cell culture. The results from ARPE-19 cell cultures could be extrapolated and tested further by in vivo studies. Like other tissues and cells, exosomes of RPE cells might have promising roles as diagnostic and therapeutic targets and in further research to elucidate the mechanism of AMD pathogenesis. For example, the proteins that are exported in exosomes might be responsible 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 were exposed to oxidative stress, which mimics a disease condition and 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−lysosome fusion and the activation of autophagy59 and is known as principal lysosomal protease in the RPE,60 in the AH of AMD patients and controls by LC−MRM and in exosomes isolated from the AH of AMD patients and controls by LC−ESI−MS/ MS. The levels of cathepsin D in exosomes from the AH and CM as well as from the AH of three AMD patients were also measured by Western blot analysis. We suggest that autophagic activity increased as a survival mechanism in response to the oxidative conditions in AMD patients and that the upregulation of cathepsin D activity is required for the proteolytic activity needed for the breakdown of toxic materials sequestered by the autophagosomes in RPE.7 The LC−MRM analysis of cathepsin D showed that although the average level in patients was higher

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

We quantified six proteins by LC−MRM in the AH from 14 AMD patients and 6 control subjects. These proteins were selected as potential candidate biomarkers of AMD or as potential contributors to the pathogenic mechanism of AMD; they are most likely secreted by the RPE, the progressive degeneration of which is believed to be the initiating event of AMD. Understanding the adaptation or damage response of the RPE in the context of AMD could improve both diagnosis and therapy of this complex disease. Yuan and associates11 reported that secreted proteins accounted for a large proportion of the proteins found to be elevated (∼44% secreted) or decreased (∼38% secreted) in AMD tissues from cadaveric donors. A comparison of the protein lists reporting the differential expression of secreted proteins in the RPE cells of AMD and control donors48 and the secretome of RPE cell cultures in this study showed that many proteins, including actin (aortic smooth muscle), myosin-9, galectin 3-binding protein, lysozyme, metalloproteinase inhibitor, pigment epithelium-derived factor (PEDF), vitamin D590



Article

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 1 month after treatment with ranibizumab; y axis, mean value: corrected value of peak area with internal standard). The relative abundances of six proteins were elevated in the AH samples from patients before and 1 month after treatment compared with those from the control group as determined 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 at 2.10 and 1.53). (B) ROC curves of six selected proteins. A representative peptide from each protein was used in the analyses. The area under the curve (AUC) at a 95% confidence level is shown.

pharmacological manipulation of autophagy or related signaling pathways may be attractive therapeutic strategies for AMD. As Fader and associates suggested,62 the induction of autophagy may decrease the secretion of exosomes to the extracellular space as multivesicular bodies are diverted to be fused with autophagosomes and subsequently degraded intracellularly by the lysosome. Wang and associates described increased autophagy and increased exocytotic activity in aged RPE and

than that in the controls, its level was decreased in some patients. This finding might indicate variability in personal adaptive response, highlighting the potential efficacy of personalized therapy in which the status of autophagic activity is identified for each individual. High concentrations of autophagy-related proteins in the AH may be a part of the defense of the RPE against AMD; that is, the RPE produces these proteins locally to minimize disease activity.61 Thus, the 591



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markers are routinely used in prognostic and monitoring assays for several types of malignancies. 68 Our detection of cytokeratins in the AH of AMD patients suggests that these proteins may be very interesting potential biomarkers that, along with clinical data such as those provided by optical coherence tomography of macula, might be used to monitor patients with neovascular AMD.

the presence of autophagy and exosome markers in drusen of donated eyes with AMD.23 Increased exocytic activity, including the formation of endosomes and multivesicular bodies and the release of exosomes from the cell, would promote cell health by expelling damaged, toxic macromolecules, or undigested intracellular proteins into the extracellular space.63 However, the increased formation of drusen with increasing exocytic activity would aggravate the interference with the exchange of metabolites and waste products between the choriocapillaris and RPE and further compromise the function of the stressed RPE. Thus, increasing autophagy activity in a discretely controlled manner may decrease the formation of drusen in patients with AMD and prevent the progression of dry AMD and the development of neovascular AMD. Recognition of the functional status of the autophagy-lysosomal pathway and exocytic activity would help to treat AMD patients and improve therapeutic outcomes.

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CONCLUSIONS The prevalence of advanced AMD in the United States is projected to increase by 50%, to ∼3 million individuals, by the year 2020, largely due to the rapid growth of the elderly population. However, there are no therapies available to repair retinal damage in advanced neovascular AMD. In addition, there is no treatment that effectively prevents dry AMD and the progression from dry to neovascular AMD. We have shown that proteins secreted from the RPE in vivo can be obtained from the AH of patients with neovascular AMD and provided the first evidence that exosomes are present in the AH of these patients. We adopted an integrated approach to compare the AH with the CM of a well-known RPE cell culture system in vitro to discover and select potential candidate proteins for further validation. We believe that comparing the proteomes of the AH of neovascular AMD patients with those of control subjects is a powerful strategy for directly identifying the mechanisms responsible for the AMD process in vivo. Six proteins were found in the AH Exosomes or in the ARPE-19 Exosomes or both; thus, the secretion of these proteins may involve exosomal exocytosis, similar to that involved in drusen formation. Despite the large quantitative and qualitative variability of the protein contents in individual human samples,24 LC−MRM analysis of samples from 14 patients showed that these proteins were increased in patients compared with control subjects and decreased after treatment. Because the current treatment with ranibizumab is not the ideal therapy for neovascular AMD, the expression of AMD-related proteins in patients may decrease, remain unchanged, or increase further after treatment depending on the biological behavior of the disease or its response to anti-VEGF therapy, which will likely vary among patients and proteins. Whether the increased or decreased abundance of these proteins in AMD reflects the consequences or the causes of AMD remains to be determined, but identifying such proteins may enhance our understanding of the biological pathways involved in this complex disease. Our study has identified several potential biomarkers and therapeutic target proteins in AMD, such as molecular chaperone proteins (heat shock proteins) and proteins related to the autophagy-lysosomal pathway, and EMT. Further research into the biology of disease progression and the mechanistic study of disease control will be driven by these findings in the AH and its exosomes. The differential expression of these candidate proteins in both the AH and plasma will also be further verified in AMD with various disease courses versus controls to confirm their utility as molecular diagnostic markers and for personalized medicine. The proteomics-based characterization of this multifactorial disease may thus help to match a particular marker to particular target-based therapy in AMD patients with various phenotypes. Incorporating the analysis of biomarkers, including the EMT markers identified in this study, in randomized clinical trials of anti-VEGF therapy in neovascular AMD patients, may also provide biomarkers predictive of response and resistance to anti-VEGF therapies in AMD.

Cytokeratins and the Epithelial Mesenchymal Transition

The presence and increased levels of cytokeratin 8 and 14 in the AH of AMD patients have not been previously reported, although a comparative proteomic analysis of the expression of several cytokeratins, including cytokeratin 14 in patients with SLE64 and cytokeratins 1, 9, and 10 in human saliva,65 has been reported. Cytokeratin 8 and 14 expression is increased in AMD patients versus controls, as shown by LC−MRM and Western blot analysis (Figures 3 and 4). Kongara and associates66 indicated that abnormal keratin accumulation in mammary tumors may be a histologic marker of defective autophagy status and oxidative stress, and it may indicate more aggressive disease. As these authors suggested, and as previously mentioned, defects in autophagy may be associated with a more aggressive course of AMD. In contrast, Lau and associates67 have suggested that the upregulation of cytokeratin 8 may be responsible for apoptotic resistance. Cytokeratin has been extensively studied in many tissues, including the liver and various tumors.67−69 However, the study of cytokeratin 8 in RPE has been limited, although it has been identified as an RPE epithelial marker.61 Whether cytokeratin upregulation in neovascular AMD patients is an endogenous adaptive response associated with a favorable clinical course and good treatment outcomes or an indicator of more aggressive disease and poor outcomes should be determined in future studies with larger sample sizes. In human CNVM, many RPE cells represented transdifferentiated RPE.70 This finding suggests that during the course of AMD, RPE cells are de- or trans-differentiated, which might induce the deterioration of RPE function as well as AMD progression. Prolonged treatment with VEGF-A and VEGF-B was reported to induce typical epithelial−mesenchymal transition (EMT) phenotypes in a human pancreatic cell line.71 We speculate that EMT of RPE cells may also affect the sensitivity of AMD to treatment. There have been no reports regarding EMT as a possible mechanism for resistance to antiVEGF treatment in AMD. However, EMT has been increasingly reported to cause resistance to drugs, including anti-VEGF drugs, and to increase metastasis in various cancers.72 We expect that this study will lead to the validation of this EMT-related biomarker as a predictor of AMD development, progression, and responsiveness to anti-VEGF treatments. It is possible that normal epithelial cells from the AMD may undergo EMT during the process of neovascular AMD development. Specific plasma or serum cytokeratin 592



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Schaumberg, D. A.; Scholl, H. P.; Schwartz, S. G.; Scott, W. K.; Shahid, H.; Sigurdsson, H.; Silvestri, G.; Sivakumaran, T. A.; Smith, R. T.; Sobrin, L.; Souied, E. H.; Stambolian, D. E.; Stefansson, H.; SturgillShort, G. M.; Takahashi, A.; Tosakulwong, N.; Truitt, B. J.; Tsironi, E. E.; Uitterlinden, A. G.; van Duijn, C. M.; Vijaya, L.; Vingerling, J. R.; Vithana, E. N.; Webster, A. R.; Wichmann, H. E.; Winkler, T. W.; Wong, T. Y.; Wright, A. F.; Zelenika, D.; Zhang, M.; Zhao, L.; Zhang, K.; Klein, M. L.; Hageman, G. S.; Lathrop, G. M.; Stefansson, K.; Allikmets, R.; Baird, P. N.; Gorin, M. B.; Wang, J. J.; Klaver, C. C.; Seddon, J. M.; Pericak-Vance, M. A.; Iyengar, S. K.; Yates, J. R.; Swaroop, A.; Weber, B. H.; Kubo, M.; Deangelis, M. M.; Leveillard, T.; Thorsteinsdottir, U.; Haines, J. L.; Farrer, L. A.; Heid, I. M.; Abecasis, G. R. Seven new loci associated with age-related macular degeneration. Nat. Genet. 2013, 45 (4), 433−439. (3) Churchill, A. J.; Carter, J. G.; Lovell, H. C.; Ramsden, C.; Turner, S. J.; Yeung, A.; Escardo, J.; Atan, D. VEGF polymorphisms are associated with neovascular age-related macular degeneration. Hum. Mol. Genet. 2006, 15 (19), 2955−61. (4) Galan, A.; Ferlin, A.; Caretti, L.; Buson, G.; Sato, G.; Frigo, A. C.; Foresta, C. Association of age-related macular degeneration with polymorphisms in vascular endothelial growth factor and its receptor. Ophthalmology 2010, 117 (9), 1769−74. (5) Seddon, J. M.; Francis, P. J.; George, S.; Schultz, D. W.; Rosner, B.; Klein, M. L. Association of CFH Y402H and LOC387715 A69S with progression of age-related macular degeneration. JAMA 2007, 297 (16), 1793−800. (6) Suzuki, M.; Tsujikawa, M.; Itabe, H.; Du, Z. J.; Xie, P.; Matsumura, N.; Fu, X.; Zhang, R.; Sonoda, K. H.; Egashira, K.; Hazen, S. L.; Kamei, M. Chronic photo-oxidative stress and subsequent MCP1 activation as causative factors for age-related macular degeneration. J. Cell Sci. 2012, 125 (Pt 10), 2407−15. (7) Rakoczy, P. E.; Zhang, D.; Robertson, T.; Barnett, N. L.; Papadimitriou, J.; Constable, I. J.; Lai, C. M. Progressive age-related changes similar to age-related macular degeneration in a transgenic mouse model. Am. J. Pathol. 2002, 161 (4), 1515−24. (8) Ni, M.; Holland, M.; Jarstadmarken, H.; De Vries, G. Timecourse of experimental choroidal neovascularization in Dutch-Belted rabbit: clinical and histological evaluation. Exp. Eye Res. 2005, 81 (3), 286−97. (9) Decanini, A.; Nordgaard, C. L.; Feng, X.; Ferrington, D. A.; Olsen, T. W. Changes in select redox proteins of the retinal pigment epithelium in age-related macular degeneration. Am. J. Ophthalmol. 2007, 143 (4), 607−15. (10) Justilien, V.; Pang, J. J.; Renganathan, K.; Zhan, X.; Crabb, J. W.; Kim, S. R.; Sparrow, J. R.; Hauswirth, W. W.; Lewin, A. S. SOD2 knockdown mouse model of early AMD. Invest. Ophthalmol. Vis. Sci. 2007, 48 (10), 4407−20. (11) Yuan, X.; Gu, X.; Crabb, J. S.; Yue, X.; Shadrach, K.; Hollyfield, J. G.; Crabb, J. W. Quantitative proteomics: comparison of the macular Bruch membrane/choroid complex from age-related macular degeneration and normal eyes. Mol. Cell. Proteomics. 2010, 9 (6), 1031−46. (12) Noma, H.; Funatsu, H.; Yamasaki, M.; Tsukamoto, H.; Mimura, T.; Sone, T.; Hirayama, T.; Tamura, H.; Yamashita, H.; Minamoto, A.; Mishima, H. K. Aqueous humour levels of cytokines are correlated to vitreous levels and severity of macular oedema in branch retinal vein occlusion. Eye (London, U. K.) 2008, 22 (1), 42−8. (13) Macknight, A. D.; McLaughlin, C. W.; Peart, D.; Purves, R. D.; Carre, D. A.; Civan, M. M. Formation of the aqueous humor. Clin. Exp. Pharmacol. Physiol. 2000, 27 (1−2), 100−6. (14) Escoffier, P.; Paris, L.; Bodaghi, B.; Danis, M.; Mazier, D.; Marinach-Patrice, C. Pooling aqueous humor samples: bias in 2D-LCMS/MS strategy? J Proteome Res. 2010, 9 (2), 789−97. (15) Panova, I. G.; Sharova, N. P.; Dmitrieva, S. B.; Levin, P. P.; Tatikolov, A. S. Characterization of the composition of the aqueous humor and the vitreous body of the eye of the frog Rana temporaria L. Comp. Biochem. Physiol., Part A: Mol. Integr. Physiol. 2008, 151 (4), 676−81.

ASSOCIATED CONTENT

S Supporting Information *

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

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: 82-2-2030-7657. Fax:82-22030-5273. Author Contributions ○

G.-Y.K. and J.Y.B. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (2012M3A9B2028333 and 2012R1A1A11012171).

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ABBREVIATIONS AMD, age-related macular degeneration; CNV, choroidal neovascularization; RPE, retinal pigment epithelium; VEGF, vascular endothelial growth factor; AH, aqueous humor; LC− ESI−MS/MS, liquid chromatography-electrospray ionization tandem mass spectrometry; LC−MRM, liquid chromatography multiple reaction monitoring; CM, conditioned medium; Hsp70, heat shock protein 70; Hspβ-1, heat shock protein β1; Tsg101, tumor susceptibility gene 101; ESCRT, endosomal sorting complexes required for transport; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; PEDF, pigment epithelium-derived factor; EMT, epithelial-mesenchymal transition

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REFERENCES

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