This study aimed to identify novel secretory biomarkers for neovascular age-related macular degeneration (AMD) using an integrated proteomics approach. The researchers compared proteins identified in conditioned media from cultured retinal pigment epithelium (RPE) cells and exosomes derived from the conditioned media and aqueous humor of AMD patients. Liquid chromatography-mass spectrometry revealed that cathepsin D, cytokeratin 8, and four other proteins were increased in the aqueous humor of AMD patients compared to controls. This study identified potential biomarkers and therapeutic targets for AMD and demonstrated a novel method for detecting AMD-associated proteins secreted by RPE in vivo in the form of exosomes.

1. 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
■ 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
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 neo-
vascular 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
Received: July 19, 2013
Published: January 8, 2014
Article
pubs.acs.org/jpr
© 2014 American Chemical Society 581 dx.doi.org/10.1021/pr400751k | J. Proteome Res. 2014, 13, 581−595

2. 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 VEGF-
A, 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
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 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 micro-
vesicles with a diameter of 30−100 nm. They are released
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, bronchoal-
veolar 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.
■ 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
Journal of Proteome Research Article
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3. 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 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. 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)
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 uncon-
trolled 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.
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 Precip-
itation 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.
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 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 6
age (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 ± 6.8
sex (men:women) 5:4 5:4 2:1 2:1 9:5 5:1
diabetes mellitus (no.) 2 2 1 1 3 1
hypertension (no.) 5 3 1 1 4 2
a
AH: aqueous humor. b
WB: Western blot analysis. c
AMD: age-related macular degeneration. d
Before: before treatment with ranibizumab. e
After:
one month after treatment with ranibizumab.
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4. For TEM, exosomes were directly adsorbed onto Formvar-
carbon-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 JEM-
1100 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 fluorescence-
conjugated 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).
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
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 immunofluor-
escence 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.
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, EksigentTechnolo-
gies, 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,
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5. 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.
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
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.
■ 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
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.
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6. 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
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
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.
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7. Table2.ListofCommonProteinsIdentifiedbyLC−ESI−MS/MSAnalysisandExistinginAllThreeProfiles:ConditionedMediumofARPE-19CellCultures(ARPE-19CM),
ExosomesIsolatedfromtheCMofARPE-19CellCultures(ARPE-19Exosomes),andExosomesIsolatedfromtheAH(AHExosomes)(No.1-23)a
no.accessionentrezreferenceMWscoreb
coveragepeptidecommonc
/targetd
11193957503848keratin,typeIIcytoskeletal165998.94350.3249.8327common
21193957543852keratin,typeIIcytoskeletal562340.01180.2425.344common
31260125713339basementmembrane-specificheparansulfateproteoglycancoreproteinprecursor468533.11070.3339542common
4471325572335fibronectinisoform1preproprotein272157.3770.3642.6780common
5669329472alpha-2-macroglobulinprecursor163188.390.246.838common
64502027213serumalbuminpreproprotein69321.63680.37813574common
7115298678718complementC3precursor187029.3260.2622.577common
81890837822934gelsolinisoformc81890.1360.2411.115common
911061123580781collagenalpha-1(XVIII)chainisoform1precursor153671.8210.3819.3130common
10324021745vitaminD-bindingproteinisoform3precursor55041.0990.2225.617common
11156523970197alpha-2-HS-glycoprotein39315.7140.181221common
12427409071191clusterinprecursor52461.0570.3120.335common
132910452307273titinisoformnovex-2301159030.140.23common
1450318633959galectin-3-bindingprotein65289.4180.3341.4219common
154502261462antithrombin-IIIprecursor52568.9830.22813common
1645031431509cathepsinDpreproprotein44523.6630.1311.24common/target
171514733751366E3ubiquitin-proteinligaseUBR5309156.720.140.82common
184557321335apolipoproteinA-Ipreproprotein30758.9450.222.812common
19324021738amyloidbetaA4proteinisoformhprecursor84986.1410.191.73common
20347340682192fibulin-1isoformAprecursor61538.350.2313.413common
21226246554643677hypotheticalproteinLOC643677801429.110.150.32common
2245058815340plasminogenisoform1precursor90510.2320.2236common
23153266841350beta-2-glycoprotein1precursor38272.6730.16116common
24126677884627myosin-9226390.6610.2830.4133target
2545049193856keratin,typeIIcytoskeletal853671.19290.3248168target
2621368837559actin,aorticsmoothmuscle41981.8250.2818.653target
271942480723303heatshock70kDaprotein1A/1B70009.1830.237.315target
28154313103861keratin,typeIcytoskeletal1451589.540.2411.99target
a
Inaddition,thetableincludestargetproteinsforLC−MRM(nos.16,24−28).b
Score:generatedbyBioworks(3.3.1)proteinconsensusscores.c
Common:Commoninallthreeprofiles(ARPE-19CM,
ARPE-19Exosomes,andAHExosomes).d
Target:TargetproteinsforLC−MRMthatarepresentinARPE-19CMandeitherARPE-19ExosomesorAHExosomes.
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8. 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 paraquat-
treated 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.
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 inhib-
itors, coagulation and complement cascades, proteins involved
in transport and metabolism, signaling molecules, and house-
keeping proteins (e.g., glyceraldehyde 3-phosphate dehydro-
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.
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
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9. 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 AMD-
related 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.
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
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 experi-
ments 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).
■ 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
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 O
myosin-9 O O
Hsp70 O O
cathepsin D O O O
cytokeratin 8 O O
cytokeratin 14 O O
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.
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10. 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 well-
known 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.
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 inhib-
itor, pigment epithelium-derived factor (PEDF), vitamin D-
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
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 38
QEYDEAGPSIVHR 2/y8 750.86 836.47 38
2 12667788 myosin-9 SGFEPASLK 2/y6 468.25 644.36 50 26
LVWVPSDK 2/y6 472.27 731.37 26
3 194248072 heat shock protein 70 LLQDFFNGR 2/y6 555.29 755.35 50 29
4 4503143 cathepsin D QPGITFIAAK 2/y9 523.31 375.24 50 28
QPGITFIAAK 2/y6 523.31 650.39 50 28
VGFAEAAR 2/y5 410.72 517.27 50 23
VGFAEAAR 2/y6 410.72 664.34 50 23
VGFAEAAR 2/y7 410.72 721.36 50 23
5 4504919 cytokeratin 8 WSLLQQQK 2/y5 515.79 644.37 50 28
YEELQSLAGK 2/y7 569.29 716.43 50 30
6 15431310 cytokeratin 14 DAEEWFFTK 2/y8 586.77 1057.5 50 31
DAEEWFFTK 2/y6 586.77 857.42 50 31
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11. 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
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
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.
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12. 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 macro-
molecules, 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.
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 trans-
differentiated 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 anti-
VEGF 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
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.
■ 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 charac-
terization 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 neo-
vascular AMD patients, may also provide biomarkers predictive
of response and resistance to anti-VEGF therapies in AMD.
Journal of Proteome Research Article
dx.doi.org/10.1021/pr400751k | J. Proteome Res. 2014, 13, 581−595592

13. ■ 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.
■ AUTHOR INFORMATION
Corresponding Author
*E-mail: hchung@kuh.ac.kr. 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.
■ 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).
■ 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 β-
1; Tsg101, tumor susceptibility gene 101; ESCRT, endosomal
sorting complexes required for transport; GAPDH, glycer-
aldehyde 3-phosphate dehydrogenase; PEDF, pigment epithe-
lium-derived factor; EMT, epithelial-mesenchymal transition
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Journal of Proteome Research Article
dx.doi.org/10.1021/pr400751k | J. Proteome Res. 2014, 13, 581−595595