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Research Article
The Measurement of Intraocular Biomarkers in Various
Stages of Proliferative Diabetic Retinopathy Using Multiplex
xMAP Technology
Stepan Rusnak,1Jindra Vrzalova,2,3 Marketa Sobotova,1Lenka Hecova,1
Renata Ricarova,1and Ondrej Topolcan2,3
1Department of Ophthalmology, University Hospital Pilsen, Alej Svobody 80, 304 60 Plzen, Czech Republic
2Department of Nuclear Medicine, Laboratory of Immunoanalysis, University Hospital Pilsen, Dr. E. Benese 13,
305 99 Plzen, Czech Republic
3Central Radioisotopic Laboratory, Faculty of Medicine in Pilsen, Charles University in Prague, Dr. E. Benese 13,
305 99 Plzen, Czech Republic
Correspondence should be addressed to Marketa Sobotova; sobotovam@fnplzen.cz
Received December ; Accepted February
Academic Editor: Ricardo Giordano
Copyright © Stepan Rusnak et al. is is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Purpose. To determine the intraocular levels of growth factors and cytokines in patients with various degrees of severity of
proliferative diabetic retinopathy (PDR) using multiplex xMAP technology. Methods. A prospective cohort study of eyes from
patients who were divided into groups based on the severity of PDR. Patients in group number are those who presented
PDR with no need of repeated surgical intervention; patients in group number had repeated vitreous bleeding; and patients in
group number had refractory neovascular glaucoma. e concentrations of proangiogenic, antiangiogenic, inammatory, and
neurotrophic factors were measured in intraocular uid. e results were also compared with levels of factors measured in eyes
from patients prior to senile cataract surgery (control group). Results. Patients with refractory neovascular glaucoma (the highest
clinical severity group) had higher levels of interleukin (IL-) (median .; median .; 𝑃 = .00096), transforming growth
factor beta (TGF𝛽-) (median .; median .; 𝑃 = .0017), and vascular endothelial growth factor (VEGF) (median
.; median .; 𝑃 = .0454)comparedwithotherPDRpatients.Conclusions. Results of our study imply that levels of IL-,
TGF𝛽-,andVEGFcorrelatewiththeseverityofPDR.
1. Introduction
Diabetes mellitus is one of the most common endocrine
disorders in the world; it aected roughly % of the global
population ca. in the year , and it is estimated that it
will aect million people in []. Diabetic retinopathy
aects % of the patients in the diabetic population []and
is the main cause of permanent vision loss in the working
population []. Proliferative diabetic retinopathy (PDR) is
characterized by the pathological formation of retinal blood
vessels. Despite progress in diagnostics and therapies, PDR
leads to a terminal stage of therapeutically unmanageable
neovascularization that is characterized by the development
of secondary neovascular glaucoma in a number of cases.
Retinalhypoxiaisamajordrivingforceforretinalneo-
vascularization that increases hypoxia inducible factor (HIF)
levels and launches a cascade of the production of cytokines
and growth factors. Since the discovery of the proangio-
genic role of vascular endothelial growth factor (VEGF) in
PDR,changesinthelevelsofanumberofotherproan-
giogenic factors, such as those in the insulin-like growth
factor family (IGF), hepatocyte growth factor (HGF), basic
broblast growth factor (b-FGF), platelet-derived growth fac-
tor (PDGF), proinammatory cytokines, and angiopoietin,
have been demonstrated. However, the intraocular synthesis
of angiogenic factors is counterbalanced by the synthesis
of antiangiogenic factors, including 𝛾-interferon inducible
protein (IP-), the pigment epithelium-derived factor
Hindawi Publishing Corporation
Journal of Ophthalmology
Volume 2015, Article ID 424783, 6 pages
http://dx.doi.org/10.1155/2015/424783
Journal of Ophthalmology
(PEDF), transforming growth factor beta (TGF𝛽), throm-
bospondin (TSP), endostatin, angiostatin, and somatostatin
[,]. Fluorescein angiography, or more recently ultra wide-
eld uorescein angiography, is used to determine the scope
of neovascularization or ischemia [].ismethodisan
image-processing technique for angiographic mapping of the
retina. It enables the most recent stage of retinal angiogenesis
to be described, but it is not an objective risk assessment
technique. e measurement of intraocular biomarkers is
emerging as a novel possibility for patient stratication.
Because neovascularization results from an imbalance in
proangiogenic and antiangiogenic factors, a multiplex ana-
lyticaltoolformonitoringthelevelsofseveralfactorsina
small sample volume is necessary to describe this process.
A combination of immunoanalysis and ow cytometry [],
called xMAP technology, is one of the most promising
multiplex technologies in clinical research to date.
In our study, the concentration levels of epidermal
growth factor (EGF), interleukin (IL-), VEGF, tumor
necrosis factor alfa (TNF-𝛼), interleukin (IL-), IP-,
monocyte chemoattractant protein (MCP-), PDGF, TGF𝛽-
, fractalkine, interleukin (IL-), interferon gamma (IFN-
𝛾), broblast growth factor (FGF-), brain-derived neu-
rotrophic factor (BDNF), ciliary neurotrophic factor (CNTF),
andRANTESinsamplesoftheaqueoushumourfroma
group of PDR patients were measured using xMAP tech-
nology. e PDR cohort was further divided into three
subgroupsonthebasisofclinicalseverityandthesesub-
groups were compared with a control group. Our aim was
to demonstrate that a biomarker panel measurement using
multiplex immunoanalysis is applicable as a diagnostic and
prognostic method in ophthalmology.
2. Materials and Methods
2.1. Patient Cohort. Patients undergoing treatment for PDR
at the University Hospital in Pilsen during – were
enrolled in this institutional prospective cohort study.
e patients with PDR were divided into groups accord-
ing to the severity of their pathologies. Group included
eyes from patients with PDR who had no need for
repeated surgical intervention (for better understanding, this
group consists of patients with PDR and vitreous bleeding,
patients with PDR and tractional retinal detachment,
patients with PDR, vitreous bleeding, and tractional retinal
detachment, patients with PDR and exudative maculopathy,
and patient with PDR with broproliferation), group was
composed of eyes of nine patients who had repeated vitre-
ousbleeding,andgroupincludedeyesfrompatients
with refractory neovascular glaucoma, which represents the
most severe stage of the disease. e control group (group )
wascomposedofeyesfrompreoperativesenilecataract
patients. Small samples (approximately 𝜇L) of intraocular
uid from the aqueous humour of each participant were
obtained under topical anesthesia from the anterior chamber
of each eye by means of aspiration using a ne -gauge
needle that was attached to a syringe.
2.2. Multiplex Analysis. All specimens were frozen imme-
diately. Samples were stored at −∘Cuntiltheywereana-
lyzed. No more than one freeze-thaw cycle was allowed
prior to analysis. e protein concentrations in the aqueous
humour were measured using multiplex xMAP technology
on a Luminex instrument with commercially available
panels from Millipore Corporation (Billerica, MA, USA),
MILLIPLEX MAP Human Cytokine/Chemokine Panel, and
MILLIPLEX MAP TGF𝛽-. e procedures were performed
according to the manufacturer’s instructions, and the control
samples that were provided within the kits were assayed in
eachanalysis.exMAPtechnologythatwasappliedisa
combination of immunoanalysis and ow cytometry based
on bead particles that can be distinguished by internal dyes,
as described, for example, by Kellar and Iannone []. In our
study, the levels of EGF, IL-, VEGF, TNF-𝛼, IL-, IP-,
MCP-, PDGF AA, TGF𝛽-, fractalkine, PDGF AB/BB, IL-,
IFN-𝛾, FGF-, CNTF, BDNF, and RANTES were studied.
2.3. Statistical Methods. A descriptive statistic was calculated
for each of the markers. e results under the calibration
curve ranges were stated as the value of the lowest calibration
point. e Mann-Whitney Utest (independent samples)
and Kruskal-Wallis test were used to compare marker levels
between groups. Borderline signicance was determined to
be reected by 𝑃values ranging from . to ., and
signicance was reected by 𝑃values below .. MedCalc
. statistics soware was used for analysis.
3. Results
e median, lower, and upper quartile values for all of
the markers within each group are listed in Table .When
comparing the groups, signicantly higher levels of IL-
, IL-, IP-, PDGF AA, and VEGF were found among
PDR patients compared with patients in the control group.
e concentrations of TGF𝛽- were higher in PDR patients
compared with the control group. Patients in group (those
with neovascular glaucoma that was refractory to treatment)
had higher levels of IL-, TGF𝛽-, and VEGF compared with
patients in PDR group and PDR group (the nonneovas-
cular glaucoma groups). e dierences in concentrations
were all of borderline signicance (see Tables and ). No
signicant dierences in marker levels were found between
PDR group (with no complications) and PDR group
(with repeated vitreous bleeding). No dierences between
groups were found in the levels of BDNF, CNTF, EGF, and
MCP-. See Tab l e fortheresultsofthegroupcomparisons.
Boxplots of the VEGF concentrations in each of the groups
areprovidedinFigure , and boxplots of the markers for
which levels in control eyes diered signicantly from levels
in the eyes of patients in PDR group are shown in Figure .
Because the vast majority of patients had intraocular uid
concentrations of fractalkine, PDGFAB/BB, IL-, IFN-𝛾,
TNF-𝛼, FGF-, and RANTES that were below the detection
limit of the panels that were used, the results from assays for
these markers are not presented. Although the concentration
of TGF𝛽- was below the detection limit in the control group,
Journal of Ophthalmology
T : Descriptive statistics. Median values and th and th percentile values in pg/mL for all markers and groups are shown. Proliferative
diabetic retinopathy (PDR) patients were divided into groups. Group : PDR patients with no need for repeated surgical intervention; group :
PDR patients with repeated vitreous bleeding, which is a less serious complication of PDR; group : PDR patients with refractory neovascular
glaucoma, which is a serious complication of PDR; group : control group.
Groups
Median – P Median – P Median – P Median – P
BDNF . .–. . .–. . .–. . .–.
CNTF . .–. . .–. . .–. . .–.
EGF . .–. . .–. . .–. . .–.
IL- . .–. . .–. . .–. . .–.
IL- . .–. . .–. . .–. . .–.
IP- . .–. . .–. . .–. . .–.
MCP- . .–. . .–. . .–. . .–.
PDGFAA . .–. . .–. . .–. . .–.
TGF𝛽- . .-. . .–. . .–. . .–.
VEGF . .–. . .–. . .–. . .–.
BDNF: brain-derived neurotrophic factor; CNTF: ciliary neurotrophic factor; EGF: epidermal growth factor; IL: interleukin; IP-: 𝛾-interferon inducible
protein ; MCP-: monocyte chemoattractant protein ; PDGF AA: platelet-derived growth factor AA; TGF𝛽-: transforming growth factor beta ; VEGF:
vascular endothelial growth factor.
P: percentile.
T : Comparison of biomarker levels between groups. 𝑃values are listed.
Kruskal-Wallis Mann-Whitney 𝑈
×
Mann-Whitney 𝑈
×
Mann-Whitney 𝑈
×
Mann-Whitney 𝑈
×
BDNF NS NS NS NS NS
CNTF NS NS NS NS NS
EGF NS NS NS NS NS
IL- <. <. NS . .
IL- <. <.NSNSNS
IP- <. <.NSNSNS
MCP- NS NS NS NS NS
PDGFAA <. <.NSNSNS
TGF𝛽- <. . NS . .
VEGF <. <. NS . .
BDNF: brain-derived neurotrophic factor; CNTF: ciliary neurotrophic factor; EGF: epidermal growth factor; IL: interleukin; IP-: 𝛾-interferon inducible
protein ; MCP: monocyte chemoattractant protein ; PDGF AA: platelet-derived growth factor AA; TGF𝛽-: transforming growth factor beta ; VEGF:
vascular endothelial growth factor.
NS: nonsignicant.
TGF𝛽- levels in some of the PDR patients were measureable;
thus, the results are presented.
4. Discussion
Biomarkers in disease detection and management have
become important tools in modern clinical medicine, and
their application to retinal disease should be no exception.
Because multiplex analysis based on xMAP technology allows
for the analysis of tens of analytes in a small sample volume
(– 𝜇L), this is a potent technology for introducing labo-
ratory medicine into ophthalmology.
In this study, we have conrmed that the patients with
PDR have higher intraocular concentrations of proangio-
genic, antiangiogenic, and inammatory cytokines compared
with nonPDR patients. Intraocular levels of IL-, IL-, IP-,
PDGF AA, TGF𝛽-, and VEGF were increased in patients
with PDR. Today, many studies compare the intraocular
concentrations of various cytokines in PDR patients versus
patientswhodonothavePDR.Maieretal.foundthat
mean cytokine levels of IP-, MCP-, and VEGF in the
vitreous humour were signicantly higher compared to those
of normal controls []. Murugeswari et al. documented that
levels of IL-, IL-, MCP-, and VEGF in the vitreous were
Journal of Ophthalmology
10000
1000
100
10
1
0123
Group
VEGF
F : Vascular endothelial growth factor levels for each group.
Group : proliferative diabetic retinopathy (PDR) patients with no
need for repeated surgical intervention; group : PDR patients with
repeated vitreous bleeding, which is a less serious complication of
PDR; group : PDR patients with refractory neovascular glaucoma,
which is a serious complication of PDR; group : control group.
10000
1000
100
10
1
IL-6IL-8IP-10 PDGF AA VEGF
ds
0
1
Group =0or group =1
TGF𝛽-1
Box-and-whisker
F : Levels of biomarkers for which signicant dierences
between the levels in control group and those in proliferative
diabetic retinopathy group were found. IL: interleukin; IP-: 𝛾-
interferon inducible protein ; PDGF AA: platelet-derived growth
factor AA; VEGF: vascular endothelial growth factor; TGF𝛽-:
transforming growth factor beta .
signicantly higher in PDR patients compared with levels
in macular hole patients. Conversely, the vitreous level of
PEDF was signicantly reduced in patients with PDR [].
Yoshimuraetal.performedacomprehensiveanalysisof
mediators in the vitreous uids in PDR patients and in
patientswithotheroculardiseases,andtheyfoundelevated
levels of VEGF, MCP-, IL-, and IL- compared with control
patients []. We found similar results in this study, but
we have not demonstrated that the concentration of MCP-
increases in patients with PDR. However, we have shown
that higher intraocular concentrations of PDGF AA and
nonmeasurablevaluesofPDGFAB/BBcanbeseeninPDR
patients. Contrary to our result, Freyberger et al. published
results showing that PDGF AB levels are elevated in patients
with PDR [].
In a clinical environment, it is essential to further stratify
the PDR patients; however, only a few studies that compare
the levels of biomarkers in PDR patients with diering disease
severities exist. Funatsu et al. divided PDR patients into
subgroups based on disease progression and regression. e
vitreous levels of VEGF and IL- were signicantly higher in
the eyes of patients in the progression group than they were
in eyes with PDR regression. Multivariate logistic regression
analysis showed that higher vitreous levels of VEGF were
associated with the progression of PDR following vitreous
surgery. A high vitreous level of VEGF was identied as a
signicant risk factor in determining the outcome of vitreous
surgery in patients with PDR []. Freyberger et al. studied
patients with PDR, four of whom had rubeosis iridis,
which is an indicator of very high vasoproliferative activity.
Signicantly elevated concentrations of PDGF AB were found
among individuals with PDR; even higher levels were found
in conjunction with rubeosis iridis []. In our study, patients
with neovascular glaucoma that was refractory to treatment
showed higher levels of IL-, TGF𝛽-, and VEGF than
other PDR patients, which implies that the levels of these
three factors are correlated with the severity of PDR. No
dierences in biomarker levels were found between patients
whobelongedtogroup(thosewithrepeatedvitreous
bleeding)andthosewhobelongedtogroup(thosewhohad
no complications).
e novel multiplex technology that we proposed not
only saves time, labor, and costs of immunoanalysis, but
it also rapidly reduces the sample volume requirements
compared to a traditional immunoanalysis method (single
ELISA) while allowing the full comparability of all studied
parameters. e last two points are critical when entering
laboratory measurements into the diagnostic and risk assess-
ment process in ophthalmology. One limitation of xMAP
technologycouldbethatitislimitedinitsabilitytodetect
some factors. In the present study, we were not able to detect
fractalkine, PDGF AB/BB, IL-, IFN-𝛾,TNF-𝛼,FGF-,and
RANTES in the aqueous humour. Similarly, Yoshimura et al.
found that the intraocular concentrationsof IL-𝛽,IL-,IL-,
IL-,IL-,IL-,IFN-𝛾,TNF-𝛼,eotaxin,MIP-𝛼,RANTES,
EGF, and FGF- were lower than the detection level [].
We have chosen three works as examples of studies that
haveshownthepotencyofxMAPtechnologyinophthal-
mology. Curnow et al. measured a panel of cytokines in
the aqueous humour, and from the spectra of cytokines
that they studied, they used random forest analysis to show
that only IL-, IL-, MCP-, IL-, IL-, and TNF-𝛼are
required to distinguish between noninammatory control
and idiopathic uveitis with % classication accuracy [].
Funding et al. used xMAP technology to simultaneously
quantify and compare the concentrations of immune
Journal of Ophthalmology
mediators in aqueous humour samples from patients with
corneal rejection and patients with a noninammatory con-
dition in the anterior chamber. eir results underscore both
the complex immunological interactions of the rejection
process and the need for multiplex laboratory measurements
based on small sample volumes []. Rusnak et al. mea-
suredthelevelsofcytokinesintheaqueoushumour
in eyes that were undergoing vitrectomies for retinal
detachment with various degrees of severity of proliferative
vitreoretinopathy. According to this study MCP- and VEGF
may participate in pathogenesis of retinal detachment and
proliferative vitreoretinopathy [].
Sohn et al. have shown that multiplex measurements
of cytokine and growth factor concentrations also enable
treatment monitoring. Aer intravitreal injections of
antiangiogenic drugs (triamcinolone and bevacizumab), the
clinical eects and dierences in biomarker levels in the
aqueous humour were monitored. A more eective treatment
modality was linked to decreases in the concentrations of IL-
, IP-, MCP-, PDGF AA, and VEGF compared with those
resulting from a less eective treatment; the latter treatment
was only connected with a decrease in the concentration of
VEGF [].
e Sohn et al. study [], in conjunction with our
ndings in patients with neovascular glaucoma refractory to
treatment, shows that biomarkers have a strong potential for
use in patient stratication and in determining personalized
medical needs. Tailored treatments are necessary due to the
introduction and costs of novel treatment. e vast majority
of novel types of therapy are based on the inhibition of VEGF.
A number of anti-VEGF agents have been introduced into
clinical use and are widely used for the treatment of many
ocular diseases, but the widespread use of these agents raises
new questions. It has been proposed that anti-VEGF agents
may have negative eects on retinal cells. Animal studies have
shown that systemic neutralization of VEGF with soluble
VEGF receptors results in a reduction of the thicknesses
of both the inner and the outer nuclear layers in adult
mouse retinas. ese results indicate that endogenous VEGF
plays an important role in the maintenance and function of
neuronal cells in the adult retina and suggest that anti-VEGF
therapies should be administered with caution []. Because
of the risks associated with using anti-VEGF therapies, it is
absolutely necessary to select patients who can benet from
anti-VEGF treatment despite the risk of adverse eects. In
ourstudy,wehaveshownthatcertaincomplications,such
as neovascular glaucoma that is refractory to conventional
treatment, are correlated with high concentrations of cer-
tainbiomarkers;inthefuture,wecanusethesetojustify
more aggressive therapies. Our ndings suggest that patients
could be selected for repeat intravitreal injections of VEGF
inhibitors, corticosteroids, more aggressive panretinal laser
photocoagulation, cyclocryodestruction, or cyclophotode-
struction on the basis of biomarker concentrations. Another
study that shows that measuring protein concentrations in the
aqueous humour has potential future benets for treatment
monitoringwasconductedbyCampochiaroetal.;they
measured concentrations of VEGF, IL-, IL- beta, tumor
necrosis factor, and ranibizumab [].
Another problem in patients who have been treated
with anti-VEGF therapies is determining the concentration
of VEGF. e determination of the VEGF concentration is
inuenced by treatment with anti-VEGF inhibitors via direct
interaction in the immunoanalysis, which we veried in our
laboratory (data not presented). e interaction requires an
adjustment to the approach to determining the intraocular
concentrations of VEGF in these patients; the half-life of
anti-VEGF therapies in the eye was established as being .
days []. Only patients who had never received anti-VEGF
treatment or, in advanced cases, had received their most
recent administrations of anti-VEGF therapy more than two
months prior to aqueous humour sampling for this study
were included. With the expansion of anti-VEGF therapy, it
is clear that multifactor monitoring, that is, introducing other
biomarkers in addition to VEGF, is important. Both our study
andothersshowthatthereareseveralpossiblecandidate
biomarkers. As more PDR biomarkers are identied, a panel
of them has the potential to be eective for identifying
high-risk individuals, monitoring disease progression, and
evaluating the ecacy of therapeutic interventions.
In conclusion, the results of our study suggest that the
concentrations of IL-, TGF𝛽-, and VEGF correlate with the
severity of PDR. In future, assessment of PDR biomarkers
in intraocular uid could be eective method for treatment
monitoring and early detection of PDR progression.
Consent
Each participant signed informed consent approved by the
Institutional Review Board.
Conflict of Interests
e authors declare that there is no conict of interests
regarding the publication of this paper.
Acknowledgment
isstudywassupportedbythegovernmentgrantingagency
IGA MZ NS-.
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