Progress in brain research, volume 221

Serial Editor

Vincent Walsh
Institute of Cognitive Neuroscience
University College London
17 Queen Square
London WC1N 3AR UK

Editorial Board
Mark Bear, Cambridge, USA.
Medicine & Translational Neuroscience
Hamed Ekhtiari, Tehran, Iran.
Addiction
Hajime Hirase, Wako, Japan.
Neuronal Microcircuitry
Freda Miller, Toronto, Canada.
Developmental Neurobiology
Shane O’Mara, Dublin, Ireland.
Systems Neuroscience
Susan Rossell, Swinburne, Australia.
Clinical Psychology & Neuropsychiatry
Nathalie Rouach, Paris, France.
Neuroglia
Barbara Sahakian, Cambridge, UK.
Cognition & Neuroethics
Bettina Studer, Dusseldorf, Germany.
Neurorehabilitation
Xiao-Jing Wang, New York, USA.
Computational Neuroscience

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Contributors
Luca Agnifili
Department of Medicine and Aging Science, Ophthalmology Clinic, University
G. d’Annunzio of Chieti-Pescara, Chieti, Italy
Francesco Aiello
Moorfields Eye Hospital, NHS Foundation Trust, London, UK, and Department of
Experimental Medicine and Surgery, University of Rome Tor Vergata, Rome, Italy
Simone Altobelli
Diagnostic Imaging Section, Tor Vergata University Hospital, Department of
Biomedicine and Prevention, University of Rome Tor Vergata, Rome, Italy
Marcus Ang
Singapore Eye Research Institute, and Singapore National Eye Centre, Singapore,
Singapore
Giacinto Bagetta
Department of Pharmacy and Health and Nutritional Sciences, Section of
Preclinical and Translational Pharmacology, and University Consortium for
Adaptive Disorders and Head Pain (UCHAD), Section of Neuropharmacology of
Normal and Pathological Neuronal Plasticity, University of Calabria, Arcavacata di
Rende, Italy
Keith Barton
Glaucoma Service, Moorfields Eye Hospital; Department of Ophthalmology,
National University Health System, Singapore, Singapore; National Institute for
Health Research, Biomedical Research Centre for Ophthalmology, Moorfields
Eye Hospital, and Department of Epidemiology and Genetics, Institute of
Ophthalmology, University College, London, UK
Francesca Bertuzzi
Ophthalmology Department, Policlinico di Monza Hospital, University of MilanoBicocca, Monza, Italy
Davide Berzaghi
Ophthalmology Unit Department of Neurological, Neuropsychological,

Morphological and Movement Sciences, University of Verona, Verona, Italy
Dana Blumberg
Bernard and Shirlee Brown Glaucoma Research Laboratory, Edward S. Harkness
Eye Institute, Columbia University Medical Center, New York, NY, USA
Lorenza Brescia
Department of Medicine and Aging Science, Ophthalmology Clinic, University
G. d’Annunzio of Chieti-Pescara, Chieti, Italy
Giamberto Casini
Ophthalmology Unit, University of Pisa, Pisa, Italy

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vi

Contributors

Massimo Cesareo
Ophthalmology Unit, Department of Experimental Medicine and Surgery,
University of Rome Tor Vergata, Rome, Italy
Balwantray C. Chauhan
Department of Ophthalmology and Visual Sciences, Dalhousie University, Halifax,
NS, Canada
Francesca Chemello
Ophthalmology Unit Department of Neurological, Neuropsychological,
Morphological and Movement Sciences, University of Verona, Verona, Italy
Marco Ciancaglini
Ophthalmic Clinic Department of Surgical Science, Eye Clinic, University of
L’Aquila, L’Aquila, Italy
Elena Ciuffoletti

Ophthalmology Unit, Department of Experimental Medicine and Surgery,
University of Rome Tor Vergata, Rome, Italy
Maria Tiziana Corasaniti
Department of Health Sciences, University “Magna Graecia” of Catanzaro,
Catanzaro, Italy
Angela Di Gregorio
Ophthalmic Clinic Department of Surgical Science, Eye Clinic, University of
L’Aquila, L’Aquila, Italy
Silvio Di Staso
Ophthalmic Clinic Department of Surgical Science, Eye Clinic, University of
L’Aquila, L’Aquila, Italy
Vincenzo Fasanella
Department of Medicine and Aging Science, Ophthalmology Clinic, University
G. d’Annunzio of Chieti-Pescara, Chieti, Italy
Michele Figus
Ophthalmology Unit, University of Pisa, Pisa, Italy
Roberto Floris
Diagnostic Imaging Section, Tor Vergata University Hospital, Department of
Biomedicine and Prevention, University of Rome Tor Vergata, Rome, Italy
Michela Fresina
Department of Specialist, Diagnostics and Experimental Medicine (DIMES),
Ophthalmology Service, University of Bologna, Bologna, Italy
Francesco Garaci
Diagnostic Imaging Section, Tor Vergata University Hospital, Department of
Biomedicine and Prevention, University of Rome Tor Vergata, Rome, Italy
Julian Garcia-Feijoo
Servicio de Oftalmologı´a, Hospital Clı´nico San Carlos, Departamento de
Oftalmologı´a y ORL, Facultad de Medicina, Universidad Complutense de Madrid,

Contributors

Instituto de Investigacio´n Sanitaria del Hospital Clı´nico San Carlos (IdISSC), and
Cooperative Research Network on Age-Related Ocular Pathology, Visual and Life
Quality, Instituto de Salud Carlos III, Madrid, Spain
Sofia Garcia-Saenz
Servicio de Oftalmologı´a, Hospital Clı´nico San Carlos, Departamento de
Oftalmologı´a y ORL, Facultad de Medicina, Universidad Complutense de Madrid,
Instituto de Investigacio´n Sanitaria del Hospital Clı´nico San Carlos (IdISSC), and
Cooperative Research Network on Age-Related Ocular Pathology, Visual and Life
Quality, Instituto de Salud Carlos III, Madrid, Spain
Mario Alberto Giuliano
Ophthalmology Unit, Department of Experimental Medicine and Surgery,
University of Rome Tor Vergata, Rome, Italy
Gianluca Guidi
Ophthalmology Unit, University of Pisa, Pisa, Italy
Jost B. Jonas
Department of Ophthalmology, Medical Faculty Mannheim of the RuprechtKarls-University, Heidelberg, Germany, and Beijing Institute of Ophthalmology,
Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University,
Beijing Ophthalmology & Visual Sciences Key Laboratory, Beijing, China
Andreas Katsanos
Ophthalmology Department, University of Ioannina, Ioannina, Greece
Peng T. Khaw
National Institute for Health Research (NIHR) Biomedical Research Centre at
Moorfields Eye Hospital NHS Foundation Trust and UCL Institute of
Ophthalmology, London, UK
Fang Ko
National Institute for Health Research (NIHR) Biomedical Research Centre at
Moorfields Eye Hospital NHS Foundation Trust and UCL Institute of
Ophthalmology, London, UK

Anastasios G.P. Konstas
1st University Department of Ophthalmology; 3rd University Department of
Ophthalmology, Aristotle University, and Ophthalmica Institute, Thessaloniki,
Greece
Jeffrey M. Liebmann
Bernard and Shirlee Brown Glaucoma Research Laboratory, Edward S. Harkness
Eye Institute, Columbia University Medical Center, New York, NY, USA
Raffaele Mancino
Ophthalmology Unit, Department of Experimental Medicine and Surgery,
University of Rome Tor Vergata, Rome, Italy
Giorgio Marchini
Ophthalmology Unit Department of Neurological, Neuropsychological,
Morphological and Movement Sciences, University of Verona, Verona, Italy

vii


viii

Contributors

Jose Marı´a Martinez-de-la-Casa
Servicio de Oftalmologı´a, Hospital Clı´nico San Carlos, Departamento de
Oftalmologı´a y ORL, Facultad de Medicina, Universidad Complutense de Madrid,
Instituto de Investigacio´n Sanitaria del Hospital Clı´nico San Carlos (IdISSC), and
Cooperative Research Network on Age-Related Ocular Pathology, Visual and Life
Quality, Instituto de Salud Carlos III, Madrid, Spain
Alessio Martucci
Ophthalmology Unit, Department of Experimental Medicine and Surgery,
University of Rome Tor Vergata, Rome, Italy

Alessandra Mastropasqua
Department of Medicine and Aging Science, Ophthalmology Clinic, University
G. d’Annunzio of Chieti-Pescara, Chieti, Italy
Leonardo Mastropasqua
Department of Medicine and Aging Science, Ophthalmology Clinic, University
G. d’Annunzio of Chieti-Pescara, Chieti, Italy
Rodolfo Mastropasqua
Ophthalmology Unit Department of Neurological, Neuropsychological,
Morphological and Movement Sciences, University of Verona, Verona, Italy
Vincenzo Maurino
Moorfields Eye Hospital, NHS Foundation Trust, London, UK
Felipe A. Medeiros
Hamilton Glaucoma Center, Shiley Eye Center, and Department of
Ophthalmology, University of California, San Diego, CA, USA
Carmen Mendez-Hernandez
Servicio de Oftalmologı´a, Hospital Clı´nico San Carlos, Departamento de
Oftalmologı´a y ORL, Facultad de Medicina, Universidad Complutense de Madrid,
Instituto de Investigacio´n Sanitaria del Hospital Clı´nico San Carlos (IdISSC), and
Cooperative Research Network on Age-Related Ocular Pathology, Visual and Life
Quality, Instituto de Salud Carlos III, Madrid, Spain
Stefano Miglior
Ophthalmology Department, Policlinico di Monza Hospital, University of MilanoBicocca, Monza, Italy
Dimitrios G. Mikropoulos
3rd University Department of Ophthalmology, Aristotle University, and
Ophthalmica Institute, Thessaloniki, Greece
Filippo Missiroli
Ophthalmology Unit, Department of Experimental Medicine and Surgery,
University of Rome Tor Vergata, Rome, Italy
Lieve Moons
Research Group of Neural Circuit Development and Regeneration, Animal

Physiology and Neurobiology Section, Department of Biology, KU Leuven,
Leuven, Belgium


Contributors

Laura Morales-Fernandez
Servicio de Oftalmologı´a, Hospital Clı´nico San Carlos, Departamento de
Oftalmologı´a y ORL, Facultad de Medicina, Universidad Complutense de Madrid,
Instituto de Investigacio´n Sanitaria del Hospital Clı´nico San Carlos (IdISSC), and
Cooperative Research Network on Age-Related Ocular Pathology, Visual and Life
Quality, Instituto de Salud Carlos III, Madrid, Spain
Luigi Antonio Morrone
Department of Pharmacy and Health and Nutritional Sciences, Section of
Preclinical and Translational Pharmacology, and University Consortium for
Adaptive Disorders and Head Pain (UCHAD), Section of Neuropharmacology of
Normal and Pathological Neuronal Plasticity, University of Calabria, Arcavacata di
Rende, Italy
Marco Nardi
Ophthalmology Unit, University of Pisa, Pisa, Italy
Carlo Nucci
Ophthalmology Unit, Department of Experimental Medicine and Surgery,
University of Rome Tor Vergata, Rome, Italy
Songhomitra Panda-Jonas
Department of Ophthalmology, Medical Faculty Mannheim of the RuprechtKarls-University, Heidelberg, Germany
Maria Papadopoulos
National Institute for Health Research (NIHR) Biomedical Research Centre at
Moorfields Eye Hospital NHS Foundation Trust and UCL Institute of
Ophthalmology, London, UK
Damiana Pieragostino

Department of Medical, Oral and Biotechnological Sciences, and Analytical
Biochemistry and Proteomics Unit, Research Centre on Aging (Ce.S.I.),
University G. d’Annunzio of Chieti-Pescara, Chieti, Italy
Luciano Quaranta
Centre for the Study of Glaucoma, University of Brescia, Brescia, Italy
Federico Ricci
Ophthalmology Unit, Department of Experimental Medicine and Surgery,
University of Rome Tor Vergata, Rome, Italy
Robert Ritch
Einhorn Clinical Research Center, New York Ear Eye and Ear Infirmary of Mt.
Sinai, New York, NY, USA
Rossella Russo
Department of Pharmacy and Health and Nutritional Sciences, Section of
Preclinical and Translational Pharmacology, University of Calabria, Arcavacata di
Rende, Italy

ix


x

Contributors

Paolo Sacchetta
Department of Medical, Oral and Biotechnological Sciences, and Analytical
Biochemistry and Proteomics Unit, Research Centre on Aging (Ce.S.I.),
University G. d’Annunzio of Chieti-Pescara, Chieti, Italy
Federico Saenz Frances
Servicio de Oftalmologı´a, Hospital Clı´nico San Carlos, Departamento de
Oftalmologı´a y ORL, Facultad de Medicina, Universidad Complutense de Madrid,

Instituto de Investigacio´n Sanitaria del Hospital Clı´nico San Carlos (IdISSC), and
Cooperative Research Network on Age-Related Ocular Pathology, Visual and Life
Quality, Instituto de Salud Carlos III, Madrid, Spain
Enrique Santos-Bueso
Servicio de Oftalmologı´a, Hospital Clı´nico San Carlos, Departamento de
Oftalmologı´a y ORL, Facultad de Medicina, Universidad Complutense de Madrid,
Instituto de Investigacio´n Sanitaria del Hospital Clı´nico San Carlos (IdISSC), and
Cooperative Research Network on Age-Related Ocular Pathology, Visual and Life
Quality, Instituto de Salud Carlos III, Madrid, Spain
Orazio Schillaci
Diagnostic Imaging Section, Department of Biomedicine and Prevention, Tor
Vergata University Hospital, University of Rome Tor Vergata, Rome, Italy
Alon Skaat
Goldschleger Eye Institute, Sheba Medical Center, Sackler Faculty of Medicine,
Tel Aviv University, Tel Aviv, Israel
Chelvin C.A. Sng
Glaucoma Service, Moorfields Eye Hospital, London, UK; Department of
Ophthalmology, National University Health System, and Singapore Eye Research
Institute, Singapore, Singapore
Ingeborg Stalmans
Department of Neurosciences, Laboratory of Ophthalmology, KU Leuven, and
Department of Ophthalmology, University Hospitals Leuven (UZ Leuven), Leuven,
Belgium
Andrew J. Tatham
Princess Alexandra Eye Pavilion, and Department of Ophthalmology, University of
Edinburgh, Edinburgh, Scotland, UK
Miguel A. Teus
Universidad de Alcala´, Alcala´ de Henares, Madrid, Spain
Nicola Toschi
Diagnostic Imaging Section, Department of Biomedicine and Prevention, Tor

Vergata University Hospital, University of Rome Tor Vergata, Rome, Italy
Gian Marco Tosi
Ophthalmology Section, Department of Medicine, Surgery and Neuroscience,
University of Siena, Siena, Italy


Contributors

Paris G. Tranos
Ophthalmica Institute, Thessaloniki, Greece
Tine Van Bergen
Department of Neurosciences, Laboratory of Ophthalmology, KU Leuven,
Leuven, Belgium
Sarah Van de Velde
Department of Neurosciences, Laboratory of Ophthalmology, KU Leuven,
Leuven, Belgium
Evelien Vandewalle
Department of Neurosciences, Laboratory of Ophthalmology, KU Leuven, and
Department of Ophthalmology, University Hospitals Leuven (UZ Leuven), Leuven,
Belgium
Jayme R. Vianna
Department of Ophthalmology and Visual Sciences, Dalhousie University, Halifax,
NS, Canada
Robert N. Weinreb
Hamilton Glaucoma Center, Shiley Eye Center, and Department of
Ophthalmology, University of California, San Diego, CA, USA
Andrea Zampieri
Ophthalmology Unit Department of Neurological, Neuropsychological,
Morphological and Movement Sciences, University of Verona, Verona, Italy
Linda M. Zangwill

Hamilton Glaucoma Center, Shiley Eye Center, and Department of
Ophthalmology, University of California, San Diego, CA, USA

xi


Preface: New Trends in Basic and
Clinical Research of Glaucoma:
A Neurodegenerative Disease of the
Visual System Part B
Glaucoma is a degenerative disease of the visual system characterized by death of
retinal ganglion cells and of their axons that form the optic nerve. This chronic
disease is clinically associated with specific defects of the visual field progressively leading to blindness. Unfortunately, glaucoma still remains one of the major
causes of irreversible blindness worldwide. High intraocular pressure (IOP) is the
principal risk factor associated with onset and progression of the disease, and
therefore, current available treatments are based on the reduction of IOP through
medical, laser, or surgical strategies. Several clinical trials have demonstrated that
neuronal damage progresses in a high percentage of patients with glaucoma, even
though their IOP has been significantly reduced. These data confirm that other
important factors contribute to the development and progression of the disease
and that new therapeutic strategies should be found to prevent the disease onset
and progression.
In the recent years, much studies have provided new insights into fundamental
clinical aspects of glaucoma, and in this volume leaders in the field have presented their innovative data. Topics include emerging risk factors for glaucoma
onset and progression, criteria for the early diagnosis, and the assessment
of disease progression based on the use of advanced technologies. A section of
the volumes is dedicated to the pathophysiology, diagnosis, and treatment of
specific form of glaucoma including pediatric, normal tension, angle-closure,
uveitic, and corneal surgery-induced. Particularly interesting are the data presented on the involvement of the central area of the visual system in glaucoma
and the evidence documenting links between glaucoma and vascular and neurodegenerative disease of the central nervous system, thus supporting the hypothesis

that glaucoma may be influenced or may share common pathogenic mechanisms
with diseases of the CNS. This hypothesis is further supported by the observation
that the cerebrospinal fluid pressure may play a role in the pathogenesis of
glaucoma.
Topics also include studies on innovative therapeutic strategies from the evaluation of 24-h efficacy of topical medication to the improvement of surgical techniques.
Finally, a chapter discusses the impact of glaucoma disability on the patient’s
quality of life.
We would like to acknowledge the outstanding contribution of all the authors to
the success of this volume of Progress in Brain Research dedicated to glaucoma and

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Preface: New trends in basic and clinical research of glaucoma

to the collaboration of the staff of Elsevier. In particular, we would like to acknowledge the professional and skillful support of Mrs Shellie Bryant and Mrs Poppy
Garraway. Finally, we would also thank the referees who have contributed to our
editorial work.
The Editors
Giacinto Bagetta and Carlo Nucci


CHAPTER

Molecular biomarkers
in primary open-angle
glaucoma: from noninvasive
to invasive

1

Luca Agnifili*,1, Damiana Pieragostino†,{, Alessandra Mastropasqua*,
Vincenzo Fasanella*, Lorenza Brescia*, Gian Marco Tosi},
Paolo Sacchetta†,{, Leonardo Mastropasqua*
*Department of Medicine and Aging Science, Ophthalmology Clinic, University G.
d’Annunzio of Chieti-Pescara, Chieti, Italy
†
Department of Medical, Oral and Biotechnological Sciences, University
“G. d’Annunzio” of Chieti-Pescara, Chieti, Italy
{
Analytical Biochemistry and Proteomics Unit, Research Centre on Aging (Ce.S.I.), University
“G. d’Annunzio” of Chieti-Pescara, Chieti, Italy
}
Ophthalmology Section, Department of Medicine, Surgery and Neuroscience,
University of Siena, Siena, Italy
1
Corresponding author: Tel.: +39-0871-358489; Fax: +39-0871-358794,
e-mail address:

Abstract
Glaucoma, the first cause of irreversible blindness worldwide, is a neurodegenerative disease
characterized by the progressive loss of retinal ganglion cells. There are different subtypes of
glaucoma, all expression of a common optic neuropathy; primary open-angle glaucoma
(POAG) is the most diffuse subtype in western countries. To date, unfortunately, several questions still remain unsolved in the glaucoma management, such as the availability of powerful
methods for screening high-risk populations, early diagnosis, timely detection of damage progression, and prediction of response to therapy. Over the last years, biomarkers have gained
immense scientific and clinical interest to solve these issues, with countless molecules that
have been candidate as potential biomarkers. In the present review, we summarize the current
knowledge about the most robust molecular biomarkers proposed in POAG, distinguishing

noninvasive from minimally invasive, and invasive biomarkers, according to the procedure
adopted to collect fluid samples.

Progress in Brain Research, Volume 221, ISSN 0079-6123, />© 2015 Elsevier B.V. All rights reserved.

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2

CHAPTER 1 Candidate biomarkers for primary open-angle glaucoma

Keywords
Primary open-angle glaucoma, Biomarkers, Proteomics, Tears, Aqueous humor, Vitreous
body, Serum, Cerebrospinal fluid

1 INTRODUCTION: WHAT IS A BIOMARKER?
A biological marker (biomarker) has been defined as a biochemical, molecular, or
cellular alteration that is measurable in biological media such as tissues, cells, or
fluids (Hulka and Garrett, 1993). A biomarker represents an indicator of either normal or pathologic processes, or of a response to therapeutic intervention.
Biomarkers have gained immense scientific and clinical interest in all fields of
medicine, since they are potentially useful along the whole spectrum of the disease.
Before the onset of a disease, biomarkers could be used for screening and risk assessment; at diagnosis, biomarkers can determine staging, grading, and selection of initial therapy; during the course of the disease, biomarkers can be used to detect
progression, to monitor response to therapy, or to select any additional therapy
(Bhattacharya et al., 2013; Biomarkers Definitions Working Group, 2001). According to this, biomarkers have been differentiated in biomarkers of exposure, used in
risk prediction or susceptibility to disease (Galasko, 2001), and biomarkers of disease, used in screening, diagnosis, and monitoring of the disease progression.
Advances in sensitivity and accuracy of genomic, metabolomic, and proteomic
techniques have generated many candidate molecular biomarkers with potential clinical value. Hence, a platform that integrates data from genomics, proteomics, and
metabolomics is essential for integrative and complete discovery of biomarkers.
Nonetheless, since most diseases are induced by the functional dysregulation of protein interactions, proteomics, which electively studies protein interactions, has progressively gained a huge consideration in the identification of potential molecular

biomarkers (Liotta et al., 2001). The commonly used approach to identify protein
biomarkers is to initially select biomarker candidates and then pursue validation
studies in larger patient populations to verify their value.
In fact, the clinical validation phase after discovery of candidate molecules remains a major standing challenge common for the biomarker discovery through different approaches.
Immunoassays, multiplex assays (Hanash et al., 2008; Matt et al., 2008), and
mass spectrometry (MS)-based multiple reaction monitoring assays (Anderson
and Hunter, 2006) provide attractive options to quantitate candidate molecules in
large numbers of samples. MS is the gold standard approach for proteomic or metabolomic biomarker discovery. Afterward, protein profiles could be validated with
specific antigen microarrays.
In ophthalmology, to date, biomarkers are generally unsatisfactory and have still a
limited use in clinical practice. Biomarkers have been studied in ocular specimens such
as tears, for the early lachrymal gland dysfunction in dry eye, or also for nonocular


1 Introduction: what is a biomarker?

pathological conditions (a tear protein similar to the human mammoglobin similar
was found increased in the breast cancer) (Molloy et al., 1997), aqueous humor
(AH) for myopia (Duan et al., 2008), and vitreous for diabetic retinopathy and proliferative vitreo-retinopathy (Capeans et al., 1998; Danser et al., 1989). In nonocular
specimens, such as plasma, biomarkers have been candidate for the age-related macular degeneration or diabetic retinopathy, but with a very limited utility (Gu et al.,
2009; Nguyen et al., 2009). Molecular biomarkers obtained from easily accessible
samples are the most suitable for potential clinical application; nevertheless, as the
research in cancer biomarkers showed, the more locally obtained from the disease site,
the greater the opportunity to discover biomarkers with high clinical significance.
Global quantitative proteomic analyses of ocular tissues involved in the early
stages of the disease appear promising for identifying biomarker candidates. Subsequently, candidate biomarkers considered of high priority can be targeted for validation in blood and ocular easily accessible fluids from larger glaucoma/control
study populations using SRM/MRM (select reaction monitoring/multiple reaction
monitoring)-based technology.
In primary open-angle glaucoma (POAG), the most diffuse subtype of glaucoma
in Western populations, potential biomarkers can be evaluated in blood serum or in

more proximal fluids, such as tears or AH, where biomarkers can be even more
sensitive and specific. Glaucoma biomarkers may provide advancements in understanding the characteristics of the disease with potential clinical applications in
the epidemiology and prevention, in early diagnosis, in assessing the risk profile
for optic neuropathy progression (prediction of conversion from ocular hypertension
to POAG, and early detection of damage progression in patients already diagnosed
with POAG), and in monitoring the response to treatment.
Potential protein biomarkers have been studied in several molecular pathways
involved in the pathogenesis of glaucoma, such as stress response, apoptosis,
DNA repair, cell adhesion, tissue remodeling, transcription regulation, multidrug resistance, and energy metabolism (Golubnitschaja and Flammer, 2007). Among all
possible biomarkers, particular emphasis must be reserved to those having a key role
in the key pathogenetic pathways.
The major difficulties to obtain robust molecular biomarkers in glaucoma are associated with the individual variations, the presence of a great dynamic spectrum of
possible concentrations of specific molecules, and the ever-increasing collection of
molecular species (Pinazo-Duran et al., 2013). Moreover, general limitations to techniques of analysis include lack of common operating procedures for proper banking
of biological tissues, analytical insensitivity of underpinning technologies, lack of
standards, multiplexed complicated assays, and an ever-changing regulatory landscape (Liotta and Petricoin, 2011).
It is extremely difficult and time expensive to report all molecules that have been
candidate during the last decades as biomarkers for glaucoma; therefore, in the present review, we will summarize the current knowledge about the most studied and
robust molecular biomarkers of POAG, distinguishing noninvasive from minimally
invasive, and invasive biomarkers (Fig. 1).

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4

CHAPTER 1 Candidate biomarkers for primary open-angle glaucoma

FIGURE 1
Schematic representation of body fluid samples that are potentially source of molecular

biomarkers in primary open-angle glaucoma. Green (dark gray in the print version) text:
noninvasive source of biomarkers; yellow (light gray in the print version) text: minimally
invasive source of biomarkers; red (dark gray in the print version) text: invasive source
of biomarkers.

2 DEFINING BIOMARKERS
2.1 PROTEOMICS TECHNOLOGIES FOR BIOMARKER IDENTIFICATION
The increased sensitivity and accuracy of genomic, proteomic, and metabolomic
techniques have brought about the potential to identify molecular entities that may
serve as potentially useful markers, including markers for early detection of a disease;
markers that will predict severity of a disease; markers that will predict the rate of disease progression; and markers that will serve as predictors of response to treatment.
Proteomics is at the center of a number of these activities, since proteins are either
the molecular therapeutic disease target or the biomarkers used for early disease detection and monitoring.
The discovery process could be very varied and can follow different routes
depending on the need and the technologies available (Fuzery et al., 2013;


2 Defining biomarkers

FIGURE 2
Workflow for biomarker discovery process, in which is described the discovery and validation
phases. The principal approaches (top-down and bottom-up) are indicated and the
funnel represents the decrease of the number of potential biomarkers during the validation
steps.

Mischak et al., 2010). The workflow (described in Fig. 2) for biomarker discovery
can be divided into two important steps: discovery and validation. As indicated by
the funnel, the number of potential biomarkers decreases during the discovery
process and with the increase of patients analyzed. During the discovery phase,
two major approaches can be distinguished: gel-based approach and gel-free approach. The first approach is based on protein separation by polyacrylamide gels

and individuation of differential proteins and, afterward, identification of the differential expressed proteins by MS after enzymatic digestion. The second workflow bypasses the gel, favors MS approach also for the step of biomarkers individuation
(Pieragostino et al., 2015). Both workflows can be considered “top down” approaches, while the last challenge of proteomics are the “bottom up” approaches,
where steps of identification and quantification are performed together.
Emerging options for performing “bottom up” proteomics use hybrid instruments
as the Q-ToF (Andrews et al., 2011; Elias et al., 2005) and Q-Orbitrap (Michalski
et al., 2011). MS-based quantitative data are obtained by stable isotope labeling
or label-free approaches (Link et al., 1999; Wolters et al., 2001). All these approaches aim to highlight differential expressed proteins between conditions analyzed to obtain potential biomarkers of disease or treatment. The required
instrumentation and procedures are highly articulated, due to the necessity of analyze
complex mixture of samples. Statistical approaches and bioinformatics tools are
needed to unravel the enormous amount of data obtained.

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CHAPTER 1 Candidate biomarkers for primary open-angle glaucoma

3 NONINVASIVE BIOMARKERS IN PRIMARY OPEN-ANGLE
GLAUCOMA
A noninvasive procedure is a conservative diagnostic or therapeutic approach, which
does not require incision into the body or the removal of tissue. Thus, noninvasive
biomarkers are those obtained in a noninvasive way. In glaucoma, different fluids
can be hypothetically noninvasively sampled, such as tears, saliva, urine, and sweat;
nonetheless only tears and, much less, the urine, have been studied as a source of
potential biomarkers.

3.1 TEARS BIOMARKERS
Tears are a source of nourishment for ocular surface tissues and a vehicle to remove
local waste products, metabolized drugs, and inflammatory mediators produced in

several ophthalmic diseases.
Among all body fluids, tears certainly represent one of the most easily and noninvasively accessible since are collected by using the Schirmer test paper or a microcapillary glass. Generally, the Schirmer test paper is easier to perform with respect to
microcapillary glass, more comfortable, and yields higher volumes of tears. Therefore, in different biomarker studies Schirmer test papers are preferred.
The complex molecular repertoire available in tears is currently intensively investigated, with the aim to recognize biomarkers of different ocular diseases, including
glaucoma (Pieragostino et al., 2015). Overall, even though many studies were focused
on glaucoma biomarker discovery, only few studies were conducted on tears.
The discovery of potential biomarkers in tears could provide essential information concerning the pathophysiology of the disease and the effects of topical medications. In the first case, tears may hypothetically host glaucoma-related proteins
directly coming from the AH, after scleral percolation in the uveoscleral outflow
pathway. The existence of a trans-scleral AH percolation has been widely documented also in vivo (Agnifili et al., 2012; Ciancaglini et al., 2009; Mastropasqua et al.,
2010, 2014). In the second case, which was more extensively studied, tears provide
markers strictly to drug-induced inflammatory processes.
Pavlenko et al. analyzed the tear levels of endothelin-1 (ET-1), which is a protein
involved in retinal ganglion cell (RGC) damage and in the TM dysfunction in POAG,
by stimulating vasoconstriction, contraction of smooth-like muscle cells, and by inducing neurotoxicity (Choritz et al., 2012; Emre et al., 2005; Pavlenko et al., 2013).
The authors showed a significant increase (two to three times) of this protein in
POAG patients with respect to healthy controls. Borovic et al. focused on the activity
of renin-angiotensin (SRA) and Kinin–Kallikrein (SKK) systems in tears, blood, and
AH (Borovic et al., 2009), reporting increased levels of kallikrein and angiotensinconverting enzyme activity in patients with POAG compared to normal subjects.
Slepova et al. (2012) have investigated markers of Fas-mediated apoptosis, reporting
the association of POAG onset and progression with interruption of Fas-mediated


3 Noninvasive biomarkers in primary open-angle glaucoma

apoptosis. Our research group documented altered tear levels of Lysozyme C,
Lipocalin-1, Protein S100, Immunoglobulins and Prolactin-Inducible Protein in patients with medically treated POAG by using an untargeted approach. Moreover, we
found a differential pattern of phosphorylated Cystatin-S that distinguished POAG
from healthy subjects and patients with pseudoexfoliative glaucoma (PEXG). By
interpreting these results, we found that both POAG and PEXG presented activated
inflammatory pathways, directly related to the disease and/or induced by medical

therapy (Pieragostino et al., 2012). Thus, due to the potential biases induced by
the use of medications, we were unable to indicate robust pathogenetic biomarkers
of glaucoma.
To overcome this limitation, a further study was set to elucidate the influence of
therapy on tears protein pattern. In this study, we found that a subgroup of 12 upregulated proteins in naı¨ve to therapy POAG patients were downregulated in patients
controlled with prostaglandin analogs (PGA) (Pieragostino et al., 2013). In the same
field of research, Lopilly Park et al. (2012) tried to identify potential tear-film-based
proteins and their effect on conjunctiva and cornea in patients with POAG receiving
PGA. The proteomic analysis was done to compare the pooled tear samples from
each group (treated vs. nontreated patients). The authors documented that the topical
use of PGA resulted in an altered balance between metalloproteinases (MMP) and
tissue metalloproteinases inhibitor, which may be triggered by inflammatory cytokines. The consequence is increased matrix degradation and decreased stromal collagens in the cornea.
Considering other medications, patients treated with BAK preserved b-blockers
showed higher levels of IL-1b compared to patients treated with the preservative-free
formulations. Thus, IL-1b was intended as a marker of BAK-induced inflammation
(Manni et al., 2005). A modification of tear cytokines concentrations was also
reported by Malvitte et al. (2007), who found proinflammatory cytokines such as
IL-1b, IL-6, IL-12, and tumor necrosis factor alpha (TNF-a) significantly increased
in tears of long-term-treated patients compared with healthy controls.
In patients under long-term medical therapy, different protein expression was
documented with respect to healthy controls:tear levels of S100-A8, S100-A9,
and mammaglobin B were significantly increased in the medicated group compared
with levels in the nonmedicated group (Wong et al., 2011).
Chong et al. (2010) determined the tear cytokine profile from medically treated
glaucoma patients and found that the monocyte chemoattractant protein 1 was significantly elevated in treated compared to nontreated eyes. Openkova et al. (2013)
found higher tear concentrations of malonic dialdehyde in POAG patients with respect to control subjects, along with a decreased activity of catalase and an increased
concentration of nitric oxide and its metabolites in both blood serum and tears.
Finally, Grus et al. (2005) analyzed autoantibody patterns in tear fluid of glaucoma patients, showing 17 autoantibody reactivities significantly altered in the glaucoma group compared with controls. Of note, these altered autoantibodies were
identical to antibody markers previously found altered also in serum samples
(Gramlich et al., 2013).

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CHAPTER 1 Candidate biomarkers for primary open-angle glaucoma

Overall, when critically interpreting the above mentioned studies, all data
seems to indicate an increase tear inflammatory status in POAG patients, for the
most part related to therapy. Therefore, tears could be a source of potential biomarkers indicating the activation of inflammatory pathways, either as a primitive pathogenetic expression of the disease or as a consequence of the medical
therapy.

3.2 URINE BIOMARKERS
The possibility of finding potential biomarkers of glaucoma in the urine, as for other
fluids not in contact with the eye, is low. Nevertheless, urine is the final step of the
blood clearance pathway of cellular metabolism byproducts, including those of
the eye.
To date, studies on urinary biomarkers in patients with POAG are still lacking,
and just a couple of studies evaluated the urine concentrations changes in some molecules that may play a role in the optic neuropathy. An interesting compound is the
citrate, a neuroprotective agent (already considered as a potential serum biomarker of
RGC injury), which was found with slight lower urine concentrations in patients with
glaucoma compared to healthy subjects (Fraenkl et al., 2011).
Yuki and coworkers investigated the 8-hydroxy-20 -deoxyguanosine (8-OHdG),
an agent involved in the control of the general oxidative stress, reporting levels
significantly higher in the serum and lower in the urine in patients with NTG compared to healthy controls (Yuki et al., 2010). The urinary 8-OHdG was proposed as a
potential indicator of increased systemic oxidative stress in patients with NTG.

4 MINIMALLY INVASIVE BIOMARKERS IN PRIMARY
OPEN-ANGLE GLAUCOMA

A minimally invasive procedure is defined as one that is carried out by entering
the body through the skin or through a body cavity or anatomical opening, but with
the smallest damage possible to these structures. The collection of blood samples
is the most diffuse way to obtain biomarkers with a safe and minimally invasive
procedure. The blood serum is the biggest reservoir of signaling molecules; many
metabolites are secreted from different types of cells in blood that represents a
universal way of communication between cells.

4.1 SERUM BIOMARKERS
4.1.1 Protein Biomarkers
Even though the potentiality is very high, the identification of biomarkers from
plasma or serum poses many challenges (Anderson and Anderson, 2002).
First, high-abundant proteins such as albumin, immunoglobulin, transferrin,
fibrinogen, apolipoprotein (APO), and haptoglobin constitute more than 95% of
the total protein mass of blood and mask the detection of low-abundant proteins. This


4 Minimally invasive biomarkers in primary open-angle glaucoma

seems to be particularly challenging for glaucoma biomarker discovery, since the
representation of eye-derived proteins in the large and complex pool of human blood
may be even more limited.
Second, marker proteins may often undergo modifications. However, in some
cases, modified proteins may be of greater interest than intact proteins (such as
the modified proteins of HbA1c for diabetes), even though such modified proteins
may undergo degradation during sample collection, transportation, or storage.
Third, one of the biggest challenges in proteomic biomarker discovery is the lack
of general means to amplify proteins. Different strategies have been proposed to
overcome this limitation, such as the enrichment of peptides, the enhancement of
N-terminal peptides, the reverse phase protein array, and biomarker-harvesting

nanoparticle technologies (Paweletz et al., 2001; Tamburro et al., 2011).
Fourth, to candidate a robust serum biomarker, it is mandatory to have a strong
correlation between serum and comparative AH samples, in both patients with
POAG and healthy controls. A previous study verified this aspect documenting a
strong positive correlation in autoantibody reactivities in 70% of POAG patients
and 60% of controls subjects (Boehm et al., 2012).
Circulating biomarkers should be searched not only through proteins but also in
leukocytes. In fact, during the course of a disease, one of the natural sensors affected
by the incredible number of metabolites presented in blood serum are circulating
leukocytes. Thus, in addition to serum analysis, the evaluation of isolated leukocytes
has been suggested as another practical application in population screening of
high-risk subjects for glaucoma.
The followings are some of the countless potential candidate serum biomarkers
proposed in glaucoma.

4.1.1.1 Autoimmunity Markers
Naturally occurring autoantibodies are effectors of the innate immune system, which
have regulatory functions and participate in several physiological activities (Poletaev
and Osipenko, 2003). Autoantibodies represent a large fraction of serum immunoglobulins in healthy subjects (Li et al., 2006). Most of the works that benefits for
molecular biomarker discovery in glaucoma comprises studies of autoantibodies
and their target antigens (Tezel, 2013). However, to date, the role of autoantibodies
in glaucoma is still unclear. Whether they have a causative effect or appear as an
epiphenomenon of the disease, or have beneficial functions, have not been clarified.
Glaucomatous patients frequently exhibit abnormal immune T-cell subsets and
increased titers of serum antibodies reacting with retina and optic nerve antigens,
indicating that immune system plays a critical role in the initiation and/or progression of the glaucomatous optic neuropathy (Yang et al., 2001a). Therefore, serum
antibodies to retina and optic nerve proteins might be considered as potential indicators of POAG and other subtypes of disease (Grus et al., 2006; Maruyama et al.,
2000; Reichelt et al., 2008; Tezel and Fourth ARVO/Pfizer Ophthalmics Research
Institute Conference Working Group, 2009; Wax et al., 2001).

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CHAPTER 1 Candidate biomarkers for primary open-angle glaucoma

The evidence that different subtypes of glaucoma present a similar serum antibody production, suggests that these antibodies may reflect a common native
response to tissue injury necessary for cell debris cleaning and tissue healing. In
sustain of this hypothesis, histopathological studies documented immunoglobulin
deposition in the glaucomatous human retina (Wax et al., 1998) and antibodymediated damage of RGCs (Tezel and Wax, 2000). Consistently, recent in vivo
studies suggested an antibody-mediated RGC loss in eyes with glaucoma
(Joachim et al., 2012). Upregulated antibodies are explained as an initiation of autoaggressive processes to RGC, which lead to the optic nerve damage.
However, not only increased antibody levels but also downregulations could be
observed in glaucoma patients and play a significant pathogenic role (Boehm et al.,
2012). The downregulations could reflect a loss of the natural protective autoimmunity and a disbalance of naturally occurring autoantibodies encouraging inflammatory neurodegenerative processes (Schwartz-Albiez et al., 2009).
The potential usefulness of serum antibodies as biomarkers for glaucoma is supported by the unique antibody pattern among patients (which exhibits specificity and
sensitivity of 93% to detect glaucoma) (Boehm et al., 2012) and the similarities in
complex antibody profiles among different ethnic populations (Grus et al., 2006;
Wax et al., 2001).
Several techniques, including serological identification of antigens by recombinant expression cloning (SEREX), serological proteome analysis (SERPA), protein
arrays, and open reading frame phage display, have been used to identify diseaseassociated antigens and their cognate autoantibodies (Boehm et al., 2012; Grus
et al., 2006; Wax et al., 2001). However, while the protein arrays used for profiling
the antibody response are limited to a number of prechosen antigenic proteins,
MS-based techniques present a high-throughput analytical approach for de novo
discovery of biomarker candidates and also enable the analysis of modified proteins
as potential biomarkers (Hanash et al., 2008; Liotta et al., 2003).
A multitude of autoantibodies have been studied, and several serum antibodies
were proposed as candidate biomarkers in POAG or are going to be candidate. Circulating antibodies against heat shock proteins (HSP), antiphosphatidylserine, g-enolase,
glycosaminoglycans, neuron-specific enolase (NSE), glutathione S-transferase (GST),

vimentin, myelin basic protein, glial fibrillary acidic protein (GFAP), retinaldehydebinding protein, or retinal S-antigen are just examples (Gramlich et al., 2013).
In a study of Maruyama et al., approximately 20% of POAG patients presented a
serum antibody against NSE, and the maximum IOP levels in patients with anti-NSE
antibody was statistically lower than those without the antibody. This supported a
major role of anti-NSE in NTG with respect to POAG (Maruyama et al., 2000).
The same study group suggested that serum autoantibody against NSE found in
patients with glaucoma induces retinal dysfunction in vivo.
Grus et al. (2006) found that glaucomatous patients from Germany and United
States have a large similarity in serum autoantibody profile and characteristic
differences from healthy subjects. In addition, the authors identified the alpha-fodrin
as a candidate antibody biomarker in both study populations. The presence of


4 Minimally invasive biomarkers in primary open-angle glaucoma

alpha-fodrin autoantibodies were confirmed by ELISA, which found significantly
higher titer of anti-alpha-fodrin in patients with normal pressure glaucoma, than
in age-matched healthy subjects or POAG patients.
Recently, by means of immuno-proteomics, Tezel et al. (2012) delineated a
variety of new antigens targeted by IgG in glaucomatous sera. The authors found
greater abundances of methionine oxidation of the 50 identified serum proteins in
samples isolated from the glaucomatous sera compared with age-matched control
sera. Among these proteins, the apoptosis-inducing facto, the cyclic AMPresponsive element-binding protein, ephrin type-A receptor, and huntingtin protein
were indicated as potential biomarkers of disease, since exhibited higher serum
ELISA titers in glaucomatous patients. In the same study, a significant increase in
serum protein methionine oxidation was found in glaucomatous blood samples compared to normal sera. Also the increased protein oxidation could be intended a
glaucoma-specific biomarker.
In another work, Tezel (2014) identified potential glaucoma-related biomarkers
by direct proteomics analysis of serum samples, reporting 22 specific proteins present only in glaucomatous patients. These biomarkers included immune mediators
and components of cell death signaling. The same authors documented increased titers of circulating antibodies against HSP (including alpha-crystallins, HSP27, and

human and bacterial HSP60), which are known to induce RGC loss and participate in
the development of the glaucomatous optic neuropathy (Tezel et al., 1998; Wax
et al., 2001).
Gonzalez-Iglesias et al. (2014) identified a panel of 35-top-ranked serum proteins
that were found in different concentrations in patients with glaucoma compared to
healthy controls. The signaling network of these proteins correlated to an immunological and inflammatory pathway. In this study, the APO A4 yielded the best performance in correctly classifying POAG from healthy cases and presented an 81%
efficacy in discriminating POAG from PEXG. Other proteins, including complement
C3, transferrin, vitronectin, alpha-1 antitrypsin (SERPINA)-1, fibulin-1, and complement factor H, also classified eyes affected with POAG, but with lower discriminatory power than APOA4.
Notably, the authors identified high serum levels of several proteins that were previously reported also increased in other body fluids such as AH and tears. Pieragostino
et al. (2012) found tear levels of transferrin, APOA1, Ig mu chain C region (IGHM), and
Ig gamma-1 chain C region (IGHG1) in higher concentration in POAG patients compared to normal subjects. Similarly, serum transthyretin (TTR) was found in higher concentrations in the AH of patients with POAG (Duan et al., 2010; Grus et al., 2008).
Given the potential important of autoantibodies, it was proposed also a patent
related to a method to diagnose glaucoma based on the composition of autoantibodies
against ocular antigens in serum (US20060166268). In this case, the authors evaluated
antibodies against human vimentin, human glial fibrillary acid protein (GFAP),
anti-Ro/SS-A (Sjogren syndrome A; also commonly called Ro antigens), chondroitin
sulfate and heparin (which bind to human optic nerve head proteoglycans), gammaenolase, and GST. Increased titers of autoantibodies to GST in some patients with

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CHAPTER 1 Candidate biomarkers for primary open-angle glaucoma

POAG may represent a generalized response to tissue stress and/or damage as a
consequence of the glaucomatous neurodegeneration process (Yang et al., 2001b).

4.1.1.2 Inflammatory Markers
The activation of inflammatory pathways represents a potential key moment in the

development and progression of glaucoma (Vohra et al., 2013). In a rodent model of
experimental glaucoma, Walsh et al. (2009) found that elevation of IOP-induced
changes in protein expression profiles and produced inflammatory biomarkers in retina, RGC, vitreous body, and also in extraocular tissues, such as the serum.
The serum amyloid-A1 (SAA1) and SAA2, which are inflammatory markers
upregulated in the retina, were found reduced in sera of IOP-responsive eyes. These
modifications are important because inflammatory processes play a relevant role in
the pathogenesis of POAG (Tezel et al., 2007; Zhou et al., 2005). It was hypothesized
that the reduced levels of SAA1 and SAA2 in the serum is a response to the stimulation of migration of SAA-positive microglial cells to the retina. This phenomenon
promotes a systemic downregulation of SAA to prevent the development of an immune response toward inflammatory processes linked to glaucoma. These results
suggest that these proinflammatory reactants could be strong candidates biomarkers
of hypertensive glaucoma.
Adenosine triphosphate (ATP)-binding cassette (ABC) transporters are proteins
involved in the translocation of substances across cell membranes. The ABC 1, particularly, has been identified as a leukocyte factor that controls the recruitment of
inflammatory cells and regulates processes induced by chronic vascular dysregulation. In a previous study (Yeghiazaryan et al., 2005), which analyzed ABC 1 expression rates in circulating leukocytes using protein quantification, the expression rates
this protein were significantly increased in leukocytes of glaucoma patients compared to healthy subjects. The authors proposed that a significantly enhanced expression of ABC 1 in circulating leukocytes might be considered as a potential predictive
and diagnostic marker of glaucoma.
Also the interleukin 6 (IL-6), an important inflammatory factor, displayed significantly higher values in both the AH and plasma samples of the POAG patients compared to healthy controls (Sorkhabi et al., 2010).

4.1.1.3 Neurodegenerative/Apoptotic Biomarkers
The existence of neurodegenerative or apoptotic mechanisms, especially in progressing patients, have been extensively postulated and studied in POAG (Galvao et al.,
2013). Therefore, also in this field of research, potential biomarkers have been
proposed.
Brain-derived neurotrophic factor (BDNF), which is a key factor in the RGC survival due to its antiapoptotic property (Quigley et al., 2000), was investigated as a
potential biomarker of disease.
In a study of Ghaffariyeh et al. (2011), the authors found that serum levels of
BDNF were significantly reduced in patients with early stage POAG compared to
controls. The lower serum BDNF concentration presented a significant negative


4 Minimally invasive biomarkers in primary open-angle glaucoma

correlation with pattern standard deviations. Serum BDNF was proposed a useful
biochemical marker for the early detection of POAG, and a reliable, cost-effective
method for diagnosis, screening, and assessing the progression of POAG. The serum
modifications of BDNF levels are worthy, since are in line with the modifications of
BDNF in the retina, where this substance is markedly reduced (Quigley et al., 2000).
Other proapoptotic molecules were studied, such as the poly-adenyl-ribose polymerase 1 (PARP-1) and caspase 3 (Cas 3) protease (that promote the disorganization and
destruction of apoptotic cells), which significantly increased their expression in
the AH and plasma of POAG patients compared to healthy controls (Sorkhabi
et al., 2010).
Citrate, a major component in mitochondrial metabolism and involved in the
mechanisms of neuroprotection, was found significantly decreased in patients with
glaucoma, compared to control cases. Fraenkl et al. (2011), setting the cut-off limit at
110 mmol/L, reported that plasma citrate levels evaluation would have a sensitivity
of 66.7% and a specificity of 71.4% to detect glaucoma giving the possibility to use
them eventually as a biomarker.
RGC neurodegeneration can be also promoted by modification of the extracellular matrix within the optic nerve head. Increased expression rates of MMP-9 and
MT1-MMP in circulating leukocytes of glaucomatous patients, indicative of an increased enzymatic activity leading to extensive tissue remodeling, and neurodegeneration in the optic nerve were found (Golubnitschaja et al., 2004).
Finally, ET-1 levels were found increased in plasma of patients with POAG. This
compound seems to play an important role in the pathogenesis of disease, since was
found increases in serum, AH and tears of patients with glaucoma (Emre et al., 2005),
and could be a potential robust biomarker of disease.

4.1.1.4 Oxidative Stress Markers
Changes of the oxidative metabolism have been widely reported in patients with
glaucoma and proposed as a complementary mechanism in the TM and optic nerve
damage (Bagnis et al., 2012). The oxidative stress may induce tissue damage in direct
way, or indirectly by stimulating neurotoxicity and immune activity, by influencing
complement regulatory molecules or inducing glial dysfunction (Tezel, 2006).
Overall, the serum of POAG patients presents a significant increase in protein

carbonyls, which is expression of oxidatively modified proteins. This oxidative modification stimulates the autoantibody production by changing antigenic features of
proteins and, therefore, may serve as biomarkers of disease.
Several evidences showed that the nitric oxide (NO) pathway and endothelial
dysfunction are implicated in the glaucoma risk (Bagnis et al., 2012). NO is an essential metabolite that acts as an antioxidant and antiapoptotic factor and plays a
physiological role in the IOP regulation (Drago and Bucolo, 2010). The asymmetric
dimethylarginine (ADMA) is an endogenous inhibitor of the NO synthase (NOS),
whereas symmetric dimethylarginine (SDMA) is a competitive inhibitor of cellular
uptake of L-arginine, the substrate for NOS. Javadiyan et al. (2012) reported elevated
levels of serum ADMA and SDMA in patients with advanced glaucoma, which

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CHAPTER 1 Candidate biomarkers for primary open-angle glaucoma

supported a clinical link between the NO pathway and poor disease outcomes. Thus,
also ADMA/SDMA might have potential as a biomarker for glaucoma progression.
Also NO(2)(-) and cyclic guanosine monophosphate (an indirect indicator of NO)
plasma levels were found significantly decreased in glaucoma patients compared
with controls, suggesting the presence of an increased oxidative stress (Galassi
et al., 2004).
Nucci et al. studied other oxidative stress-related compounds reporting significantly increased levels of serum and AH malondialdehyde levels in glaucomatous
patients with respect to control group. In contrast, the control group presented significantly higher serum and AH total antioxidant capacity than did the glaucoma
group in both the blood, and higher levels ATP/adenosine diphosphate (Nucci
et al., 2013).
A particular field of study is the gene–diet interactions, since diet can be considered as either a risk or a protective factor depending on the contribution of vitamins
or other antioxidant compounds (Zhou et al., 2005). Nutritional elements are involved in modifications of the trabecular meshwork (vitamin C and glutathione)
and may play a role in the development of the glaucomatous optic neuropathy

(vitamin E), by modifying the oxidative status. Moreover, vitamins A and C might
be involved also in the progression of glaucomatous optic neuropathy (Tam et al.,
2010). Zanon-Moreno et al. (2013) found that patients with POAG had statistically
significant lower plasma vitamin E and C concentrations and higher plasma
glutathione peroxidase activity compared to healthy subjects. All these evidences
highlighted a reduced oxidative control in patients with glaucoma.

4.1.1.5 Biomarkers of Trabecular Meshwork Dysfunction
As strongly demonstrated, the anatomical modifications of the TM represent the
most important aspect in the development of glaucoma, since leads to the progressive
elevation of the IOP (Inoue and Tanihara, 2013). Several substances may interact
with the TM, negatively affecting its physiology and anatomy.
SAA, an acute-phase APO, plays critical roles in inflammation and tissue repair;
increased levels of SAA may contribute to changes of the TM that lead to the IOP
elevation. Based on this role, Alcon and Novartis filled a series of patents
(US20087357931, US20120064532) proposing a method for diagnosing glaucoma
modulating the expression of SAA (Clark, 2012).
The 3a-hydroxysteroid dehydrogenase (3a-HSD) is an enzyme that metabolizes
steroids in TM, playing a significant role in the regulation of IOP. Moreover, it was
found increased also in optic nerve head astrocytes, in response to elevated IOP in a
monkey model of experimental glaucoma (Agapova et al., 2003). In a previous study,
the mean 3a-HSD activity of POAG-derived peripheral blood lymphocytes (PBL)
was found significantly reduced in glaucomatous patients, this suggesting a potential
role of this factor in the IOP modulation (Weinstein et al., 1996). Since a decrease in
the 3a-HSD activity in PBL may reflect a similar decrease in the ocular enzyme, the
decreased 3a-HSD activity in the readily obtainable PBL could serve as a biomarker
for POAG or as a risk predictor for the disease.