Progress in brain research, volume 221

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

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Mark Bear, Cambridge, USA.

Medicine & Translational NeuroscienceHamed Ekhtiari, Tehran, Iran

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First edition 2015

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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

Milano-Bicocca, 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

Giamberto Casini

Ophthalmology Unit, University of Pisa, Pisa, Italy

v

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Morphological and Movement Sciences, University of Verona, Verona, ItalyMarco Ciancaglini

Ophthalmic Clinic Department of Surgical Science, Eye Clinic, University ofL’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

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 ofBiomedicine and Prevention, University of Rome Tor Vergata, Rome, ItalyJulian Garcia-Feijoo

Servicio de Oftalmologı´a, Hospital Clı´nico San Carlos, Departamento de

Oftalmologı´a y ORL, Facultad de Medicina, Universidad Complutense de Madrid,

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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

Mario Alberto Giuliano

Ophthalmology Unit, Department of Experimental Medicine and Surgery,

University of Rome Tor Vergata, Rome, Italy

Gianluca Guidi

Jost B Jonas

Department of Ophthalmology, Medical Faculty Mannheim of the

Ruprecht-Karls-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

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

Giorgio Marchini

vii Contributors

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Jose Marı´a Martinez-de-la-Casa

Oftalmologıá y ORL, Facultad de Medicina, Universidad Complutense de Madrid,Instituto de Investigacioń Sanitaria del Hospital Clıńico San Carlos (IdISSC), andCooperative Research Network on Age-Related Ocular Pathology, Visual and LifeQuality, 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

Leonardo Mastropasqua

Rodolfo Mastropasqua

Morphological and Movement Sciences, University of Verona, Verona, ItalyVincenzo 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

Stefano Miglior

Ophthalmology Department, Policlinico di Monza Hospital, University of Bicocca, Monza, Italy

Milano-Dimitrios G Mikropoulos

3rd University Department of Ophthalmology, Aristotle University, and

Ophthalmica Institute, Thessaloniki, Greece

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Laura Morales-Fernandez

Luigi Antonio Morrone

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

Carlo Nucci

Songhomitra Panda-Jonas

Department of Ophthalmology, Medical Faculty Mannheim of the

Ruprecht-Karls-University, Heidelberg, Germany

Maria Papadopoulos

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

Robert Ritch

Einhorn Clinical Research Center, New York Ear Eye and Ear Infirmary of Mt

Sinai, New York, NY, USA

Rossella Russo

Preclinical and Translational Pharmacology, University of Calabria, Arcavacata di

Rende, Italy

ix Contributors

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Paolo Sacchetta

Department of Medical, Oral and Biotechnological Sciences, and AnalyticalBiochemistry and Proteomics Unit, Research Centre on Aging (Ce.S.I.),University G d’Annunzio of Chieti-Pescara, Chieti, Italy

Federico Saenz Frances

Enrique Santos-Bueso

Orazio Schillaci

Diagnostic Imaging Section, Department of Biomedicine and Prevention, TorVergata University Hospital, University of Rome Tor Vergata, Rome, ItalyAlon 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 ofOphthalmology, National University Health System, and Singapore Eye ResearchInstitute, Singapore, Singapore

Ingeborg Stalmans

Department of Neurosciences, Laboratory of Ophthalmology, KU Leuven, andDepartment of Ophthalmology, University Hospitals Leuven (UZ Leuven), Leuven,Belgium

Ophthalmology Section, Department of Medicine, Surgery and Neuroscience,University of Siena, Siena, Italy

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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,

Andrea Zampieri

Linda M Zangwill

xi Contributors

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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

progres-sively 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

pre-sented 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

pre-sented on the involvement of the central area of the visual system in glaucoma

and the evidence documenting links between glaucoma and vascular and

neuro-degenerative 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

evalua-tion of 24-h efficacy of topical medicaevalua-tion 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 ofProgress in Brain Research dedicated to glaucoma and

xxiii

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to the collaboration of the staff of Elsevier In particular, we would like to edge the professional and skillful support of Mrs Shellie Bryant and Mrs PoppyGarraway Finally, we would also thank the referees who have contributed to oureditorial work.

acknowl-The EditorsGiacinto Bagetta and Carlo Nuccixxiv Preface: New trends in basic and clinical research of glaucoma

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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: l.agnifili@unich.it

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

ques-tions 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

pro-gression, 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, http://dx.doi.org/10.1016/bs.pbr.2015.05.006

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Primary open-angle glaucoma, Biomarkers, Proteomics, Tears, Aqueous humor, Vitreousbody, Serum, Cerebrospinal fluid

A biological marker (biomarker) has been defined as a biochemical, molecular, orcellular alteration that is measurable in biological media such as tissues, cells, orfluids (Hulka and Garrett, 1993) A biomarker represents an indicator of either nor-mal or pathologic processes, or of a response to therapeutic intervention

Biomarkers have gained immense scientific and clinical interest in all fields ofmedicine, 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 assess-ment; at diagnosis, biomarkers can determine staging, grading, and selection of ini-tial therapy; during the course of the disease, biomarkers can be used to detectprogression, to monitor response to therapy, or to select any additional therapy(Bhattacharya et al., 2013; Biomarkers Definitions Working Group, 2001) Accord-ing to this, biomarkers have been differentiated in biomarkers of exposure, used inrisk prediction or susceptibility to disease (Galasko, 2001), and biomarkers of dis-ease, used in screening, diagnosis, and monitoring of the disease progression.Advances in sensitivity and accuracy of genomic, metabolomic, and proteomictechniques have generated many candidate molecular biomarkers with potential clin-ical value Hence, a platform that integrates data from genomics, proteomics, andmetabolomics is essential for integrative and complete discovery of biomarkers.Nonetheless, since most diseases are induced by the functional dysregulation of pro-tein interactions, proteomics, which electively studies protein interactions, has pro-gressively gained a huge consideration in the identification of potential molecularbiomarkers (Liotta et al., 2001) The commonly used approach to identify proteinbiomarkers is to initially select biomarker candidates and then pursue validationstudies in larger patient populations to verify their value

In fact, the clinical validation phase after discovery of candidate molecules mains a major standing challenge common for the biomarker discovery through dif-ferent approaches

re-Immunoassays, multiplex assays (Hanash et al., 2008; Matt et al., 2008), andmass spectrometry (MS)-based multiple reaction monitoring assays (Andersonand Hunter, 2006) provide attractive options to quantitate candidate molecules inlarge numbers of samples MS is the gold standard approach for proteomic or meta-bolomic biomarker discovery Afterward, protein profiles could be validated withspecific antigen microarrays

In ophthalmology, to date, biomarkers are generally unsatisfactory and have still alimited use in clinical practice Biomarkers have been studied in ocular specimens such

astears, for the early lachrymal gland dysfunction in dry eye, or also for nonocular

2 CHAPTER 1 Candidate biomarkers for primary open-angle glaucoma

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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), andvitreous for diabetic retinopathy and

prolif-erative vitreo-retinopathy (Capeans et al., 1998; Danser et al., 1989) In nonocular

specimens, such as plasma, biomarkers have been candidate for the age-related

mac-ular degeneration or diabetic retinopathy, but with a very limited utility (Gu et al.,

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

Subse-quently, candidate biomarkers considered of high priority can be targeted for

vali-dation 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

under-standing 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

re-sistance, 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

as-sociated 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

tech-niques 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

land-scape (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

pre-sent 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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2 DEFINING BIOMARKERS

2.1 PROTEOMICS TECHNOLOGIES FOR BIOMARKER IDENTIFICATION

The increased sensitivity and accuracy of genomic, proteomic, and metabolomictechniques have brought about the potential to identify molecular entities that mayserve 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 dis-ease 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 eitherthe molecular therapeutic disease target or the biomarkers used for early disease de-tection and monitoring

The discovery process could be very varied and can follow different routesdepending on the need and the technologies available (Fuzery et al., 2013;

FIGURE 1

Schematic representation of body fluid samples that are potentially source of molecularbiomarkers 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: minimallyinvasive source of biomarkers; red (dark gray in the print version) text: invasive source

of biomarkers

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Mischak et al., 2010) The workflow (described inFig 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

ap-proach The first approach is based on protein separation by polyacrylamide gels

and individuation of differential proteins and, afterward, identification of the

differ-ential expressed proteins by MS after enzymatic digestion The second workflow

by-passes the gel, favors MS approach also for the step of biomarkers individuation

ap-proaches, while the last challenge of proteomics are the “bottom up” apap-proaches,

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

ap-proaches aim to highlight differential expressed proteins between conditions

ana-lyzed 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

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

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3 NONINVASIVE BIOMARKERS IN PRIMARY OPEN-ANGLE GLAUCOMA

A noninvasive procedure is a conservative diagnostic or therapeutic approach, whichdoes not require incision into the body or the removal of tissue Thus, noninvasivebiomarkers are those obtained in a noninvasive way In glaucoma, different fluidscan 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 ofpotential biomarkers

3.1 TEARS BIOMARKERS

Tears are a source of nourishment for ocular surface tissues and a vehicle to removelocal waste products, metabolized drugs, and inflammatory mediators produced inseveral ophthalmic diseases

Among all body fluids, tears certainly represent one of the most easily and invasively accessible since are collected by using the Schirmer test paper or a micro-capillary glass Generally, the Schirmer test paper is easier to perform with respect tomicrocapillary glass, more comfortable, and yields higher volumes of tears There-fore, in different biomarker studies Schirmer test papers are preferred

non-The complex molecular repertoire available in tears is currently intensively tigated, with the aim to recognize biomarkers of different ocular diseases, includingglaucoma (Pieragostino et al., 2015) Overall, even though many studies were focused

inves-on glaucoma biomarker discovery, inves-only few studies were cinves-onducted inves-on tears.The discovery of potential biomarkers in tears could provide essential informa-tion concerning the pathophysiology of the disease and the effects of topical medi-cations In the first case, tears may hypothetically host glaucoma-related proteinsdirectly coming from the AH, after scleral percolation in the uveoscleral outflowpathway The existence of a trans-scleral AH percolation has been widely documen-ted alsoin vivo (Agnifili et al., 2012; Ciancaglini et al., 2009; Mastropasqua et al.,

2010, 2014) In the second case, which was more extensively studied, tears providemarkers strictly to drug-induced inflammatory processes

Pavlenko et al analyzed the tear levels of endothelin-1 (ET-1), which is a proteininvolved in retinal ganglion cell (RGC) damage and in the TM dysfunction in POAG,

by stimulating vasoconstriction, contraction of smooth-like muscle cells, and by ducing 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 inPOAG patients with respect to healthy controls Borovic et al focused on the activity

in-of renin-angiotensin (SRA) and Kinin–Kallikrein (SKK) systems in tears, blood, and

AH (Borovic et al., 2009), reporting increased levels of kallikrein and converting enzyme activity in patients with POAG compared to normal subjects

angiotensin-Slepova et al (2012)have investigated markers of Fas-mediated apoptosis, reportingthe association of POAG onset and progression with interruption of Fas-mediated

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apoptosis Our research group documented altered tear levels of Lysozyme C,

Lipocalin-1, Protein S100, Immunoglobulins and Prolactin-Inducible Protein in

pa-tients 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

upre-gulated proteins in naı¨ve to therapy POAG patients were downreupre-gulated 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

cyto-kines The consequence is increased matrix degradation and decreased stromal

col-lagens in the cornea

Considering other medications, patients treated with BAK preservedb-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

reported byMalvitte 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

sig-nificantly elevated in treated compared to nontreated eyes.Openkova et al (2013)

found higher tear concentrations of malonic dialdehyde in POAG patients with

re-spect 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

coma patients, showing 17 autoantibody reactivities significantly altered in the

glau-coma 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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Overall, when critically interpreting the above mentioned studies, all dataseems to indicate an increase tear inflammatory status in POAG patients, for themost part related to therapy Therefore, tears could be a source of potential bio-markers indicating the activation of inflammatory pathways, either as a primi-tive pathogenetic expression of the disease or as a consequence of the medicaltherapy.

3.2 URINE BIOMARKERS

The possibility of finding potential biomarkers of glaucoma in the urine, as for otherfluids not in contact with the eye, is low Nevertheless, urine is the final step of theblood clearance pathway of cellular metabolism byproducts, including those ofthe 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 mol-ecules that may play a role in the optic neuropathy An interesting compound is thecitrate, a neuroprotective agent (already considered as a potential serum biomarker ofRGC injury), which was found with slight lower urine concentrations in patients withglaucoma 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 levelssignificantly higher in the serum and lower in the urine in patients with NTG com-pared to healthy controls (Yuki et al., 2010) The urinary 8-OHdG was proposed as apotential indicator of increased systemic oxidative stress in patients with NTG

OPEN-ANGLE GLAUCOMA

A minimally invasive procedure is defined as one that is carried out by enteringthe body through the skin or through a body cavity or anatomical opening, but withthe smallest damage possible to these structures The collection of blood samples

is the most diffuse way to obtain biomarkers with a safe and minimally invasiveprocedure The blood serum is the biggest reservoir of signaling molecules; manymetabolites are secreted from different types of cells in blood that represents auniversal way of communication between cells

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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

immuno-globulins 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

progres-sion of the glaucomatous optic neuropathy (Yang et al., 2001a) Therefore, serum

antibodies to retina and optic nerve proteins might be considered as potential

indi-cators 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

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The evidence that different subtypes of glaucoma present a similar serum body production, suggests that these antibodies may reflect a common nativeresponse to tissue injury necessary for cell debris cleaning and tissue healing Insustain of this hypothesis, histopathological studies documented immunoglobulindeposition in the glaucomatous human retina (Wax et al., 1998) and antibody-mediated damage of RGCs (Tezel and Wax, 2000) Consistently, recentin vivostudies suggested an antibody-mediated RGC loss in eyes with glaucoma(Joachim et al., 2012) Upregulated antibodies are explained as an initiation of auto-aggressive processes to RGC, which lead to the optic nerve damage.

anti-However, not only increased antibody levels but also downregulations could beobserved in glaucoma patients and play a significant pathogenic role (Boehm et al.,

2012) The downregulations could reflect a loss of the natural protective nity and a disbalance of naturally occurring autoantibodies encouraging inflamma-tory neurodegenerative processes (Schwartz-Albiez et al., 2009)

autoimmu-The potential usefulness of serum antibodies as biomarkers for glaucoma is ported by the unique antibody pattern among patients (which exhibits specificity andsensitivity of93% to detect glaucoma) (Boehm et al., 2012) and the similarities incomplex antibody profiles among different ethnic populations (Grus et al., 2006;Wax et al., 2001)

sup-Several techniques, including serological identification of antigens by nant expression cloning (SEREX), serological proteome analysis (SERPA), proteinarrays, and open reading frame phage display, have been used to identify disease-associated antigens and their cognate autoantibodies (Boehm et al., 2012; Grus

recombi-et al., 2006; Wax recombi-et al., 2001) However, while the protein arrays used for profilingthe antibody response are limited to a number of prechosen antigenic proteins,MS-based techniques present a high-throughput analytical approach for de novodiscovery 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 antibodieswere proposed as candidate biomarkers in POAG or are going to be candidate Circu-lating antibodies against heat shock proteins (HSP), antiphosphatidylserine, g-enolase,glycosaminoglycans, neuron-specific enolase (NSE), glutathioneS-transferase (GST),vimentin, myelin basic protein, glial fibrillary acidic protein (GFAP), retinaldehyde-binding 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 aserum antibody against NSE, and the maximum IOP levels in patients with anti-NSEantibody was statistically lower than those without the antibody This supported amajor 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 inpatients with glaucoma induces retinal dysfunctionin vivo

States have a large similarity in serum autoantibody profile and characteristicdifferences from healthy subjects In addition, the authors identified the alpha-fodrin

as a candidate antibody biomarker in both study populations The presence of

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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

AMP-responsive 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

com-pared 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

pre-sent only in glaucomatous patients These biomarkers included immune mediators

and components of cell death signaling The same authors documented increased

ti-ters 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

immuno-logical and inflammatory pathway In this study, the APO A4 yielded the best

per-formance 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

com-plement factor H, also classified eyes affected with POAG, but with lower

discrim-inatory power than APOA4

Notably, the authors identified high serum levels of several proteins that were

pre-viously 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

com-pared to normal subjects Similarly, serum transthyretin (TTR) was found in higher

con-centrations 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),

gamma-enolase, and GST Increased titers of autoantibodies to GST in some patients with

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POAG may represent a generalized response to tissue stress and/or damage as aconsequence 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 thedevelopment and progression of glaucoma (Vohra et al., 2013) In a rodent model ofexperimental glaucoma, Walsh et al (2009) found that elevation of IOP-inducedchanges in protein expression profiles and produced inflammatory biomarkers in ret-ina, RGC, vitreous body, and also in extraocular tissues, such as the serum.The serum amyloid-A1 (SAA1) and SAA2, which are inflammatory markersupregulated in the retina, were found reduced in sera of IOP-responsive eyes Thesemodifications are important because inflammatory processes play a relevant role inthe pathogenesis of POAG (Tezel et al., 2007; Zhou et al., 2005) It was hypothesizedthat the reduced levels of SAA1 and SAA2 in the serum is a response to the stimu-lation of migration of SAA-positive microglial cells to the retina This phenomenonpromotes a systemic downregulation of SAA to prevent the development of an im-mune response toward inflammatory processes linked to glaucoma These resultssuggest that these proinflammatory reactants could be strong candidates biomarkers

of hypertensive glaucoma

Adenosine triphosphate (ATP)-binding cassette (ABC) transporters are proteinsinvolved in the translocation of substances across cell membranes The ABC 1, par-ticularly, has been identified as a leukocyte factor that controls the recruitment ofinflammatory cells and regulates processes induced by chronic vascular dysregula-tion In a previous study (Yeghiazaryan et al., 2005), which analyzed ABC 1 expres-sion rates in circulating leukocytes using protein quantification, the expression ratesthis protein were significantly increased in leukocytes of glaucoma patients com-pared to healthy subjects The authors proposed that a significantly enhanced expres-sion of ABC 1 in circulating leukocytes might be considered as a potential predictiveand diagnostic marker of glaucoma

Also the interleukin 6 (IL-6), an important inflammatory factor, displayed icantly higher values in both the AH and plasma samples of the POAG patients com-pared to healthy controls (Sorkhabi et al., 2010)

sur-In a study of Ghaffariyeh et al (2011), the authors found that serum levels ofBDNF were significantly reduced in patients with early stage POAG compared tocontrols The lower serum BDNF concentration presented a significant negative

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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

polymer-ase 1 (PARP-1) and casppolymer-ase 3 (Cas 3) protepolymer-ase (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

110mmol/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

extracellu-lar matrix within the optic nerve head Increased expression rates of MMP-9 and

MT1-MMP in circulating leukocytes of glaucomatous patients, indicative of an

in-creased enzymatic activity leading to extensive tissue remodeling, and

neurodegen-eration 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

mod-ification 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

es-sential 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 ofL-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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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 comparedwith controls, suggesting the presence of an increased oxidative stress (Galassi

et al., 2004)

Nucci et al studied other oxidative stress-related compounds reporting cantly increased levels of serum and AH malondialdehyde levels in glaucomatouspatients with respect to control group In contrast, the control group presented sig-nificantly higher serum and AH total antioxidant capacity than did the glaucomagroup in both the blood, and higher levels ATP/adenosine diphosphate (Nucci

in-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 statisticallysignificant lower plasma vitamin E and C concentrations and higher plasmaglutathione peroxidase activity compared to healthy subjects All these evidenceshighlighted 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 themost important aspect in the development of glaucoma, since leads to the progressiveelevation of the IOP (Inoue and Tanihara, 2013) Several substances may interactwith 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 IOPelevation Based on this role, Alcon and Novartis filled a series of patents(US20087357931, US20120064532) proposing a method for diagnosing glaucomamodulating the expression of SAA (Clark, 2012)

The 3a-hydroxysteroid dehydrogenase (3a-HSD) is an enzyme that metabolizessteroids in TM, playing a significant role in the regulation of IOP Moreover, it wasfound increased also in optic nerve head astrocytes, in response to elevated IOP in amonkey 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 potentialrole of this factor in the IOP modulation (Weinstein et al., 1996) Since a decrease inthe 3a-HSD activity in PBL may reflect a similar decrease in the ocular enzyme, thedecreased 3a-HSD activity in the readily obtainable PBL could serve as a biomarkerfor POAG or as a risk predictor for the disease

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4.1.2 Genetic Biomarkers

The search for genetic biomarkers of glaucoma represents a crucial field of research

Previous genetic linkage studies identified mutations inMYOC (myocilin), OPTN

(optineurin), andWDR36 for late onset glaucoma; high-risk variants in these genes

are predominantly observed in familial cases of glaucoma, but their frequency in

spo-radic patients from the general population is low (3–5%) (Allingham et al., 2009) In

the last years, major advances in the understanding of genomic architecture, along

with remarkable progresses in bioinformatics and statistics allowed high-resolution

genomic analysis possible Thus, genome-wide association studies identified several

new genes potentially linked to glaucoma, which should be viewed as

disease-associated risk factors rather than causative loci

The cyclin-dependent kinase inhibitor 2B antisense-noncoding RNA

(CDKN2B-AS1) genomic region, on chromosome 9p21.3, is a genetic susceptibility locus for

several age-related complex diseases, including glaucoma

Two of the most important anatomical parameters defining the optic disc, such

as the area and the vertical cup–disc ratio (VCDR), are highly heritable and were found

strictly linked to POAG (Ramdas et al., 2010) Besides the association between the

CDKN2B-AS1 genomic region polymorphisms and the VCDR, a strong association

was also found between theCDKN2B-AS1 genomic region polymorphisms POAG

in US Caucasians, Asian, and African-derived populations (Burdon et al., 2011;

CDKN2B-AS1, reporting the association also in normal tension glaucoma, which is an open-angle

glaucoma subtype in which the IOP falls in normal ranges (Wiggs et al., 2012)

Other genetic biomarkers, related to glaucoma quantitative traits, were reported

associated with IOP, VCDR, and with POAG, such asTMCO1 (transmembrane and

coiled-coil domains 1),SIX1/6 (sine oculis homeobox homolog), ATOH7 (atonal

ho-molog 7) Genetic variation in and aroundTMCO1 has been reported significantly

associated with IOP and POAG (Burdon et al., 2011; Sharma et al., 2012), even

though, to date, the function of this gene is not well elucidated and it is not yet known

how it is linked to glaucoma

The SIX1 and CDKN2B genes, which present the strongest association with

VCDR, and theATOH7 gene, which is expressed in the optic nerve during

embryo-genesis (Brown et al., 1998) and presents a strong association with the optic disc area,

were found significantly associated with POAG (Ramdas et al., 2011)

Single-nucleotide polymorphisms between the CAV1 (caveolin 1) and CAV2

(caveolin 2) genes (chromosome 7q31), which are expressed in the TM and RGC,

were found significantly associated with POAG, especially in women and in cases

with early paracentral visual field defects (Loomis et al., 2014; Thorleifsson et al.,

2010; Wiggs et al., 2011)

In the field of genetic biomarkers, we could also include the detection of cellular

DNA damage: circulating leukocytes isolated from patients with glaucoma showed

significantly enhanced DNA damage compared to healthy subjects (Moenkemann

et al., 2005) Comparative comet assay analysis revealed patterns of comets typical

for glaucoma; therefore, the comet assay profiling of DNA damage in circulating

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leukocytes was proposed as a potential tool for minimally invasive molecular nostics of glaucoma (Golubnitschaja and Flammer, 2007).

diag-Besides the DNA damage, also the DNA-repair machinery alteration is involved

in the pathogenesis of glaucoma and might represent a potential biomarker of ease In fact, concomitant upregulation ofp53 (stress-regulated gene) and downre-gulation ofXPGC (essential member of DNA-repair machinery) has been found incirculating leukocytes of glaucoma patients (Golubnitschaja-Labudova et al., 2000).Numerous other potential genes are under investigation or are going to be studied inthe upcoming future

dis-In closing, validation studies of potential serum biomarker candidates should clude patients with different types and stages of glaucoma, with different demo-graphics and comorbidities, nonglaucoma-related neurodegenerative diseases, andhealthy controls This appears mandatory to candidate a pattern of integrated bio-markers potentially applicable in the wide spectrum of manifestation of the disease

in-In fact, a set of biomarkers rather than a single molecule may potentially be moreuseful as a predictive, diagnostic, and/or prognostic clinical tool

GLAUCOMA

An invasive procedure is a diagnostic or therapeutic procedure that requires entry of

a body cavity, interruption of normal body functions, and removal of tissues Thus,

an invasive biomarker is obtained in an invasive way Besides the biopsy of tissueocular tissues (i.e., conjunctiva, trabecular meshwork, etc.), invasive biomarkers forglaucoma are those obtainable from the AH or vitreous, and from nonocular fluidstaking direct contact with the eye structures, such as the cerebrospinal fluid (CSF)

5.1 AQUEOUS HUMOR BIOMARKERS

The AH is an intraocular fluid containing proteins secreted from the anterior segmentstructures and is responsible for the supply of nutrients and removal of metabolicwastes from the avascular tissues (Klenkler and Sheardown, 2004) Protein levels in

AH are modified in many diseases affecting the anterior segment of the eye, includingPOAG (Duan et al., 2008) The AH proteome profile in patients with glaucoma is verydifferent when compared with age- and sex-matched controls, this suggesting thatchemical modifications of this fluid could be strongly involved in the development

of this disease (Izzotti et al., 2010; Sporn and Roberts, 1990; Yu et al., 2010).Because the AH is in direct contact with the most critical site involved in the path-ogenesis of glaucoma, that is the TM, it represents a precious medium where searchfor candidate biomarkers of TM dysfunction Generally, the more convincing can-didate biomarkers for POAG are those inducing extracellular matrix alterations inthe TM (Duan et al., 2008), such as cytokines stimulating the TM cell activity(Alvarado et al., 2005) and other molecules promoting synthesis, degradation, andmodification of the TM matrix (Fuchshofer and Tamm, 2009) In addition, the

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AH may contain also markers of RGC neurodegeneration, inflammation, immune

response, and oxidative stress

Unfortunately, the AH sampling is a very invasive procedure since requires a

sur-gical approach; therefore, the possibility to analyze the molecular composition of

AH, and to search biomarkers, is limited to patients undergoing intraocular surgery,

such as glaucoma or cataract surgery

5.1.1 Trabecular Meshwork Damage Biomarkers

From a biological point of view, the anterior chamber is a space surrounded by an

endothelium, and a path by which the AH travels; thus, it looks like a vessel and

be-haves like a vessel Also the TM is constituted by endothelial cells, which control the

permeability of the Schlemm’s canal In patients with POAG, the population of TM

endothelial are significantly reduced compared to age-matched healthy subjects

(Alvarado et al., 1984)

In a recent study on patients with POAG, Saccà et al found increased AH levels

of inflammatory proteins (ELAM1), proteins regulating the cholesterol metabolism

(APO B and APO E) or involved in the muscle cell differentiation (myotrophin and

myogenin), stress response proteins (HSP 60, 90), and protein involved in the signal

transduction (Sacca et al., 2012) These changes reflected an unbalance of adhesion

molecules produced by endothelial cells, the recruitment of inflammatory cells and

cytokines targeting endothelial cells, vascular smooth muscle cells, and the

extracel-lular matrix These changes ultimately lead to apoptosis and degeneration of the TM

Interestingly, these molecular modifications within the anterior chamber resemble

the events that occur to vessel endothelia during atherosclerosis

In a study on AH proteome,Izzotti et al (2010)reported a list of 31 proteins with

more than twofold significant variance in expression in POAG versus control

sub-jects Among these proteins, six were mitochondrial proteins and their increased

levels were indicative of severe damage of cells in tight contact with AH, such as

those constituting the TM Five proteins were directly involved in apoptotic

mech-anisms, either mitochondrial-dependent (caspases such as BAX and BIK) or

mitochondrial-independent (TNFs) Six proteins were components of the

intercellu-lar junction, contributing to the maintenance of cell–cell adhesion (catenins,

junc-tional plaque protein, dynein, and cadherins), and their increase was intended as

expression of a severe loss of the TM integrity Other proteins were involved in

sev-eral neural functions, especially the neural survival, and in the oxidative stress Very

interestingly, the AH protein concentration correlated with the IOP

POAG patients The most commonly overexpressed protein was the cochlin, a

com-ponent of the extracellular matrix, which could interact with the fibrillar collagen and

promote the collagen degradation

Prostaglandin H2D-isomerase and caspase-14, which induces apoptosis in a

va-riety of cell types, are potentially involved also in the TM modification in POAG

since both molecules were found in increased levels in the AH, possibly mediating

the apoptosis of the TM cells (Duan et al., 2008)

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A critical role in the pathogenesis of POAG is played by a particular group ofcytokines, which are the transforming growth factors (TGF) This is a family of mul-tifunctional polypeptides that regulate critical cell mechanisms such as proliferation,migration, differentiation, cytokine production, synthesis of extracellular matrix,wound healing, immunosuppression, andin vivo angiogenesis Among TGF-b iso-forms, only three isoforms namelyb1,b2, andb3are expressed in human ocular tis-sues, and TGF-b2is regarded as the major isoform in the eye (Nishida et al., 1995;

AH of patients with POAG compared to normal subjects (Inatani et al., 2001;Jampel et al., 1990) TGF-b2seems to be strongly involved in the structural changes

of TM in patients with POAG since this cytokine promotes the increase in fibrillarextracellular matrix deposition in the juxtacanalicular region of the TM (Fuchshofer

et al., 2009) In support of this, it was demonstrated that minimizing TGF-b2levels inthe AH, may help to prevent the aging process in the TM in POAG (Yu et al., 2010).Besides this direct effect, TGF-b2may also act indirectly through the induction of theexpression of the angiopoietin-like (ANGPTL) 7 protein, a member of the ANGPTLfamily, which are important regulators of the angiogenesis ANGPTL7 levels werefound increased in the AH of POAG eyes, this appearing an adjunctive mechanismleading to collagen changes in the TM (Kuchtey et al., 2008) Based on theseevidences, AH levels of TGF-b2 appear a robust invasive candidate biomarkerfor POAG

Besides TGF-b2, others immune-inflammatory response proteins (cytokines)are markedly modified in the glaucomatous AH, this supporting an abnormal activity

of the immune system in patients with POAG (Pinazo-Duran et al., 2013) TheTNF-a, the endothelial leukocyte adhesion molecule 1 (ELAM-1), IL-1a, IL-6,and IL-8 levels resulted significantly increased in the AH and outflow pathways

of POAG patients compared to control subjects (Liton et al., 2005; Sacca et al.,2012; Tezel, 2008)

All these findings suggest that the innate immunity and inflammatory pathwaysare strongly involved in the pathogenesis and progression of the glaucoma-relatedoptic neuropathy, as occur in other age-related diseases such as AMD (Licastro

ET-1 has been found in elevated concentrations in the AH of glaucoma patients,and some evidence also suggests that ET-1 correlates and directly influence IOPvalues, by causing contraction of TM In addition, ET-1 has a neurotoxic activity

on RGC, either directly or indirectly by inducing vasoconstriction Thus, ET-1

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was candidate as a potential pathogenetic biomarker for TM and RGC damage

(Choritz et al., 2012)

The erythropoietin (EPO), besides the regulation of red cell production, acts as a

neuroprotective factor through the inhibition of apoptosis, the reduction of glutamate

and reactive oxygen species (ROS) levels, the reduction of proinflammatory cytokines,

and the promotion of vascular autoregulation EPO levels were found significantly

in-creased in the serum and AH of patients with POAG, when compared with healthy

subjects (Cumurcu et al., 2007; Mokbel et al., 2010); such increased levels may be

intended as a compensatory neuroprotective mechanism stimulated by an ischemia-,

hypoxia-, or ROS-induced optic nerve damage Thus, also EPO was proposed as a

potential biomarker of RGC damage and visual field loss

Soluble CD44 (sCD44) is a cytotoxic protein that negatively affect the survival of

TM and RGC; its levels were found significantly increased in the AH of patients with

POAG (Choi et al., 2005), and correlated with the severity of visual field loss in all

stages of disease (Mokbel et al., 2010) Thus, also sCD44 concentrations in the AH

could be proposed a possible protein biomarker of TM damage and visual field loss

(Nolan et al., 2007)

5.1.3 Oxidative Stress Biomarkers

The oxidative stress and antioxidant status in AH were found associated with early

stages of glaucoma and with the progressing disease (Liu et al., 2007;

Zanon-Moreno et al., 2011) Generally, decreased antioxidant defenses along with increasing

prooxidants in the AH have been reported to play a potential pathogenic role in

glau-coma (Ferreira et al., 2004; Nucci et al., 2013; Zanon-Moreno et al., 2008)

Particu-larly, superoxide dismutase (SOD) and glutathione synthase (GS) concentrations

were significantly lower in POAG patients than in controls, whereas the NOS and

GS expression were higher in glaucomatous patients than in controls The

overproduc-tion of NO through inducible NOS can form toxic products and change the metabolism

of the TM Of note, prooxidants were found to positively correlate with

proinflam-matory and proapoptotic molecules, and with the visual field progression in POAG

patients (Pinazo-Duran et al., 2013) Therefore, GS, NOS, SOD, and GST could be

potentially useful AH markers of glaucomatous damage (Bagnis et al., 2012)

Some studies reported increased levels of selected amino acids in the AH of

pa-tients with POAG (Ghanem et al., 2012) This is the case of the homocysteine, which

is involved in the RGC apoptosis (but also in extracellular matrix alterations,

oxida-tive stress, and ischemic vascular dysregulation), and hydroxyproline, which is a

marker of increased collagen turnover These amino acids could potentially have

a significant role in the pathogenesis of glaucoma

The role and implication of myocilin in glaucoma has been extensively studied,

and has been significantly linked to elevation of the IOP Increased levels of myocilin

were found in the AH of animal and human eyes affected with POAG, being this

protein widely considered as a marker of the glaucomatous state Because serum

levels of myocilin are unchanged in patients with glaucoma, this modification is

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tissue specific and in POAG is expression of critical TM alterations (Abu-Amero

et al., 2012; Howell et al., 2010)

Transferrin regulates the growth and maintenance of many cellular types of theanterior segment of the eye, and is implicated in inflammatory processes Elevatedtransferrin concentrations were observed in the AH of POAG patients, as expression

of an inflammatory response occurring in conditions of elevated IOP (Duan et al.,

2008) Because of this, also transferrin has been incriminated in the pathophysiologicchanges occurring in the anterior chamber during glaucoma

TTR, which normally vehiculates thyroxine and retinol in the bloodstream, mayalso induce extracellular polymerization of insoluble protein fibrils, called amyloiddeposits In patients with glaucoma, TTR could be poured in the AH from the ciliarypigment epithelium or directly by degenerating RGC (Dwork et al., 1990; Tripathi

et al., 1992) The accumulation of TTR in the anterior chamber may induce itations of protein fibrils and cause a mechanical barrier for the AH outflow withfinal elevation of the IOP (Grus et al., 2008) Increased levels of TTR were docu-mented in patients with Alzheimer’s disease and in the AH of POAG eyes, indicatingthat this protein might play a significant role in the pathogenesis of some neurode-generative conditions

precip-In closing, the AH is a reservoir of numerous potential biomarkers, which areexpression of alteration of the most important pathogenetic pathways of glaucoma,especially those related to the TM damage and RGC degeneration

5.2 VITREOUS BODY BIOMARKERS

The major advantage in the analysis of the vitreous body in glaucoma is due to thefact this structure is juxtaposed to RGC Therefore, the vitreous may act as a potentialreservoir of marker proteins directly leaked from injured or dying RGC, as a conse-quence of the IOP-induced loss of membrane permeability

The major con is that vitreous can be sampled exclusively by invasive dures, such as vitreous biopsy (vitreous tap) or during vitrectomy performed for dif-ferent surgical indications This is an important limiting factor that also limits thenumber of studies available in literature

proce-In an experimental mouse model of hyperbaric glaucoma, Western blots of thevitreous showed a 25-fold increase of catalase content in the hyperbaric compared

to healthy eyes, whereas XIAP (a member of an inhibitor of apoptosis (IAP) genefamily) content increased by ninefold (Walsh et al., 2009) The increase of catalaselevels can be explained by the attempt to reduce the levels of ROS induced by thehigh IOP levels On the other hand, the increased levels of XIAP could be intended as

a stimulation of an intrinsic pathway to prevent the cell death phenomena induced byapoptotic caspases Of note, the increase in the levels of these proteins in the vitreous

of hyperbaric eyes was greater than those observed in the retina, thus confirming theaccumulation of these proteins in the vitreous

Studies on vitreous concentrations of glutamate, an amino acid strongly involved

in the RGC neurotoxic damage, produced conflicting results (Dreyer et al., 1996;Honkanen et al., 2003)

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In closing, vitreous body appears a potential interesting source of glaucoma

biomarkers, even though the invasive procedure of sampling limits its analysis

5.3 CEREBROSPINAL FLUID BIOMARKERS

In the recent years, the biomechanical mechanisms of the CSF have stimulated a

growing interest in the field of glaucoma The orbital CSF pressure, in fact,

repre-sents the true counter-pressure against the IOP across the lamina cribrosa; recent

studies suggested that a low CSF pressure could be associated with glaucomatous

optic neuropathy in normal pressure glaucoma (Morgan et al., 2008) However, also

the chemical composition of CSF may provide insight in the knowledge of

patho-physiological processes occurring in patients with glaucoma

The molecular composition of CSF is nearly identical to that of the AH, as both

fluids are produced by carbonic anhydrase-catalyzed reactions and represent an

ultrafiltrate of blood, with more proteins and less ascorbate in the CSF than in the

be modified during the course of glaucoma because it is in direct contact with the

optic nerve, which hosts axons of degenerating RGC Therefore, identification of

potential biomarkers in the CSF appears very intriguing Nonetheless, to date, due

to the high invasivity of the procedure to sample CSF (rachicentesis), dedicated

studies on potential CSF biomarkers for POAG are lacking The literature search

for CSF and glaucoma biomarkers did not show published studies

IS TRANSLATION FEASIBLE?

In glaucoma, as in other fields of ophthalmology and medicine, the transfer of

biomarkers from discovery to clinical practice is a process with numerous pitfalls

and limitations, mostly due to structural and scientific factors The most

impor-tant pitfalls consist in the lack of making different selections before initiating the

discovery phase, in the lack in biomarker characterization and validation strategies,

and in the robustness of analysis techniques used in clinical trials (Drucker and

Krapfenbauer, 2013)

To become a clinically approved test, a potential biomarker should be confirmed

and validated using hundreds of specimens and should be reproducible, specific, and

sensitive In fact, the discovery phase is based on specimens derived from a small

well-characterized cohort; to proceed in the validation phase, larger heterogeneous

cohorts are needed for appropriate statistical power The validation process,

espe-cially concerning statistics, is a crucial step to eliminate false positivity and calculate

the sensitivity and specificity of potential biomarkers Large cohorts of patients are

also required to consider the different subtypes and stages of glaucoma, subjects with

different demographics and comorbidities, and the effects of each single drug or

combination of medications The presence of healthy controls is also mandatory

to validate initial results

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Because of the etiological complexity of glaucoma, the existence of differentsubtypes of disease and the interpatient heterogeneity, multiple biomarkers couldbetter reach the sensitivity and specificity required, rather than a single biomarker.Therefore, a panel of biomarkers, representing different fluids and each of whichexpression of the most important pathogenetic steps, could be the ideal solution.

In this way, one may hypothetically have a “cocktail” of biomarkers indicating a highrisk of having glaucoma, or biomarkers diagnosing glaucoma at the very early stage,

or pondering the risk of the optic neuropathy progression However, assembling andvalidating such a biomarker panel is even more challenging

Nevertheless, because of the need of powerful and validated screening strategies,early stage diagnosis, timely detection of damage progression, and new availablemedications, also in glaucoma the biomarker field is speeding up toward clinicaltranslation

To date, no definitive molecule is the biomarker for preventing blindness fromPOAG Certainly, a single “ideal” glaucoma biomarker that covers all aspects ofthe disease does not exist

Considering the “social nature” of glaucoma, its prevalence, and the huge impact

of this disease in the worldwide irreversible blindness, the identification of patients atrisk of glaucoma, a very early diagnosis, an early detection of the damage progres-sion, and the prediction of the response to treatment are urgent issues to be satisfiedwith potential molecular biomarkers

In the management of glaucoma, biomarkers could aid research of potentialnew therapies with patient selection, identifying subjects who most probablyrespond to medications from those who are refractory However, once defined,one must consider that during the course of disease biomarkers may significantlychange, because of the effect of therapy and the modification of the phenotype ofthe disease Previous studies clearly documented that glaucoma medications maydefinitely mask alterations in crucial biomarkers (i.e., markers of the nitric oxidepathway) (Drucker and Krapfenbauer, 2013) To date, although advances have beendone in the functional and structural assessment of progressing damage, idealinstruments to evaluate and estimate the optic neuropathy progression early arenot definitely available Also in this field, molecular biomarkers could certainlyhelp clinicians in detecting RGC loss in time and in planning timely interventions

In vivo live imaging with biomarkers directly imaged in the eye, could allowidentification of living, dying, or injured RGC before they become evident withroutinely used morpho-functional examinations All these requirements will be bestsatisfied in case of biomarkers obtainable in an ease and noninvasive way, such as

by sampling tears

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