ARUMUGAM BALASUBRAMANIAN First Name: SIVARAM

THE UNIVERSITY OF NEW SOUTH WALES Thesis/Dissertation Sheet

Surname or Family name: ARUMUGAM BALASUBRAMANIAN

First name: SIVARAMAN Other name/s:

Abbreviation for degree as given in the University calendar: PhD

School: OPTOMETRY AND VISION SCIENCE Faculty: SCIENCE

Title: CHANGES TO THE TEAR FILM PROTEOME IN KERATOCONUS

Abstract 350 words maximum: (PLEASE TYPE) Keratoconus (KC) is a progressive degenerative disease of the eye which causes an irregularly shaped cornea leading to severe impairment of vision. Despite basic and clinical studies KC remains a poorly understood disease. This project was designed to investigate the levels of proteins, proteases and cytokines in the tears of people with KC. Basal tears were collected from normal controls (C); people with KC and from people who had undergone corneal collagen cross-linking (CXL) for the treatment of KC. Corneal curvature of each subject was mapped. Proteomic technologies including gel electrophoresis, ELISA, mass spectrometry, antibody arrays and activity assays were used to examine the changes in tear proteins, proteases and cytokines between the different subject groups. There was approximately 2-fold decrease in total protein levels, lactoferrin, secretory IgA between KC and C tears, but the level of albumin was not significantly reduced. The tear protein changes correlated to the severity of the disease. Increased levels of cathepsin B (2.7-fold) and decreased levels of cystatin S (2.1-fold) and cystatin SN (2-fold), polymeric immunoglobulin receptor (9.4-fold), fibrinogen alpha chain (8.2-fold) were observed in KC compared to C. Keratin type-1 cytoskeletal-14 and keratin type- 2 cytoskeletal-5 were present only in the tears of KC. Tears of people with KC had 1.9 times higher levels of proteolytic activity and over-expression of several matrix metalloproteinases (MMP) -1, -3, -7, -13 and interleukins (IL) -4, -5, -6, -7, -8 and tumour necrosis factor (TNF) -α, -β compared to tears from C. No significant difference in MMPs were observed between C and CXL groups, although the expression levels of TNF-α was 1.5 times increased in CXL compared to C. The activities of tear proteases in CXL were not significantly different compared to either KC or C. The tears of people with KC appear to have an altered tear protein profile, higher levels of proteases and cytokines that might reflect the pathological events in KC corneas. The novel findings reported in this thesis might lead the way to the development of non-invasive diagnostic and prognostic tests for KC or determine the success of CXL using tear proteomics.

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THIS SHEET IS TO BE GLUED TO THE INSIDE FRONT COVER OF THE THESIS

CHANGES TO THE TEAR FILM PROTEOME

IN KERATOCONUS

SIVARAMAN A. BALASUBRAMANIAN MBBS, DO

A thesis submitted in fulfilment of the requirements for the degree of

Doctor of Philosophy

School of Optometry and Vision Science

The University of New South Wales, Sydney, Australia

and

Brien Holden Vision Institute, Sydney, Australia

October 2012

Certificate of Originality

CERTIFICATE OF ORIGINALITY

‘I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgement is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis.

I also declare the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project’s design and conception or in style, presentation and linguistic expression is acknowledged.’

Signed………………………………………………

25 Oct 2012

Date ………………………………………………...

‘I hereby grant the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all proprietary rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation.

I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation

Abstract International (this is applicable to doctoral theses only).

I have either used no substantial portions of copyright material in my thesis or I have obtained permission to use copyright material; where permission has not been granted I have applied/will apply for a partial restriction of the digital copy of my thesis or dissertation.'

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25 Oct 2012

Date ………………………………………………...

AUTHENTICITY STATEMENT

‘I certify that the Library deposit digital copy is a direct equivalent of the final officially approved version of my thesis. No emendation of content has occurred and if there are any minor variations in formatting, they are the result of the conversion to digital format.’

Signed………………………………………………

25 Oct 2012

Date ………………………………………………...

Changes to the tear film proteome in keratoconus ii Acknowledgements

ACKNOWLEDGEMENTS

First and foremost I would like to thank my supervisor Prof. Mark Willcox for his endless support, advice and encouragement. I count myself lucky to have had Mark

Willcox as my supervisor, who always has a “ready to help” attitude. I really appreciate his guidance and the complete freedom given to me in this work conducted over the years. You have made the course of my PhD, a truly enriching learning experience.

Thanks also to my supervisor Assoc/Prof. David Pye for his useful advice on the clinical aspects of the work.

I am very grateful to Prof. Brien Holden for being so generous in providing me with a scholarship to pursue PhD. I thank God for the accidental chat with Brien outside the research building which eventually presented me with a scholarship. Special thanks to the Assoc/Prof. Eric Papas for being my trouble shooter and often bailing me out of problems which could have greatly affected the progress of my PhD. Eric Papas is one of the most humane person I have ever met. Cheers to my friend Dr. Mahesh Bandara for sharing with me some of his valuable lab tricks and presentation tips.

I do not have words to express my gratitude to Dr. Mohan Rajan and Dr. Sujatha Mohan for motivating me to stay focused on my project. I would like to take this opportunity to thank Ms. Ananthalakshmi for helping me to recruit volunteers for the study.

I would like to extend my appreciation to Dr. Thomas Naduvilath for his invaluable guidance for the statistical analysis, Dr. Valerie Wasinger for her help with mass spectrometry and Dr. Judith Flanagan for proof reading this thesis.

I am grateful to all the volunteers who took part in this study and spared their valuable time for me.

Changes to the tear film proteome in keratoconus iii Acknowledgements

Last but not least my family especially my beloved wife for her unfailing support and encouragement. I am indebted towards my Dad and Mom for their unconditional love and affection.

Changes to the tear film proteome in keratoconus iv

DEDICATION

To my wife Kaavya

Changes to the tear film proteome in keratoconus v Abstract

ABSTRACT

Keratoconus (KC) is a progressive degenerative disease of the eye which causes an irregularly shaped cornea leading to severe impairment of vision. Despite basic and clinical studies KC remains a poorly understood disease. This project was designed to investigate the levels of proteins, proteases and cytokines in the tears of people with

KC.

Basal tears were collected from normal controls (C); people with KC and from people who had undergone corneal collagen cross-linking (CXL) for the treatment of KC.

Corneal curvature of each subject was mapped. Proteomic technologies including gel electrophoresis, ELISA, mass spectrometry, antibody arrays and activity assays were used to examine the changes in tear proteins, proteases and cytokines between the different subject groups.

There was approximately 2-fold decrease in total protein levels, lactoferrin, secretory

IgA between KC and C tears, but the level of albumin was not significantly reduced.

The tear protein changes correlated to the severity of the disease.

Increased levels of cathepsin B (2.7-fold) and decreased levels of cystatin S (2.1-fold) and cystatin SN (2-fold), polymeric immunoglobulin receptor (9.4-fold), fibrinogen alpha chain (8.2-fold) were observed in KC compared to C. Keratin type-1 cytoskelatal-

14 and keratin type-2 cytoskeletal-5 was present only in the tears of KC. Tears of people with KC had 1.9 times higher levels of proteolytic activity and over-expression of several matrix metalloproteinases (MMP) -1, -3, -7, -13 and interleukins (IL) -4, -5, -6, -

7, -8 and tumour necrosis factor (TNF) -α, -β compared to tears from C. No significant difference in MMPs were observed between the C and CXL groups, although the expression levels of TNF-α was 1.5 times increased in CXL compared to C. The activity of tear proteases in CXL were not significantly different compared to either KC or C

Changes to the tear film proteome in keratoconus vi Abstract subjects.

The tears of people with KC appear to have an altered tear protein profile, higher levels of proteases and cytokines that might reflect the pathological events in KC corneas. The novel findings reported in this thesis might lead the way to the development of non- invasive diagnostic or prognostic tests for KC or determine the success of CXL using tear proteomics.

Changes to the tear film proteome in keratoconus vii Table of contents

TABLE OF CONTENTS

LIST OF FIGURES…………………………………………………………………xi LIST OF TABLES ...... xiii TERMINOLOGY AND ABBREVIATIONS ...... xiv 1. CHAPTER 1: INTRODUCTION ...... 1 1.1. The Eye ...... 2 1.2. The Cornea ...... 2 1.3. Keratoconus ...... 5 1.3.1. Incidence and prevalence ...... 7 1.3.2. Histopathology ...... 8 1.3.3. Risk factors ...... 9 1.3.4. Genetics of KC ...... 9 1.3.5. Diagnosis ...... 11 1.3.5.1. Clinical signs and symptoms ...... 11 1.3.5.2. Topography ...... 12 1.3.6. Management ...... 14 1.3.6.1. Contact lenses ...... 14 1.3.6.2. Surgery ...... 14 1.3.7. Public health importance ...... 17 1.4. Tear film ...... 17 1.4.1. Structure of the tear film ...... 18 1.4.2. Functions ...... 19 1.4.3. Types of tears ...... 19 1.4.4. Tear collection methods ...... 20 1.4.5. Tear proteins and changes with disease ...... 21 1.5. Proteases ...... 24 1.5.1. KC and MMPs ...... 28 1.5.2. KC and Cathepsins ...... 29 1.6. Inflammatory molecules ...... 30 1.6.1. KC and Inflammatory Molecules ...... 32 1.7. Tear proteomics ...... 37 1.8. Thesis overview ...... 41 2. CHAPTER 2: LEVELS OF LACTOFERRIN, SECRETORY IMMUNOGLOBULIN A AND SERUM ALBUMIN IN THE TEAR FILM OF KERATOCONUS PATIENTS ...... 45 2.1. Introduction ...... 46 2.2. Materials and methods ...... 48 2.2.1. Ethics approval...... 48 2.2.2. Recruitment of Subjects ...... 48 2.2.3. Collection of Basal tears ...... 51 2.2.4. Corneal topography ...... 52 2.2.5. SDS-PAGE ...... 54

Changes to the tear film proteome in keratoconus viii Table of contents

2.2.6. Total protein concentration of tears ...... 55 2.2.7. Quantification of lactoferrin, sIgA and serum albumin in tears ...... 56 2.2.8. Statistical Analysis ...... 57 2.3. Results ...... 57 2.3.1. SDS-PAGE ...... 57 2.3.2. Total tear protein concentration ...... 59 2.3.3. Tear lactoferrin, sIgA and serum albumin levels ...... 59 2.4. Discussion...... 65 3. CHAPTER 3: IDENTIFICATION OF DIFFERENTIALLY EXPRESSED TEAR PROTEINS IN KERATOCONUS ...... 69 3.1. Introduction ...... 70 3.2. Materials and Methods ...... 71 3.2.1. Subjects ...... 71 3.2.2. Corneal topography ...... 72 3.2.3. Tear collection ...... 72 3.2.4. Evaluation of tear proteins ...... 72 3.2.5. Total protein level ...... 73 3.2.6. Protein fractionation ...... 73 3.2.7. Sample preparation for LC-MS/MS ...... 77 3.2.7.1. C18 Stage tip ...... 77 3.2.7.2. Acetone precipitation ...... 77 3.2.8. Trypsin digestion ...... 77 3.2.9. LC-MS/MS using LTQ-FT instrument ...... 78 3.2.10. Search of the database ...... 79 3.2.11. Statistical methods ...... 79 3.3. Results ...... 80 3.3.1. Corneal topography ...... 80 3.3.2. Total tear protein level ...... 80 3.3.3. Tear protein profile ...... 80 3.3.4. Differentially expressed proteins in KC compared to C ...... 90 3.3.5. Ontology Analysis ...... 92 3.4. Discussion...... 94 4. CHAPTER 4: PROTEASES AND INFLAMMATORY MOLECULES IN THE TEARS OF PEOPLE WITH KERATOCONUS ...... 98 4.1. Introduction ...... 99 4.2. Materials and Methods ...... 101 4.2.1. Subjects ...... 102 4.2.2. Tear collection ...... 104 4.2.3. Corneal topography ...... 104 4.2.4. Total tear proteins ...... 104 4.2.5. Proteases and cytokines in tears ...... 105 4.2.6. Activity of proteases ...... 106 4.2.7. Statistical methods ...... 107 4.3. Results ...... 107 4.3.1. Total tear protein concentration ...... 107 4.3.2. Tear Proteases ...... 109

Changes to the tear film proteome in keratoconus ix Table of contents

4.3.3. Tear Cytokines ...... 114 4.3.4. Proteolytic activity of tears ...... 114 4.3.5. ROC analysis of proteolytic activities ...... 117 4.4. Discussion...... 119 5. CHAPTER 5: EFFECTS OF EYE RUBBING ON THE CONCENTRATION OF PROTEASES AND CYTOKINES IN THE TEARS ...... 124 5.1. Introduction ...... 125 5.2. Materials and Methods ...... 129 5.2.1. Subjects ...... 129 5.2.2. Corneal topography ...... 130 5.2.3. Eye rubbing technique ...... 130 5.2.4. Tear collection ...... 131 5.2.5. Total tear protein concentration ...... 131 5.2.6. Quantification of MMP-13, IL-6 and TNF-α ...... 131 5.2.7. Protease activities ...... 132 5.2.8. Statistical methods ...... 132 5.3. Results ...... 132 5.3.1. Total tear protein concentration ...... 132 5.3.2. Tear levels of MMP-13, IL-6 and TNF-α ...... 132 5.3.3. Tear proteolytic activities ...... 133 5.4. Discussion...... 133 6. CHAPTER 6: SUMMARY, CONCLUSIONS AND FUTURE DIRECTIONS 137 6.1. Significance of research ...... 138 6.2. Novel findings ...... 138 6.3. Summary ...... 140 6.4. Implication of research ...... 141 6.5. Limitation of the research and recommendations for future studies ...... 142 6.6. Conclusions ...... 144 REFERENCES ...... 146 APPENDIX………………………………………………………………………….176

Changes to the tear film proteome in keratoconus x List of Figures

LIST OF FIGURES

Figure 1.1: Cross-section of the human eye…………………….…………………...... 2 Figure 1.2: Layers of the cornea...... 3 Figure 1.3: Normal cornea (left) and KC (right) showing anterior protrusion of the cornea...... 7 Figure 1.4: Topography maps of (a) normal cornea (b) mild KC (c) moderate KC (d) severe KC...... 13 Figure 1.5: CXL using UV-A/riboflavin (left) and PKP (right)...... 16 Figure 1.6: Layers of the tear film...... 19 Figure 2.1: (a) Tear sample collection using a thin polished glass micro capillary tube placed at the lateral canthus of the eye (b) Polished glass micro capillary tube (c) Rubber pump to expel the tears...... 52 Figure 2.2: Examination of the corneal curvature using Medmont corneal topographer...... 54 Figure 2.3: Standard curve plot of serial dilutions of BSA...... 56 Figure 2.4: 1D SDS-PAGE of tears from KC patients and C...... 58 Figure 2.5: Comparison of the densitometry of the bands between C (Lane 2) and KC (Lane 3)...... 59 Figure 2.6: Negative correlation between total tear protein levels and corneal curvature...... 63 Figure 2.7: Negative correlation between tear lactoferrin levels and corneal curvature.63 Figure 2.8: Negative correlation between tear sIgA and corneal curvature...... 64 Figure 2.9: Insignificant correlation between serum albumin and corneal curvature. . 64 Figure 3.1: Schematic diagram showing the work flow to analyse tear proteins in the present study...... 72 Figure 3.2: The Microflow 10 (MF-10) separation system ...... 76 Figure 3.3: Tear proteins which were relatively (a) up-regulated or (b & c) down- regulated in KC compared to C...... 91 Figure 3.4: Cellular compartments of the proteins altered in KC...... 92 Figure 3.5: Biological processes of the proteins altered in KC...... 93 Figure 3.6: Functions of the proteins altered in KC...... 93 Figure 4.1: Illustration of the interplay between proteases and cytokines based on previous reports...... 101 Figure 4.2: The Allegro Oculyzer examination set up...... 104 Figure 4.3: Correlation analysis between total protein levels and keratometry in 80 eyes...... 108 Figure 4.4: Correlation analysis between total protein levels and keratometry in 60 eyes (without CXL group)...... 109 Figure 4.5: Insignificant correlation between collagenolytic activity and keratometry reading in C, KC and CXL groups...... 115 Figure 4.6: Significant positive correlation between collagenolytic activity and keratometry reading in C and KC groups...... 115 Figure 4.7: Insignificant correlation between gelatinolytic activity and keratometry

Changes to the tear film proteome in keratoconus xi List of Figures reading in C, KC, CXL groups...... 116 Figure 4.8: ROC analysis of the protease activities in C and KC groups (a) Gelatinase activity (b) Collagenase activity...... 118 Figure 4.9: Diagram showing the relative expression of MMPs and cytokines in the tears of controls (C), keratoconus (KC) and cross-linked (CXL) groups...... 122 Figure 5.1: KC patients use their (a) finger tips or (b) knuckle to generate pressure localized to the cornea in a circular motion...... 126 Figure 5.2: Different methods adopted by allergic patients for eye rubbing using (a) back of the hand, (b) palm and (c) finger pad involving the caruncle ...... 127

Changes to the tear film proteome in keratoconus xii List of Tables

LIST OF TABLES

Table 1.1: Clinical signs of KC...... 12 Table 1.2: Classification of MMPs...... 27 Table 1.3: Summary of studies that have been conducted to investigate MMPs, cathepsins and inflammatory molecules in KC...... 34 Table 1.4: Examples of tear proteins up or down regulated in systemic diseases or ocular diseases ...... 39 Table 1.5: Purpose and study groups with total number of subjects in each chapter…44 Table 2.1: Demographics of C and KC subjects...... 50 Table 2.2: Percentage (%) of KC subjects in each group based on disease severity. .. 57 Table 2.3: Levels of total protein, lactoferrin and sIgA in the tears of C and KC subjects...... 61 Table 2.4: Influence of gender, age, contact lens wear and atopy on the level of tear proteins...... 62 Table 3.1: Demographics of the study...... 71 Table 3.2a: List of total tear proteins identified in C and KC...... 81 Table 3.2b: Proteins in 1 to 5 kDa fraction……………………………………………85 Table 3.2c: Proteins in 5 to 25 kDa fraction…………………………………………..86 Table 3.2d: Proteins in 25 to 50 kDa fraction………………………………………...87 Table 3.2e: Proteins in 50 to 75 kDa fraction………………………………………...88 Table 3.2f: Proteins in 75 to 150 kDa fraction……………………………………….89 Table 3.3: Altered tear proteins in KC compared to C...... 91 Table 4.1: Demographics of the study...... 103 Table 4.1: Layout of the custom G-series array…………………………………….106 Table 4.3: Expression of proteases and cytokines in tears of the three groups...... 110 Table 4.4a: Correlations between tear proteases/cytokines and keratometry (D) of control (C) and keratoconus (KC) group...... 111 Table 4.4b: Correlation between MMPs:TIMPs and keratometry (D) in C, KC and CXL groups...... 112 Table 4.5: Gelatinase and collagenase activities in tear samples...... 113 Table 4.6: Substrate specificity of collagenases (MMP-1,-8,-13), gelatinases (MMP-2,- 9), stromelysin (MMP-3) and matrilysin (MMP-7)...... 116 Table 0.2: Substrate specificity of collagenases (MMP-1,-8,-13), gelatinases (MMP-2,- 9), stromelysin (MMP-3) and matrilysin (MMP-7)………………………………..117 Table 5.1: Demographics of the study……………………………………………...130 Table 5.2: Levels of total protein, inflammatory mediators, proteolytic activities in the tear samples...... 133

Changes to the tear film proteome in keratoconus xiii Terminology and Abbreviations

TERMINOLOGY AND ABBREVIATIONS

µm micrometres

1-D one-dimensional

ABC atopic blepharoconjunctivitis

AKC atopic keratoconjunctivitis

ANG angiogenin

AQP5 aquaporin 5

AR after rubbing

BCA Bicinchoninic acid

BR before rubbing

BSA Bovine serum albumin

BSCVA best spectacle-corrected visual acuity

C control

CaCl2 calcium chloride

CATS cathepsin S

CLEK collaborative longitudinal evaluation of keratoconus

COL4A3 collagen type IV alpha 3

COL4A4 collagen type IV alpha 4

COL6A1 collagen type VI alpha 1

COL8A1 collagen type VIII alpha 1

CRB1 crumbs homolog 1

CXL corneal collagen cross-linking

D dioptre

DDA Data Dependant Acquisition

DALK Deep Anterior Lamellar Keratoplasty dH2O distilled water

Changes to the tear film proteome in keratoconus xiv Terminology and Abbreviations

EACA epsilon aminocaproic acid

ECM extracellular matrix

EDTA disodium ethylene-diaminetetra-acetic acid

EGF epidermal growth factor

ELISA enzyme-linked immunosorbent assays

ET-1 endothelin-1

FGF fibroblast growth factor

FIU fluorescence Intensity Units

FT ICR Fourier Transform Ion Cyclotron Resonance

GAG glycosaminoglycans

HCL hydrochloric acid

HGF hepatocyte growth factor

HPLC High Performance Liquid Chromatography

HREA Human Research Ethics Advisory Panel

IGF insulin-like growth factor

IL interleukins

IL1B interleukin 1 beta

KC keratoconus kDa kilodaltons

KGF keratinocyte growth factor kVHrs kilovolt hours

LAR leukocyte common antigen related protein

LASIK laser assisted in-situ keratomileusis

LC liquid chromatography

LIF leukemia inhibiting factor

LOX lysyl oxidase

LTQ-FT MS Linear Ion Trap Quadrupole Fourier Transform mass spectrometer

Changes to the tear film proteome in keratoconus xv Terminology and Abbreviations m/z Mass to Charge ratio

MCP -1 monocyte chemoattractant protein

M-CSF macrophage colony stimulating factor

MES 2-N-morpholino-ethanesulfonic acid

MF10 Microflow 10 mg milligrams min minutes mL millilitres mm millimetres mM millimolar

MMP matrix metalloproteinase mRNA messenger ribonucleic acid

MS mass spectrometry

MT1-MMP membrane type matrix metalloproteinase

NaCl sodium chloride

NaN3 sodium azide ng nanograms nm nanometres

PAGE polyacrylamide gel electrophoresis

PDGF platelet-derived growth factor pg picograms pH measure of acidity or basicity

PKP penetrating keratoplasty ppm parts per million

PRK photorefractive keratectomy

ROC receiver operating curve rpm revolutions per minute

Changes to the tear film proteome in keratoconus xvi Terminology and Abbreviations

SD standard deviation

SDS sodium dodecylsulfate sIgA secretory immunoglobin A

SOD1 superoxide dismutase 1

SPSS statistical package for the social sciences

TGF transforming growth factor

TIMP tissue inhibitors of matrix metalloproteinase

TNF tumour necrosis factor

UNSW University of New South Wales

UV ultra-violet

V volts v/v volume per volume

VKC vernal keratoconjunctivitis

VSX1 visual homeo box 1 w/v weight per volume

Changes to the tear film proteome in keratoconus xvii Chapter 1: Introduction

1. CHAPTER 1: INTRODUCTION

Changes to the tear film proteome in keratoconus 1 Chapter 1: Introduction

1.1. The Eye

The eye is a vital sensory organ of the visual system. It detects and focuses light from the surrounding objects and the light entering the eye is converted into electrical signals, which the brain translates into images. Anatomically, the eye is divided into anterior and posterior segments (Figure 1.1). The anterior segment consists of structures such as the cornea, iris, ciliary body, crystalline lens and aqueous humour. The posterior segment forms the back two-thirds of the eye consisting of choroid, vitreous humour, retina and the optic nerve. These structures work in a co-ordinated manner to provide vision (Snell & Lemp 1998).

Figure 1.1: Cross-section of the human eye. Modified from www.healthyeyes.org.uk

1.2. The Cornea

The clear and transparent layer in front of the eye is the cornea (Figure 1.1). Often termed the “window of the eye”, it helps to focus light entering the eye onto the retina.

The anterior surface of the cornea is the most important refractive surface of the eye.

Along with the lens, the cornea refracts light and, by itself, accounts for about two-

Changes to the tear film proteome in keratoconus 2 Chapter 1: Introduction thirds of the eye’s total optical power (Meek et al. 2003). In humans, the cornea has a diameter of about 11.5 mm, a thickness of 0.5-0.6 mm in the centre and 0.6-0.8 mm at the periphery (Dartt et al. 2011). The horizontal diameter (11-12 mm) is greater than the vertical diameter (9-11 mm) of the human adult cornea (Brightbill et al. 2009). The extracellular matrix (ECM) of the cornea consists of 70% collagen fibres (Kaufman et al. 2003).

Figure 1.2: Layers of the cornea. Modified from www.hybridcornea.org

The human cornea is composed of five layers (Figure 1.2).

(a) The corneal epithelium is the outermost layer of the cornea consisting of non-

keratinized stratified squamous epithelial cells. The thickness of the epithelium is

about 50 µm with a superficial layer of squamous cells, a middle layer of wing cells

and a layer of inner basal cells (Ehlers et al. 2010). The surface epithelial cells

function as a barrier between the extracellular space of the cornea and the tear film

(Beuerman & Pedroza 1996). The basal cells adhere to a basement membrane

which is composed of mainly type IV collagen. Epithelial cell turn over occurs

approximately every seven days by shedding of cells into the tear film (Hanna et al.

Changes to the tear film proteome in keratoconus 3 Chapter 1: Introduction

1961). The outer layer of the epithelium is kept moist by the tear film and any

irregularity or oedema of the epithelium disrupts the smoothness of the air-tear film

interface, causing a reduction in visual acuity.

(b) The Bowman’s layer is an acellular layer with condensation of short and small

irregularly arranged collagen type I fibrils. The thickness of Bowman’s layer is 8-12

µm. This layer is often considered as a remnant of the primary acellular stroma and

does not regenerate following injury (Beuerman & Pedroza 1996).

thickness. The stroma is essentially made up of regularly arranged type I collagen

fibrils and accounts for over 90% of the dry weight of the cornea (Wilson 1970;

Jester et al. 1999). The diameter of the collagen fibrils ranges from 22-32 nm

(Hogan et al. 1971; Giraud et al. 1975). These fibrils are placed parallel to each

other forming flat lamellar bundles. There are around 200-250 of such lamellae each

measuring 2 µm in thickness (Dartt et al. 2011). The collagen lamellae are arranged

obliquely in the anterior one third of the stroma, and moving posteriorly, these

collagen lamellae run parallel to the surface of the cornea (Komai & Ushiki 1991).

The arrangement of collagen fibrils is vital to maintain the shape, strength and

transparency of the human cornea. The collagen is surrounded by glycoproteins and

proteoglycans, which are responsible for the regular spacing of the collagen fibrils.

Glycosaminoglycans (GAG) are important constituents of the proteoglycans

regulating the collagen fibril organization. The important GAGs of the corneal

stroma are keratin sulphate, dermatan sulphate and chondroitin sulphate (Bron

2001). Keratocyte is the predominant cell type found in the corneal stroma and it is a

spindle shaped, specialised connective tissue cell involved in the synthesis of

collagens and proteoglycans. These keratocytes produce enzymes that degrade and

Changes to the tear film proteome in keratoconus 4 Chapter 1: Introduction

replace the older collagen fibrils. Moreover, keratocytes also play an important role

in collagen repair and scar formation following corneal injury (Wang & Swartz

2009). The turnover of the collagen molecules in the cornea is around 2-3 years

(Krachmer et al. 2005).

(d) Descemet’s membrane is a layer mainly composed of types IV and VIII collagen

and is about 5-20 µm depending on age. It serves as the modified basement

membrane of the corneal endothelium. The Descemet’s membrane contains no cells

and does not regenerate after injury (Wang & Swartz 2009).

(e) The corneal endothelium is the innermost layer of the cornea adhering to the

posterior surface of Descemet’s membrane. It is a monolayer of squamous or

cuboidal mitochondria-rich cells. These cells help in regulating the fluid and solute

transport in the stromal compartments of the cornea. The cells of the human

endothelium cannot regenerate and therefore stretch to compensate for the loss of

dead cells, impacting the fluid regulation of the cornea. These imbalances in fluid

regulation or stromal hydration can cause swelling of the stroma, thereby reducing

the transparency of the cornea.

For optimal vision, the cornea must efficiently transmit incident light by

maintaining its transparency. Avascularity, or the lack of blood vessels, is an

essential element of corneal transparency (Maurice 1957). In summary, the layers of

the cornea play fundamental roles such as focusing the incident light onto the retina

by refraction, maintaining the structural integrity of the eye and protecting the eye

from infective organisms, noxious substances and ultra-violet (UV) radiation.

1.3. Keratoconus

Ectasia or bulging of the cornea is a pathological condition that occurs due to structural

Changes to the tear film proteome in keratoconus 5 Chapter 1: Introduction changes in the cornea. These changes are mainly associated with thinning of the layers of the cornea. The most common type of ectatic corneal condition is keratoconus followed by pellucid marginal degeneration, keratoglobus and Terrien’s marginal degeneration (Rabinowitz 1998).

Keratoconus (KC) is typically described as a bilateral, asymmetric and non- inflammatory corneal ectasia (Nichols et al. 2004). The name keratoconus is derived from a Greek word meaning kerato-horn or cornea; conus-cone (Horner 1869). This degenerative disease of the eye is characterized by progressive thinning of all or specific locations of the cornea giving rise to a cone shaped cornea instead of the normal spherical shape (Figure 1.3). Structural changes caused by myopia (near-sightedness) and irregular astigmatism (irregular corneal curvature) during the development of KC lead to defective vision (Krachmer et al. 1984). KC is a progressive disease that affects both eyes, although only one eye might be affected initially (Lee et al. 1995). The severity of the disease is usually more pronounced in one eye compared to the other.

Progression of KC is associated with an increase in the level of myopia or spherical myopic refractive error, higher order irregular astigmatism of the cornea, and deterioration of best spectacle-corrected visual acuity (BSCVA) (Suzuki et al. 2007).

The cause of KC is not certain and its outcome is unpredictable.

Changes to the tear film proteome in keratoconus 6 Chapter 1: Introduction

Figure 1.3: Normal cornea (left) and KC (right) showing anterior protrusion of the cornea. Photo courtesy of Dr. Phillip Reese (normal cornea) and School of Optometry & Vision science, UNSW, Sydney, Australia (KC).

1.3.1. Incidence and prevalence

There have been varied reports on the incidence and prevalence of KC, ranging from 1 to 430/2000 people in a population, due to various diagnostic methods used within different populations (Applebaum 1936; Hofstetter 1959; Franceschetti 1965; Palimeris et al. 1981; Ihalainen 1986; Kennedy et al. 1986). The disease mostly affects people during the productive period of their lives (less than 40) (Ertan & Muftuoglu 2008).

Due to the possible links between myopia and KC (Burdon et al. 2008; Baird et al.

2010) and, recent advances in the diagnosis of KC by widespread use of corneal topography, the reported incidence and prevalence rates of KC are expected to increase over the next few years (Romero-Jimenez et al. 2010). More cases of KC are being identified during the pre-operative assessment for surgical correction of refractive errors such as laser assisted in-situ keratomileusis (LASIK) (Oshika & Klyce 1998).

Population-based prevalence studies of KC are yet to be reported in the literature. It had previously been suggested that males and females, and people from all ethnic backgrounds are equally affected but studies have shown that people of Asian origin are more susceptible to KC (Pearson et al. 2000; Georgiou et al. 2004), and men are

Changes to the tear film proteome in keratoconus 7 Chapter 1: Introduction predominately affected with a male to female ratio of 3:1 (Weed & McGhee 1998).

Fink et al. determined gender differences in patient history of the disease, and in vision and ocular symptoms among people with KC (Fink et al. 2005).

1.3.2. Histopathology

Thinning of the cornea in KC results from structural changes in the layers of the cornea.

Classic histopathologic features include stromal thinning, iron deposition in the epithelial basement membrane and folds in the posterior cornea. The disease usually starts by dissolving the Bowman’s layer of the cornea (Sawaguchi et al. 1998). The breaks in Bowman’s layer cause anterior clear spaces in the cornea (Pataa et al. 1970;

Shapiro et al. 1986). These spaces are then filled by relatively unstructured stromal collagen, keratocytes and epithelial cells leading to the formation of scar tissue.

Degeneration of the epithelial cells and the basement membrane is accompanied by deposition of a brown pigment called Fleischer’s ring. This ring is composed of ferritin deposits and usually surrounds the margins of the cone in a KC cornea (Teng 1963;

McPherson & Kiffney 1968). Vogt first described the presence of folds or vertical stress lines in the deeper layers of the stroma anterior to Descemet’s membrane (Vogt

1919). These stress lines are called Vogt’s striae, probably caused by stretching of the collagen lamellae in the areas of corneal protrusion (Hollingsworth & Efron 2005). The endothelial layer of the cornea is affected during the late stages of KC leading to acute hydrops. Acute hydrops is a condition marked by severe stromal oedema, usually accompanied by rupture of Descemet’s membrane. In some severe cases, acute hydrops may lead to perforation of the cornea (Ingraham et al. 1991; Rubsamen & McLeish

1991).

Changes to the tear film proteome in keratoconus 8 Chapter 1: Introduction

1.3.3. Risk factors

KC is a complex multifactorial disease. Association of KC with environmental factors such as constant eye rubbing (Coyle 1984; McMonnies 2008), contact lens wear (Gasset et al. 1978), allergy (Lowell & Carroll 1970; Bawazeer et al. 2000) and genetic disorders such as Down’s syndrome (Cullen & Butler 1963), Ehlers-Danlos syndrome

(Robertson 1975) and Marfan’s syndrome (Maumenee 1981) have been suggested.

However, there are no substantial data available to establish the involvement of these factors in the progression or initiation of the disease.

1.3.4. Genetics of KC

The regulation of many genes has been shown to be altered in KC. Studies have suggested the involvement of a genetic component (Rabinowitz et al. 1992; Wang et al.

2000; Edwards et al. 2001; Bisceglia et al. 2009). Several genes such as visual homeo box 1 (VSX1) (Bisceglia et al. 2005), superoxide dismutase 1 (SOD1) (Udar et al.

2006), interleukin 1 beta (IL1B) (Kim et al. 2008), crumbs homolog 1 (CRB1)

(McMahon et al. 2009), aquaporin 5 (AQP5) (Rabinowitz et al. 2005), lysyl oxidase

(LOX) (Nielsen et al. 2003), collagen type IV alpha 3 (COL4A3) and collagen type IV alpha 4 (COL4A4) (Stabuc-Silih et al. 2009) have been proposed to be involved in KC, but these findings have not been replicated or confirmed by further studies. The role of other candidate genes such as collagen type VI alpha 1 (COL6A1), collagen type VIII alpha 1 (COL8A1), matrix-metalloproteinase genes (MMP9, MMP2) were dismissed as causative genes after investigations (Fullerton et al. 2002; Rabinowitz et al. 2005; Udar et al. 2006). Genome- wide association studies have identified linkage at the chromosomal regions 1p36.23-36.21 and 8q13.1-q21.11 and 14q24.3 (Burdon et al.

2008).

Changes to the tear film proteome in keratoconus 9 Chapter 1: Introduction

Family studies have reported that up to 50% of people with KC have atleast one close relative affected by the disease (Gonzalez & McDonnell 1992). The relatives of KC have a higher risk (15-67 times) of developing the disease when compared to those relatives without KC (Wang et al. 2000).

In a study on identical twins, Hammerstein postulated an autosomal dominant inheritance with incomplete penetrance in KC (Hammerstein 1972). To date, 19 pairs of monozygotic twins affected with KC have been described in the literature where-in both twins were affected with either similar (Bechara et al. 1996) or different levels of severity of the disease (Parker et al. 1996). This strongly supports the involvement of a genetic component in the development of KC. However, a study on two pairs of twins failed to detect KC in both twins (McMahon et al. 1999).

KC is inherited as a polygeneic disorder and environmental risk factors could influence the phenotype (Rabinowitz 1998). However, another study reported the variable expression of the disease in monozygotic twins where neither of the twin had a history of atopy or eye rubbing (Weed et al. 2006).

Another theory is that keratocytes in KC corneas might be explicitly sensitive to apoptotic mediators due to a possible genetic predisposition (Edwards et al. 2001).

Myopia is an important feature of KC and these two conditions could share a common genetic basis (Burdon et al. 2008; Baird et al. 2010). The genetic component of KC is complex as evidenced by the genetic heterogeneity and phenotypic diversity of the disease (Edwards et al. 2001). The wide range of results reported in various genetic studies of KC might indicate the contribution of multiple genes in the development and progression of KC.

Changes to the tear film proteome in keratoconus 10 Chapter 1: Introduction

1.3.5. Diagnosis

1.3.5.1. Clinical signs and symptoms

KC, in its early stages can present as a case of common refractive error to clinicians.

Clinical signs depend on the severity of the disease. A range of clinical signs of KC can be observed using various diagnostic methods (Table 1.1). Symptoms are highly variable and partly depend on the stage of progression of the disease. The early stages of the disease can be asymptomatic, while in the advanced stage there is significant distortion of vision accompanied by profound visual loss with sometimes excruciating pain. The collaborative longitudinal evaluation of keratoconus (CLEK) study established that KC patients suffer more ocular pain compared to patients with other refractive errors such as myopia (Zadnik et al. 1998). This pain is often aggravated by discomfort caused by rigid contact lenses used for correcting vision in KC. The early detection of KC is difficult as slit-lamp corneal changes are absent or too subtle to detect and keratometry can be normal. KC can also present without clinical signs and this type of KC is termed forme-fruste or subclinical KC (Rabinowitz 1998).

Changes to the tear film proteome in keratoconus 11 Chapter 1: Introduction

Table 1.1: Clinical signs of KC.

External signs Munson’s sign- the visible protrusion of the lower eyelid while looking down, due to the cone shaped cornea pushing the lid out Rizzuti’s phenomenon- conical reflection on the nasal cornea when a penlight is shone from the temporal side Slit-lamp findings Stromal thinning Posterior stress lines which disappears with gentle digital pressure (Vogt’s striae) Iron ring (Fleischer’s ring) Scarring- epithelial or sub-epithelial Retroillumination signs Scissoring of retinoscopy reflex Oil droplet sign (Charleaux sign) Photokeratoscopy signs Compression of mires infero-temporally (egg-shaped mires) Compression of mires inferiorly or centrally

Videokeratography signs Localized increased surface power Inferior superior dioptric asymmetry Relative skewing of the steepest radial axes above and below the horizontal meridian

1.3.5.2. Topography

Corneal topography, or photokeratoscopy, or videokeratography is a non-invasive medical imaging technique widely used to study the curvature of the cornea and detect videokeratography signs in KC (Table 1.1). Screening of KC using videokeratography maps (Figure 1.4) has been reported to be the only effective method available to identify early stages of KC (Maguire & Bourne 1989). Although these techniques might have a greater sensitivity for the detection of KC, interpretation of the topographic maps is

Changes to the tear film proteome in keratoconus 12 Chapter 1: Introduction sometimes difficult (Dastjerdi & Hashemi 1998). KC is grouped into different stages based on the steepening of the corneal curvature. The assessment of the corneal curvature is expressed as keratometry readings in dioptres (D). According to the steepest simulated keratometry reading (K2), KC is classified as mild (<45D), moderate

(45-52D) or severe (>52D) (Zadnik et al. 1998). KC has numerous presentations such as inferior, temporal, superior, central and horse-shoe shaped corneal steepening.

Examples of topography maps of normal and different stages of KC corneas with inferior steepening are shown in Figure 1.4. The clinical signs and the topography maps aid in the diagnosis of KC and also differentiate KC from other types of corneal ectasias.

Figure 1.4: Topography maps of (a) normal cornea (b) mild KC (c) moderate KC (d) severe KC.

Changes to the tear film proteome in keratoconus 13 Chapter 1: Introduction

1.3.6. Management

1.3.6.1. Contact lenses

At an early stage, KC is managed by prescribing glasses or contact lenses. Once diagnosed, contact lenses are the mainstay of therapy for the distorted vision caused by

KC and represent the management of choice for 90% of KC patients (Rabinowitz 1998).

Lens fitting for KC is difficult because of the irregular and constantly changing corneal shape. Soft lenses of toric design are often used during the early stage of KC but are usually later replaced by complex rigid gas permeable lenses including multicurve spherical-based lenses, aspheric lenses, and bi-aspheric lenses. A hybrid lens, which has a rigid central portion for obtaining best optics and a soft hydrophilic peripheral skirt can also be used (Rabinowitz et al. 1991; Yeung et al. 1995). The visual performance in people with KC can be improved by wearing scleral contact lenses (ScCL). Previously,

ScCL were used in advanced stages of KC but with recent advances in ScCL, mild stages of KC can also be managed successfully (Pullum et al. 2005). However, as the disease progresses, the contact lenses might not provide adequate visual correction and can become highly intolerable for KC patients. Contact lens intolerance becomes the main indication for the use of surgical interventions to treat KC (Lim et al. 2000).

1.3.6.2. Surgery

Surgical techniques commonly used in the management of KC are corneal collagen cross-linking (CXL) and penetrating keratoplasty (PKP) (Figure 1.5). Other procedures such as deep lamellar keratoplasty (DALK) (Funnell et al. 2006), epikeratoplasty

(Wagoner et al. 2001), intrastromal corneal rings or Intacs (Colin et al. 2000),

Descemet-stripping endothelial keratoplasty (DSEK) (Lee et al. 2011) are also considered in the treatment of KC. Epidemiological studies report that approximately

Changes to the tear film proteome in keratoconus 14 Chapter 1: Introduction

20% of KC patients progress to a stage where they require PKP (Kennedy et al. 1986).

The outcomes of treating advanced stages of KC by PKP have been encouraging

(Sutton et al. 2008).

Wollensak et al. introduced the technique of corneal collagen cross-linking (CXL), using UV-A radiation coupled with riboflavin (Vitamin B2) to slow or stop the progression of KC (Wollensak et al. 2003). In brief, this technique exposes the cornea to

UV-A radiation (370 nm) for 30 minutes. The central 7 mm of the corneal epithelium is removed with a blunt knife before irradiation. Riboflavin 0.1% solution (10 mg riboflavin-5-phosphate in 10 ml dextran-T-500 20% solution) is applied for 5 minutes before and during the procedure. The use of riboflavin considerably increases the absorption of UV-A in the corneal stroma by acting as a photosensitiser or a photomediator. This procedure is considered minimally invasive and an alternative to other surgical techniques used in the management of KC (Wollensak et al. 2003).

The use of UV-A/riboflavin has a direct effect on the corneal stroma by making it rigid.

Collagen cross-links are induced in the stromal layer of KC, similar to the effect of photo-polymerization of polymers (Hettlich et al. 1992). These cross-links are achieved by increasing the covalent bonding within the collagen fibrils and proteoglycans in the corneal stroma (McCall et al. 2010). An approximate 70% increase in the corneal rigidity has been shown in experimental studies on rabbit and porcine corneas after UV-

A/riboflavin treatment (Wollensak et al. 2004). A significant increase in the diameter of collagen fibrils in rabbit corneas (Wollensak et al. 2004) and an increased resistance of corneal collagen to enzymatic digestion in porcine corneas have been demonstrated following cross-linking (Spoerl et al. 2004). Cross-linking in human corneas with KC has been shown to increase the diameter of collagen fibres and proliferation of

Changes to the tear film proteome in keratoconus 15 Chapter 1: Introduction keratocytes (Mencucci et al. 2010). A minimum central corneal thickness of 400 µm is a pre-requisite for CXL to avoid complications such as loss of endothelial cell density or cataract formation (Wollensak et al. 2003). However, instillation of hypotonic riboflavin has been considered during CXL in thinner corneas (Kohlhaas et al. 2006).

UV-A/riboflavin induced cross-linking of the corneal collagen in KC has shown promise in the treatment of keratoconus. However, the precise molecular mechanisms of

CXL are not fully established and long-term results are still being awaited to establish its efficacy.

For conditions in which the use of contact lenses or CXL is unsuitable, PKP appears to be the only option available for KC patients. Nevertheless, the PKP procedure is not exempt from complications and limitations. Studies have reported increased rates of graft rejection (Lim et al. 2000; Epstein et al. 2006; Lass et al. 2010), reduction of corneal sensitivity (Rao et al. 1985) and more importantly, recurrence of KC after PKP

(Kremer et al. 1995). Managing a case of KC is a great challenge for clinicians because the management options for KC currently available restore the refractive error or the distorted vision caused by the disease without treating the underlying cause.

Figure 1.5: CXL using UV-A/riboflavin (left) and PKP (right). CXL picture reproduced with permission from the article (Wollensak et al. 2003) in the American Journal of Ophthalmology (Confirmation number: 2866740453853).

Changes to the tear film proteome in keratoconus 16 Chapter 1: Introduction

1.3.7. Public health importance

In many countries KC is the leading indication for PKP (Wagoner & Ba-Abbad 2009).

The mean survival rate of the corneal grafts in 95% of KC patients is 5 years and 90% have a graft survival of over 10 years (Williams et al. 2012). KC can be prone to recurrences after PKP (Bechrakis et al. 1994) and a recent study based on the Australian corneal graft registry, has estimated that a person undergoes a maximum of 5 corneal transplants due to KC (Rebenitsch et al. 2011). The loss of quality of life and the life time costs incurred for managing KC was equal to, or greater than, other eye disorders such as age related macular degeneration or myopia (Rebenitsch et al. 2011). Impaired vision caused by KC poses a significant public health burden with a typical onset during early adulthood (around 16 years of age) extending through peak education, prime earning, child-bearing and child-rearing years (Kymes et al. 2004).

1.4. Tear film

The tear film rests on the most anterior surface of the eye. Oxygen from the atmosphere reaches the cornea mainly by diffusing into the tear film. It is often referred to as the

“pre-corneal tear film” since it spreads over the cornea with every blink (Wong et al.

1996). Although the thickness of the tear film has been reported to vary from about 2

µm (Nichols & King-Smith 2003) to 40 µm (Prydal et al. 1992), the widely accepted range is around 3 µm (King-Smith et al. 2004). Tears comprise the collective ocular surface secretions of the main and accessory lacrimal glands, meibomian glands, corneal epithelial cells, goblet cells and epithelial cells of the conjunctiva, as well as having contributions from the conjunctival vasculature. The secretions are controlled by complex interactions between the endocrine and autonomic nervous systems (Bromberg

1981; Schirra et al. 2006). It is believed that tear film is a three-layered structure (Figure

Changes to the tear film proteome in keratoconus 17 Chapter 1: Introduction

1.6) consisting of superficial or outer lipid layer, intermediate or middle aqueous layer, and the inner mucous layer (Holly & Lemp 1977).

1.4.1. Structure of the tear film

The lipid layer of the tear film is secreted mainly by the meibomian glands and additionally by glands of Zeiss and Moll glands (Bron & Tiffany 1998) and it is approximately 100 nm in thickness (Mishima 1965). The oily nature of this layer helps to reduce the evaporation of the underlying aqueous layer (Craig & Tomlinson 1997).

Rich innervations of parasympathetic, sympathetic and sensory nerves control the secretions of the meibomian gland (Foulks & Bron 2003).

The middle aqueous layer is secreted by the lacrimal glands and the accessory glands of

Krause and Wolfring. This sandwiched layer is the watery layer of the tear film and has been reported to be 6.5 µm-7.5 µm thick (Mishima 1965). Proteins and ions form the major composition of the aqueous layer, and maintenance of the integrity of corneal and conjunctival epithelial cells is achieved by the presence of aqueous tear layer (Mishima

1965).

The inner mucous layer is secreted by goblet cells in the conjunctiva and the crypts of

Henle. Overlaying the corneal and conjunctival epithelial cells, the mucous layer ensures the smooth sliding of eyelids. This smooth sliding movement is essential for protecting the epithelial cells from sheer forces of the eyelids during every blink. The hydrophilic nature of the mucous layer helps the tear film to adhere to the ocular surface

(Holly & Lemp 1977).

Changes to the tear film proteome in keratoconus 18 Chapter 1: Introduction

Figure 1.6: Layers of the tear film.

1.4.2. Functions

The tear film performs a major role in the maintenance of the integrity of ocular surface.

It acts as a barrier or first wall of defence for the eye against pathogens and toxic substances. Other important functions of the tear film are: lubrication of the eyelids, cornea and conjunctiva, provision of a smooth ocular surface for the refraction of light, removal of foreign material from the cornea and conjunctiva (Holly 1973).

The tear film is a complex mixture consisting of proteins, proteases, cytokines, lipids, metabolites, electrolytes and hydrogen ions (Liu et al. 1999; Eperon et al. 2004). The concentration of these constituents can be influenced by the type of tear secretion

(Fullard & Snyder 1990; Fullard & Tucker 1991), diurnal variation (Fullard & Carney

1984; Ng et al. 2001) and various tear collection methods (Stuchell et al. 1984).

1.4.3. Types of tears

Tears, based on rate of secretion, can be classified in three types: basal, reflex or psycho-emotional tears (Murube 2009). Basal tears are also referred to as unstimulated or minimally stimulated tears and these tears help in keeping the ocular surface

Changes to the tear film proteome in keratoconus 19 Chapter 1: Introduction constantly moist. Factors such as blinking or changes in outside air temperature are the factors involved in basal tear secretion. The rate at which basal tears are secreted is estimated to be around 1.2 µL/min (Pflugfelder et al. 1999) and total protein concentration 6-10 mg/mL (Gachon et al. 1982). Excessive stimulation of the ocular surface in the form of irritants such as onion vapours, tear gas, dust particles or pathological conditions such as infections or inflammations can lead to the production of reflex tears. Unlike basal tears, reflex tears have increased rate of tear secretion (>50

µL/min), lowering the protein concentration (<5 mg/mL) (Fullard & Snyder 1990;

Fullard & Tucker 1991; Sitaramamma et al. 1998). Psycho-emotional tears are seen only in humans and are caused by emotional stress, anger, pain, depression or happiness

(Murube 2009). The tears collected in the open-eye state or upon awakening are known as open and closed-eye tears respectively (Sack et al. 1992; Sitaramamma et al. 1998).

The concentration of total tear protein is maximum in closed eye tears (12-15 mg/ml) compared to open eye basal or reflex tears (Sack et al. 1992; Sitaramamma et al. 1998).

1.4.4. Tear collection methods

Use of glass micro capillary tubes, filter paper strips (Van Haeringen 1981), sponges or porous polyester rods (Jones et al. 1997) are the different means by which tears can be collected. Filter paper strips or sponges placed in the conjunctival sac can absorb the tears present in the palpebral fissure. These two techniques can produce ocular irritation and mild trauma to the conjunctival and corneal epithelial cells due to the physical presence of the collecting device (van Haeringen & Glasius 1976; van Setten et al.

1990; Choy et al. 2001). This affects the composition of tears by releasing cellular proteins into the tear film (Stuchell et al. 1984; Green-Church et al. 2008). The contamination of tears by epithelial cells and other cellular debris are minimal when

Changes to the tear film proteome in keratoconus 20 Chapter 1: Introduction tears are collected using glass micro capillary tubes. These tubes are less invasive and may be advantageous over other methods to collect tears. Tears can also be sampled by a “flush” method, where in the eye is treated with saline before collecting the tears using glass capillary tubes (Bjerrum & Prause 1994; Markoulli et al. 2011).

1.4.5. Tear proteins and changes with disease

The proteins found in the tear fluid are mainly synthesized and produced by the lacrimal gland. Neuropeptides and neurotransmitters present in the lacrimal gland stimulate the protein secretion (Dartt 1989). In an open-eye state, lactoferrin, lysozyme, lipocalin and secretory immunoglobin A (sIgA) form the major proteins in the tear film (Sack et al.

1992; Kijlstra & Kuizenga 1994). Lactoferrin, lysozyme and lipocalin constitutes 70-

85% of the total protein content (Gachon et al. 1979; Fullard & Tucker 1994). The total number of tear proteins is still under debate. There have been various reports on the total number of tear proteins ranging from 54 proteins to 491 proteins (Herber et al.

2001; Zhou et al. 2003; Li et al. 2005; de Souza et al. 2006; Zhou et al. 2006). The following details are a summary of the conventional wisdom and more recent understandings of tear film proteins will be discussed comprehensively in subsequent sections.

Lactoferrin, possessing anti-inflammatory and antimicrobial activities, plays an essential role in maintaining ocular health (Flanagan & Willcox 2009). It is produced by acinar cells of the main and accessory lacrimal glands (Gillette & Allansmith 1980). The anti- inflammatory properties of lactoferrin are partly due to its inhibition of the complement activation pathway, and the binding of lactoferrin with free iron in tears, reduces the iron necessary for bacterial growth (Kijlstra 1990). Lactoferrin might possess additional antimicrobial activities distinct from its iron sequestration. Reduced tear lactoferrin

Changes to the tear film proteome in keratoconus 21 Chapter 1: Introduction levels have been reported in dry eye (Mackie & Seal 1984), trachoma (Rapacz et al.

1988), vernal conjunctivitis, contact lens-induced giant papillary conjunctivitis (Ballow et al. 1987) and post-operative cataract surgery (Jensen et al. 1985).

Tear lysozyme exhibits anti-bacterial properties essential for ocular surface immunity. It is a glycolytic enzyme secreted by the lacrimal gland, exhibiting bactericidal action by enzymatic digestion of the mucopolysaccharides in all walls of bacteria (Herber et al.

2001). The tear lysozyme levels have been reported to be decreased in dry eye patients

(Avisar et al. 1979), during acute adenoviral conjunctivitis (Gupta et al. 1986) and herpes simplex keratitis (Saari et al. 1983) and increased during contact lens wear

(Temel et al. 1991).

Lipocalin or tear specific pre-albumin was thought to be a protein found only in the tear fluid (Bonavida et al. 1969). Subsequent investigations established that lipocalin is comparable to Von Ebner’s gland protein and, also produced in the prostrate, nasal and tracheal mucosa (Redl 2000). Tear lipocalin is the principal lipid binding protein in tears capable of binding and transporting endogenous lipid molecules (Glasgow et al. 1998).

Having both hydrophilic and hydrophobic properties, lipocalin is essential for the stability aqueous-lipid layer interface of the tear film (Benedetto et al. 1975). Excess lipids are removed by the scavenging property of the lipocalin in order to maintain the integrity of the corneal surface (Gasymov et al. 2005). Decreased levels of tear lipocalin has been reported in meibomian gland dysfunction (Yamada et al. 2005), keratitis

(Ananthi et al. 2008) and increased levels were observed in non-tolerant contact lens wearers (Glasson et al. 2003). sIgA is the major immunoglobulin in tears (Little et al. 1969), and an essential immune defence factor against infections. It is secreted into the tears by plasma cells in the

Changes to the tear film proteome in keratoconus 22 Chapter 1: Introduction adenoid or epithelial layer of the conjunctiva in addition to sub-epithelial cells in the lacrimal gland (Franklin et al. 1973). Decreased levels of tear sIgA have been reported in dry eye (Mackie & Seal 1984) and, trachoma (Sen et al. 1977), and raised levels in herpetic keratitis (Pramod et al. 1999).

Serum albumin is a plasma derived protein detectable in tears due to its leakage from conjunctival blood vessels (Fukuda et al. 1996), and not due to neural regulation of the lacrimal gland. In tears it serves as a marker for the integrity of blood tear barrier

(Strasser & Grabner 1982). Elevated levels of serum albumin in tears have been reported in dry eye (Bron & Mengher 1989; Versura et al. 2010) and acute adenovirus conjunctivitis (Gupta et al. 1988).

Tear proteins are secreted either constitutively or regulated by the flow of the aqueous component of tears. Lactoferrin, as well as lysozyme and tear lipocalin, are regulated proteins and, their concentration remains almost stable in basal and reflex tears (Fullard

& Snyder 1990; Fullard & Tucker 1991). sIgA is the main constitutively produced tear protein and its concentration decreases in reflex tears or upon stimulation of tear flow

(Fullard & Snyder 1990; Fullard & Tucker 1991).

The levels of serum derived proteins such as albumin, immunoglobin G and transferrin were decreased substantially in reflex tears compared to basal tears (Fullard & Tucker

1991). In closed eye tears, the levels of total protein, sIgA and serum albumin were increased compared to open eye and reflex tears (Sack et al. 1992). The amounts of lactoferrin, lysozyme and tear lipocalin were observed to be relatively constant between open or closed eye tears or reflex tears (Sack et al. 1992). In other words, the predominant tear proteins in open eye and reflex tears were lactoferrin, lysozyme and

Changes to the tear film proteome in keratoconus 23 Chapter 1: Introduction lipocalin and, sIgA is the major protein present in closed eye tears (Sack et al. 1992).

Apart from the major tear proteins, the tear film is comprised of an abundance of proteins, proteases and protease inhibitors in low concentration. Investigating these proteases and inflammatory molecules could be critical in providing insights into ocular pathologies (de Souza et al. 2006).

1.5. Proteases

Proteases are enzymes that cleave or break down other proteins and can be involved in the degradation of extracellular matrix proteins such as collagens or activation of cellular apoptosis (Ollivier et al. 2007). The proteases are also known as proteinases or proteolytic enzymes or peptidases and, these enzymes are collectively termed hydrolases since they hydrolyse the peptide bonds of proteins (Wilkesman & Kurz

2009).

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases. MMPs, also called matrixins, were first described by Gross and Lapiere in the tail of metamorphosing tadpoles (Gross & Lapiere 1962). They belong to a large family of proteases known as the metzincin superfamily and are capable of cleaving components of ECM such as collagen and elastin (Nagase & Woessner 1999). MMPs are produced as an inactive zymogen (Harper et al. 1971) and the conversion of the zymogen (pro-

MMP) to active MMP involves proteolysis of the propeptide that obstructs the active site of the protein (Murphy et al. 1999).

MMPs are primarily synthesized by cells of the connective tissue (Murphy et al. 1991).

Human synovial fibroblasts and endothelial cells also express MMPs (Hanemaaijer et al. 1998). In the human cornea, MMPs are secreted by epithelial cells, stromal cells and

Changes to the tear film proteome in keratoconus 24 Chapter 1: Introduction neutrophils (Fini et al. 1992). Twenty three MMPs have been identified and they participate in wound healing, differentiation, apoptosis, host defence and angiogenesis

(Woessner 2002; Nagase et al. 2006; Murphy & Nagase 2008). The various types of

MMPs and their substrate specificity are illustrated in Table 1.2. Raised levels of MMP-

1, -3 -9 and -10 have been reported in tears of vernal keratoconjunctivitis (VKC) sufferers (Leonardi et al. 2009) and MMP-8 in the tears of atopic blepharoconjunctivitis

(ABC) subjects (Maatta et al. 2008).

MMPs are inhibited by tissue inhibitors of matrix metalloproteinase (TIMP) which comprise a family of four protease inhibitors TIMP-1, -2, -3 and -4. TIMPs form tight

1:1 complexes with MMPs, except for the rather weak interaction between TIMP-1 and

MT (membrane type)-MMPs. TIMPs show little discrimination between the various

MMPs (Bode et al. 1999).

In addition to MMPs, proteases such as cathepsins can degrade the ECM. Cathepsins are lysosomal (aspartyl and cysteine) and neutrophilic proteases (Skrzydlewska et al.

2005), with cathepsin G being the only neutrophilic protease, and cathepsin D, the only aspartyl protease in this group (Kenney et al. 1989). The cysteinyl group makes up the majority of cathepsins (B, C, F, H, K, L, O, S, V, W and X/Z) (Turk et al. 2000) and these have been shown to localize within cell membranes and intracellular lysosomes

(Mohamed & Sloane 2006). The substrate specificity and functions of the cathepsins change with their localization. These cathepsins are known to cleave ECM proteins such as type IV collagen (Buck et al. 1992), laminin (Ishidoh & Kominami 1995), tenascin C

(Mai et al. 2002) and cell-adhesion proteins (Gocheva et al. 2006).

The proteases present in the tear film have been suggested to be secreted mainly by the corneal and conjunctival epithelial cells (Bhuyan & Bhuyan 1970). The concentration

Changes to the tear film proteome in keratoconus 25 Chapter 1: Introduction of different proteases might be subjected to individual variation and the type of tears collected. Closed-eye tears have been shown to have high levels of proteases such as

MMP-9, cathepsin B and elastase and protease inhibitors such as alpha (α)-1 protease inhibitor, α-1 antichymotrypsin and α-2 macroglobulin. The levels and activities of these proteases and protease inhibitors are significantly decreased in reflex tears (Sathe et al. 1998; Sack et al. 2000). The amount of α-1 antitrypsin, α-2 macroglobulin have been increased in the tears of people with corneal ulcers (Prause 1983). The increased tear proteolytic activity in subjects with corneal ulcer has been reported to be due to the presence of plasmin (Salonen et al. 1987). Normal tears have detectable levels of plasmin and its activity is elevated in the tears of people presenting with allergic conjunctivitis and herpetic keratitis (Salonen et al. 1987; Berta et al. 1990).

Changes to the tear film proteome in keratoconus 26 Chapter 1: Introduction

Table 1.2: Classification of MMPs.

MMPs Enzymes Types Substrate specificity MMP - 1 (Collagenase-1), Cleaves interstitial collagen types I, II, III, VIII, X and gelatin (Madlener et al. 1998; Snoek-van Beurden & Von den Hoff 2005). Collagenases MMP - 8 (Collagenase-2), MMP-13 (Collagenase-3) Gelatinases MMP - 2 (Gelatinase A), Degrades gelatin, collagen types III, IV, V, VII, X and XI, elastin, laminin, fibronectin, and proteoglycan (Van den Steen et al. 2002). MMP - 9 (Gelatinase B)

Stromelysins MMP-3, -10, -11 Digest collagen IV and fibronectin, proteoglycans, laminin, fibronectin (Alexander & Werb 1989; Barksby et al. 2006; Shiomi et al. 2010). Matrilysins MMP-7, -26 Digest collagen IV, fibronectin and gelatin (Alexander & Werb 1989; Snoek-van Beurden & Von den Hoff 2005). Enamelysin MMP-20 Degrades amelogenin, aggrecan, cartilage oligomeric matrix protein (Murphy & Knauper 1997). Metalloelastase MMP-12 Cleaves collagen type IV, fibronectin, laminin, entactin, proteoglycans (Shipley et al. 1996). MT- MMPs MMP-14, -15, -16, -17, -24, -25 Digest collagen types I, II, III, gelatin, fibronectin, laminin, aggrecan, tenascin, perlecan, fibrin, fibrillin (Murphy & Knauper 1997; Shiomi et al. 2010). Others MMP-19, -21, -23, -27, -28 Cleave substrates such as elastin and aggrecan (Snoek-van Beurden & Von den Hoff 2005).

MMP- Matrix metalloproteinase

Changes to the tear film proteome in keratoconus 27 Chapter 1: Introduction

The following sections reporting the literature search on the role of proteases and inflammatory molecules in the aetiology of KC have been published as a review article

(Balasubramanian et al. 2010) and are presented here with permission from the journal

Current Eye Research (Confirmation number: 2873270412450).

1.5.1. KC and MMPs

Studies have demonstrated that the collagenase activity of cultured corneal buttons from KC were higher than normals (Kao et al. 1982; Rehany et al. 1982), while an increase in gelatinase activity was observed in KC corneal cells derived from KC patients (Kenney et al. 1989). However, subsequent studies showed no differences in the levels of MMP-2 and MMP-9 in KC and control corneas (Fini et al. 1992; Zhou et al. 1998) but a change in TIMP-1 level was observed. A decrease in TIMP in KC was found and an increase was seen in MMP/TIMP ratio (Kenney et al. 1994).

KC subjects with a clear cornea have been reported to have low levels of TIMPs, while upon scarring, TIMPs are over-expressed in the cornea (Smith et al. 2006). In other words, TIMP-1 may not be sufficiently expressed in the early stages of KC, and its expression increases as the disease progresses. Kenney et al. showed that the concentration of TIMP-1 and TIMP-2 were increased in scarred corneas of KC patients

(Kenney et al. 1998). The imbalance of TIMP-1 and -3 has also been shown to enhance keratocyte apoptosis in KC (Matthews et al. 2007), and studies have revealed that during KC, apoptosis is the major form of cell death of keratocytes (Kim et al. 1999;

Kaldawy et al. 2002). Apoptosis in KC may be caused by low levels of TIMPs, raised levels of leukocyte common antigen related protein (LAR), and mechanical trauma to the corneal epithelium (Cristina Kenney & Brown 2003).

Changes to the tear film proteome in keratoconus 28 Chapter 1: Introduction

As KC is a slowly progressing disease, only minute levels of enzyme activity might be needed for progression of the disease. MMP activities may not be continuously expressed and hence the over-expression of MMPs has not been demonstrated consistently (Alexander & Werb 1989). Collier et al. found that the expression of membrane type MMP (MT1-MMP) was elevated in KC corneas (Collier et al. 2000).

MT1-MMP can activate MMP-2 (Murphy et al. 1994; Sato et al. 1994) and directly degrade collagen type I, II, and III, fibronectin, tenascin, laminin-1, vitronectin and perlecan, (all parts of the ECM) (Heusel et al. 1991; Strongin et al. 1995; Ohuchi et al.

1997; Chwieralski et al. 2006). Induction of MT1-MMP expression in-vitro by concanavalin A resulted in the activation of pro-MMP-2 to form active MMP-2 (Collier et al. 2000). Therefore MT1-MMP could be involved in the matrix degradation by

MMP-2 activation and direct degradation.

A study by Sutton et al. has demonstrated increased expression of secretory frizzled- related protein 1 (SFRP1) in the epithelium of KC patients (Sutton et al. 2010) and these proteins have been linked to changes in MMP levels (Foronjy et al. 2010). The studies investigating the role of MMPs in KC are summarized in Table 1.3.

1.5.2. KC and Cathepsins

Another possible explanation for increased gelatinase activity seen in corneal cells from

KC (Kenney et al. 1989) is the activation of MMPs by proteases such as cathepsins

(Murphy et al. 1994), or direct degradation of the corneal collagen by cathepsins. In

1997, Whitelock et al. measured the mRNA level of cathepsin G in the KC cornea and demonstrated elevated levels of cathepsin G transcript (Whitelock et al. 1997). A subsequent study demonstrated increased levels of the cathepsin B and G proteins in KC corneas (Zhou et al. 1998). Cathepsin B and G are localized in the corneal epithelium

Changes to the tear film proteome in keratoconus 29 Chapter 1: Introduction

(Collier 2001) (Im & Kazlauskas 2007) and are known to degrade collagen and proteoglycans (Neurath & Walsh 1976; Alexander & Werb 1989). Regulation of these proteins is not understood but binding sites for the transcription factor Sp1 are present in the promoter regions of cathepsins B and G (Berquin & Sloane 1996) and Sp1 activity is known to be upregulated in KC (Whitelock et al. 1997).

Cathepsin K is expressed by the surface epithelial layer at the corneal-scleral interface

(Haeckel et al. 1999) and its protein levels are increased in KC cornea (Mackiewicz et al. 2006). The levels of cathepsin K in cultures of KC corneal fibroblast decreased in neutral pH and increased in low pH conditions when compared to normal corneal fibroblast cultures (Chwa et al. 2008). Thus, the increased gelatinase activity seen in KC might be due to the elevated cathepsin B and G instead of MMPs, as was thought previously (Zhou et al. 1998). Furthermore, cathepsins are said to trigger apoptotic cell death (Chwieralski et al. 2006) and, as mentioned before, apoptosis is the major form of cell death of keratocytes during KC. Studies showing the involvement of cathepsins in

KC are summarized in Table 1.3.

1.6. Inflammatory molecules

Inflammatory molecules, or cytokines, are released from cells or tissues in response to irritants, pathogens and damaged cells. The important functions of the cytokines include cell proliferation, differentiation, adhesion and migration to initiate wound healing

(Ferrero-Miliani et al. 2007). Cytokines are multipotent peptides expressed by cells of the ocular surface (Nakamura et al. 1998). Interleukins (IL) (types 1 to 35) and tumor necrosis factor (TNF-α, -β) belong to the group of cytokines regulating immune responses and inflammation. IL-1, -6, and 8 have been reported to be expressed by ocular surface cells (Nakamura et al. 1998). IL or TNF are intercellular signalling

Changes to the tear film proteome in keratoconus 30 Chapter 1: Introduction molecules acting on specific target cells mediating inflammation. The function of TNF depends on its binding to specific cell types and is known to activate the pathways for apoptosis and cellular differentiation (Balkwill 1989). Apart from ILs and TNFs, chemokines and growth factors are also included in the family of cytokines.

Chemokines or chemotactic cytokines are sub-divided into CXC (α- chemokines) and

CC (β- chemokines) groups. In vitro and in vivo studies have reported the expression of these chemokines in human corneas (Farber 1993; Tran et al. 1996; Spandau et al.

2003). Several growth factors such as epidermal growth factor (EGF), fibroblast growth factor (FGF), transforming growth factor (TGF), keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF), endothelin-1 (ET-1), leukemia inhibiting factor (LIF) and macrophage colony stimulating factor (M-CSF) have been expressed in the epithelial and fibroblast cell cultures of the human cornea (Schultz et al. 1988; Imanishi et al.

2000).

The type of tears collected influence the level and type of cytokines present in the tear fluid. The concentrations of IL-6, IL-8 and granulocyte macrophage colony-stimulating factor (GM-CSF) were found to be increased in tears collected upon awakening or closed-eye tears (Thakur et al. 1998). Sack et al. investigated the cytokine expression levels in open, closed-eye and reflex tears, and concluded that the levels of IL-8, EGF,

TGF, angiogenin (ANG) and monocyte chemoattractant protein (MCP)-1 were elevated in closed-eye tears compared to open-eye and reflex tears (Sack et al. 2005). In reflex tears, the level of IL-8 was found to be decreased compared to basal tears (Sonoda et al.

2006). However, in the same study, the concentration of IL‐1β, IL‐6, IL‐10, IL‐12 and

TNF‐α were not significantly different between basal and reflex tears.

Changes to the tear film proteome in keratoconus 31 Chapter 1: Introduction

In ocular pathologies, the levels of IL-4 and -5 were increased in tears of people with a proliferative type of atopic keratoconjunctivitis (AKC) (Uchio et al. 2000). Elevated levels of IL-8 (Leonardi et al. 2009) in the tear film have been reported in vernal VKC.

Tears of people with dry eye syndrome were shown to have elevated levels of IL-6 and

TNF–α (Yoon et al. 2007).

1.6.1. KC and Inflammatory Molecules

Although KC is considered a non-inflammatory disease, inflammatory molecules such as ILs and TNFs have been found in the tears of KC (Lema & Duran 2005). Wilson et al. proposed that the IL-1 system may be the cause of KC (Wilson et al. 1996), which is supported by KC stromal cells in culture exhibiting a 4-fold increase in binding sites for

IL-1 (Fabre et al. 1991). Studies of genetic association between unrelated Korean KC patients and IL-1α, IL-1β, IL-1 receptor antagonist gene polymorphins demonstrated IL-

1β polymorphins were associated with KC (Kim et al. 2008). Lema et al. found increased levels of IL-6 and TNF-α in the tears of KC eyes indicating a potential role of chronic inflammatory events in the pathogenesis of KC (Lema et al. 2009).

Studies have shown that ILs regulate the expression of MMPs. Cox et al. demonstrated that gingival fibroblasts grown with collagen can produce collagenolytic MMPs and cysteine proteinases when stimulated by IL-1 (Cox et al. 2006). A previous study observed a decrease in the levels of MMP-2 and -9 in corneas treated with anti-IL-1, suggesting that IL-1 upregulates the expression of MMP-2 and 9 (Xue et al. 2003).

TNF- has also been shown to upregulate the expression of MMP-9 (Li et al. 2001).

Kim et al. demonstrated that TGF- upregulates the production and activity of gelatinase MMP-9, collagenases MMP-1, -13 and stromelysins MMP-3, -10, -11 (Kim

Changes to the tear film proteome in keratoconus 32 Chapter 1: Introduction et al. 2004). TGF-β and IL-1 were not only increased in KC corneas but also in other diseased corneas (Zhou et al. 1996). TGF-β2 was also increased in the aqueous humour of the KC eyes (Maier et al. 2007). Studies examining the involvement of inflammatory molecules in KC are illustrated in Table 1.3.

Changes to the tear film proteome in keratoconus 33 Chapter 1: Introduction

Table 1.3 : Summary of studies that have been conducted to investigate MMPs, cathepsins and inflammatory molecules in KC.

Authors Findings (Kao et al. 1982) Higher collagenolytic and gelatinolytic activity in the stromal cells of KC corneas. (Rehany et al. 1982) Higher collagenase activity in whole corneal buttons of KC. (Ihalainen et al. 1986) Elevated type I and type IV collagenolytic activity in fibroblast cell cultures of KC. (Kenney et al. 1989) Increased gelatinolytic activity in stromal keratocytes of KC corneas. (Fabre et al. 1991) Cultured fibroblast of KC showed a 4-fold increase binding site for IL-1. (Fini et al. 1992) No difference in MMP-2 and MMP-9 levels in normal and KC corneal buttons. (Kenney et al. 1994) No difference in MMP-1, -2, -3 levels between normal and KC keratocyte cultures. Decrease in TIMP level and an increase in MMP/TIMP ratio in KC keratocyte cultures. (Zhou et al. 1996) Increased expression of IL-1 receptor in KC corneal buttons (Whitelock et al. 1997) Elevated levels of cathepsin G mRNA in the corneal epithelium of KC. (Kenney et al. 1998) No change in MMP-2 and MMP-9 in normal and KC corneal buttons. (Zhou et al. 1998) No difference in MMP-1, -2, -3, -9 levels in normal and KC corneal buttons. Increased expression of cathepsins B and G in KC corneal buttons. (Collier et al. 2000) Increased MT1-MMP and no change in MMP-2 levels in the epithelium and stroma of KC corneas. (Saghizadeh et al. 2001) MMP-3, MMP-10 unchanged in the epithelium and stroma of KC and normal corneas.

Changes to the tear film proteome in keratoconus 34 Chapter 1: Introduction

Table 1.3 (Continued) Authors Findings (Brookes et al. 2003) Higher expression cathepsin B and G in the keratocytes of KC corneas. (Lema & Duran 2005) Elevated levels of inflammatory molecules MMP-9, IL-6 and TNF-α in KC tears. (Mackiewicz et al. 2006) Strong expression of MMP-13 in the epithelium and anterior stromal keratocytes of KC corneas. (Seppala et al. 2006) Strong expression of EMMPRIN (extracellular matrix metalloproteinase inducer) in all the layers of KC corneas. Increased expression of MMP-1 in the epithelium and stroma of KC corneas. (Lema et al. 2009) Over-expressed IL-6 and TNF-α in the tears of subclinical and KC eyes. (Pannebaker et al. 2010) Increased tear expression of MMP-1 in KC. (Jun et al. 2011) Elevated levels of IL-6, -17 and decreased levels of IL-12 and TNF-α in KC tears.

Changes to the tear film proteome in keratoconus 35 Chapter 1: Introduction

The over-expression of MMPs and the presence of their active forms have not been found consistently and since most of the corneas available for study were in their final stages of KC, it becomes impossible to detect pathological events in the early stages of the disease. Some individuals appear to exhibit increased susceptibility to KC and hence it is possible that many events combine to produce the KC cornea.

KC appears to be associated with eye rubbing, atopy and contact lens wear (Section

1.3.3). The effect of eye rubbing on tear proteases or cytokines is unknown. Atopic eye diseases such as VKC and AKC have shown increased levels of IL-6 i.e., 5.1 ± 4.7 ng/mL and 1.2 ± 1.7 ng/mL respectively compared to 0.5 ± 4.7 ng/mL in normal subjects (Shoji et al. 2007). In VKC, tear levels of pro-MMP-1 (16.6 ± 13.8 ng/mL), and pro-MMP-9 (253 ± 186 ng/mL) were significantly higher compared to normal pro-

MMP-1 (0.5 ± 0.2 ng/mL) and normal levels of pro-MMP-9 (10.5 ± 0.2 ng/mL)

(Leonardi et al. 2003). The concentration of MMP-8 has been reported to be higher among the patients with ABC (545.6 ± 879.3 μg/L) than among the healthy controls

(50.4 ± 62.3 μg/L) (Maatta et al. 2008). Levels of IL-6 increased from non-detectable levels in non-contact lens wearers to 43.8 ± 5.3 pg/5μL in contact lens wearers (Schultz

& Kunert 2000). The contact lens wearers also had raised levels of tear IL-8 when compared with no contact lens wearers (122 ± 95 vs. 24 ± 17 ng/mL) (Thakur &

Willcox 2000).

These changes may be significant in the production of KC in certain individuals, as these conditions have been proposed to predispose to the disease, however, a cause and effect has not yet been established.

Mechanical trauma or oxidative stress caused to the cornea due to eye rubbing, UV radiation, contact lenses or allergy in genetically susceptible individuals could stimulate

Changes to the tear film proteome in keratoconus 36 Chapter 1: Introduction the release of IL-1 from the corneal epithelium. This may then diffuse into the stroma and interact with the highly expressed IL-1 receptor (Fabre et al. 1991; Bureau et al.

1993) of the stromal keratocytes triggering apoptosis (also caused directly by mechanical damage to the cornea).

The study of proteases in post-UV cross-linked KC corneas might be crucial in determining the role of protease inhibitors in the long-term management of KC.

Detection of KC before ‘corneal insult’ is essential and assessing the tears for biomarkers could be valuable. Proteases might be involved in the pathogenesis of KC.

Hence diagnosis of the disease using changes in the tear film proteins and proteases would enable the use of medical or conservative treatment to control the progression of the disease.

1.7. Tear proteomics

Proteomics is the study of protein level, expression and function, and their interactions with cell, tissues or organisms (James 1997). Tear proteomics is the study of complexity of proteins present in the tear film, and analysing tear proteins might generate valuable information for increased understanding of the aetiology and pathogenesis of ocular diseases. Tear proteomics could also be used for disease diagnosis and prognosis, by providing useful biomarkers. This could also help in designing better diagnostic or prognostic devices for eye care delivery to improve ocular health. Under normal conditions, only a small volume of tear fluid (<5 µL) can be collected, and this makes proteome profiling a great challenge. The normal methods of tear proteomics include qualitative evaluation using one or two dimensional polyacrylamide gel electrophoresis coupled with mass spectrometry (MS) or liquid chromatography (LC) of trypsin-derived peptides followed by MS. Using enzyme-linked immunosorbent assays (ELISA), the

Changes to the tear film proteome in keratoconus 37 Chapter 1: Introduction individual proteins of interest can be quantified in the tear film. The summary of human tear proteins elevated in various systemic and ocular pathologies is shown in Table 1.4.

Changes to the tear film proteome in keratoconus 38 Chapter 1: Introduction

Table 1.4: Examples of tear proteins up or down regulated in systemic diseases or ocular diseases, modified from (Wu & Zhang 2007; Jacob & Ham 2008).

Tear protein Regulation Pathology Investigators Lacryglobin Up Colon and prostate cancer (Evans et al. 2001) Apolipoprotein A-1 Up Diabetic retinopathy (Kawai et al. 2002) Global protein profile Up Diabetes Mellitus Type II (Herber et al. 2001) Telopeptides Up Keratoconus (Abalain et al. 2000) Aquaporin 5 Up Dry eye (Ohashi et al. 2003) Serum albumin and ceruloplasmin Up Dry eye (Mackie & Seal 1984) Secretoglobin 2A2, serum albumin, Up Contact lens-related dry (Nichols & Green-Church 2009) glycoprotein 340 and prolactin-inducible eye protein Lysozyme, lactoferrin and lipocalin Down Dry eye (Mackie & Seal 1984) β-2 microglobulin, proline rich 4, lacritin and Down Contact lens-related dry (Nichols & Green-Church 2009) secretoglobin 1D1 eye

Changes to the tear film proteome in keratoconus 39 Chapter 1: Introduction

Table 1.4 (Continued) Tear protein Regulation Pathology Investigators Proline rich protein 3 Down Dry eye (Grus et al. 1998) Eosinophil cationic protein Up Allergic conjunctivitis Epidermal growth factor Down Sjogren’s syndrome (Pflugfelder et al. 1999) Histamine Down Vernal kerato (Abelson et al. 1995) conjunctivitis Mucin-5AC Down Sjogren’s syndrome (Zhao et al. 2001) Substance P Down Corneal hypoesthesia (Yamada et al. 2000) Lactate dehydrogenase Up External eye disease (Guo & Zhang 1995) Serum albumin precursor Down Blepharitis (Koo et al. 2005) Human α- Defensins/S100 Calcium-binding Up Pterygium (Zhou et al. 2009) proteins A8 and A9 Collagen type IX Up Pseudoexfoliation (Assouti et al. 2006) syndrome Homocysteine Up Primary open angle (Roedl et al. 2008) glaucoma Glutaredoxin-related protein Up Fungal keratitis (Ananthi et al. 2008)

Changes to the tear film proteome in keratoconus 40 Chapter 1: Introduction

1.8. Thesis overview

The first case of KC was reported in a doctoral dissertation by Burchard Mauchart in

1748 and subsequently John Nottingham clearly described and distinguished KC from other ectasias of the cornea in 1854 (Nottingham 1854). However, the cause of KC remains elusive, and whether MMPs and inflammatory molecules are the major cause for KC remains to be demonstrated.

Proteases and cytokines in KC have been a topic of substantial discussion and speculation over many years and this thesis investigates the role and interactions of proteases and inflammatory molecules in the pathogenesis of KC.

This doctoral thesis aims to examine whether changes in the tear film during the disease can be useful to aid in the understanding of the pathology of KC. The majority of studies carried out on KC are in corneas in the final stages of KC and hence it becomes impossible to detect pathological events in the early stages of the disease. Our project, studying the tear film proteome of KC might help elucidate the early manifestations of the disease that could result in improved formulations and treatment options.

Biomarkers are becoming a fundamental component of medical diagnostics. Native to the ocular region, tears are a probable source for non-invasive molecular markers for ocular diseases. Many diseases associated with biomarkers in tears have been described previously (Table 1.3). The use of proteomics and peptidomics may help identify potential biomarkers in the tear film of KC.

The central hypothesis of this thesis is that, the corneal pathology during KC is likely to cause changes in proteins, proteases and inflammatory molecules to manifest in the tears of KC compared to people without KC. As corneal collagen is mainly type I,

Changes to the tear film proteome in keratoconus 41 Chapter 1: Introduction

collagenases and gelatinases such as MMP-1,-8, -13 and MMP-2,-9 respectively may be present at higher concentrations in KC tears. Cathepsins directly and indirectly degrade the ECM and hence will be elevated in KC tears. The thinning of the cornea during KC may cause subclinical inflammation which could manifest as increased levels of tear cytokines.

The work conducted to test the hypothesis in this thesis is grouped into the following chapters:

Chapter 2 examines the major tear protein changes in different stages of KC. The influence of contact lens wear and the history of atopy and eye rubbing on the tear proteins of KC were also examined.

The contents of Chapter 2 has been published (Balasubramanian et al. 2011) and is presented here with permission (Confirmation number: 2872300440488) from the journal Experimental Eye Research. A portion of this work has been presented as a poster at the Tear film and ocular surface society (TFOS), Florence 2010 (Appendix B).

Chapter 3 identifies the differentially expressed proteins in KC tears using mass spectrometry.

Chapter 4 investigates whether the levels and activity of the MMPs and inflammatory molecules are altered in the tears of people with KC. The effect of CXL procedure on the tear film proteases and inflammatory molecules in KC have also been examined.

The contents of Chapter 4 has been published (Balasubramanian et al. 2012) and is reproduced here with permission (Confirmation number: 2872300997031) from the journal Acta Ophthalmologica. The work conducted in this chapter has also been presented as a paper at European association for Vision and Eye Research (EVER),

Changes to the tear film proteome in keratoconus 42 Chapter 1: Introduction

Crete 2011 and as a poster at the Royal Australian and New Zealand College of

Ophthalmologists (RANZCO), Canberra 2011 (Appendix B).

Chapter 5 examines the effect of eye rubbing on the expression of proteases, proteolytic activity and inflammatory molecules in the tear film.

Chapter 6 concludes with a general summary and gives recommendations for future studies.

List of the study groups with total number of subjects in each chapter is shown in Table

1.5.

Changes to the tear film proteome in keratoconus 43 Chapter 1: Introduction

Table 1.5 : Study groups with total number of subjects.

Chapter 1 Chapter 2 Chapter 3 Chapter 4

Levels of total protein, Differentially expressed tear Expression of proteases, Influence of eye rubbing on Groups lactoferrin, secretory IgA and proteins in KC using mass proteolytic activity and proteases, proteolysis and serum albumin spectrometry cytokines inflammatory molecules C KC C KC C KC CXL C Total 69 64 18 36 81 91 59 17 Men 32 30 10 19 42 47 26 7 Women 37 34 8 17 39 44 33 10 Mean age 37.98 36.23 36.60 36.73 30.77 27.62 26.82 32.58 ± ± ± ± ± ± ± ± (years) 9.21 11.98 9.85 13.12 7.60 6.51 4.83 7.95

C-Controls; KC-Keratoconus; CXL-Cross-linked

Changes to the tear film proteome in keratoconus 44 Chapter 2: Levels of lactoferrin, secretory immunoglobulin A and serum albumin in the tears of keratoconus patients

2. CHAPTER 2: LEVELS OF LACTOFERRIN, SECRETORY IMMUNOGLOBULIN A AND SERUM ALBUMIN IN THE TEAR FILM OF KERATOCONUS PATIENTS

Changes to the tear film proteome in keratoconus 45 Chapter 2: Levels of lactoferrin, secretory immunoglobulin A and serum albumin in the tears of keratoconus patients

2.1. Introduction

KC is a progressive, debilitating disease of the eye affecting the quality of vision. It occurs generally in young adults during the productive period of their lives. Studies have indicated the possible association of KC with contact lens wear (Lowell & Carroll

1970; Gasset et al. 1978) and atopy (Bawazeer et al. 2000). Very little is known about the aetiology of the disease and the prognosis is difficult due to variability in outcome.

The main clinical features of KC are thinning, ectasia and scarring of the cornea. KC is characterized by thickening of corneal nerves, deposition of iron in the epithelial basement membrane (Fleischer’s ring), Vogt’s lines and distorted corneal shape

(Edrington et al. 1995). The initial changes are thought to be related to the breaking down of corneal basement membrane (Teng 1963) and degradative enzymes such as proteases might be implicated in KC. The tear film in KC has also been found to have increased levels of collagen degradation products (Abalain et al. 2000).

The cornea is an avascular structure deriving oxygen mainly from the tear film. Changes in the tear film have been studied in various ocular diseases (van Setten et al. 1991;

Smith et al. 2001; Tomosugi et al. 2005). With its complex mixture of proteins, lipids, carbohydrates and electrolytes, the tear film offers the first line of defense against pathogens and provides a smooth refractive interface for improved optical function

(Montes-Mico 2007). The major proteins found in the tear film have been described previously in Section 1.4.5.

Lactoferrin is mainly produced by the acinar cells of the lacrimal gland (Gillette &

Allansmith 1980) and plays an essential role in maintaining the integrity of the ocular surface (Flanagan & Willcox 2009). The tear film in dry eye (Mackie & Seal 1984;

Versura et al. 2010), trachoma (Rapacz et al. 1988), vernal conjunctivitis, contact lens-

Changes to the tear film proteome in keratoconus 46 Chapter 2: Levels of lactoferrin, secretory immunoglobulin A and serum albumin in the tears of keratoconus patients induced giant papillary conjunctivitis (Ballow et al. 1987) and post-operative cataract surgery (Jensen et al. 1985) have been reported to have low levels of lactoferrin. sIgA is the major immunoglobulin in tears (Little et al. 1969), secreted by the plasma cells in the adenoid or epithelial layer of the conjunctiva and the sub-epithelial cells in the lacrimal gland (Franklin et al. 1973). The tear levels of sIgA have been reported to be decreased in dry eye (Mackie & Seal 1984), trachoma (Sen et al. 1977) and increased in herpetic keratitis (Pramod et al. 1999).

Serum albumin is secreted into the tears by leakage from the conjunctival blood vessels

(Fukuda et al. 1996), and serves as a marker for the integrity of blood tear barrier

(Rodriguez et al. 2010). Elevated levels of serum albumin in tears have been reported in dry eye (Bron & Mengher 1989; Versura et al. 2010) and acute adenoviral conjunctivitis (Gupta et al. 1988).

Proteins are secreted by regulated and constitutive secretory pathways (Burgess &

Kelly 1987) (Section 1.4.5, Chapter 1). In tears, lactoferrin is a regulated protein, i.e. its concentration remains almost constant upon stimulation of tear flow and sIgA is a constitutive protein, i.e. its concentration decreases upon stimulation of tear flow

(Fullard & Snyder 1990). Tear flow rate is a variable factor and hence by analysing the levels of regulated and constitutive proteins, the nature of the tears (basal or reflex) can be established.

Recent studies have examined the tear proteome in KC (Lema et al. 2010; Pannebaker et al. 2010; Jun et al. 2011), and found differential expression of zinc-α2-glycoprotein, lactoferrin, immunoglobulin kappa chain, IL-6, MMP-1, keratins and mammaglobin B.

However, to date no studies have examined whether there are changes to other major tear proteins in KC and whether the constitutive, regulated or plasma-derived proteins

Changes to the tear film proteome in keratoconus 47 Chapter 2: Levels of lactoferrin, secretory immunoglobulin A and serum albumin in the tears of keratoconus patients are differentially affected by or contribute to KC. The present study examines the level of the regulated protein lactoferrin, the constitutive protein sIgA and the serum-derived protein albumin in tears of KC patients. In addition, the factors that may contribute to differences found in the levels of these proteins were examined.

2.2. Materials and methods

2.2.1. Ethics approval

Ethics was approved by the Human Research Ethics Advisory Panel (HREA) at the

University of New South Wales (UNSW), Sydney, Australia (approval no: 084081). All the procedures conducted in this study were in accordance with the 2000 Declaration of

Helsinki. Written informed consent was obtained from all study subjects prior to the start of the study (Appendix A).

2.2.2. Recruitment of Subjects

People of either gender, who were diagnosed with keratoconus (KC) or who did not have keratoconus (C) were recruited into this study during their routine visits to the

Optometry clinic at the University of New South Wales. The diagnosis of KC was confirmed by the investigator based on corneal topography maps which will be discussed in subsequent sections.

A total of 54 individuals (C=28, KC=26) were selected to study the total tear protein and lactoferrin levels. A further 46 subjects (C=26, KC=20) and 33 subjects (C=15,

KC=18) were recruited to study the sIgA levels and albumin levels in tears respectively.

Analysis of protein by one-dimensional (1D) gel electrophoresis was performed using

16 subjects (C=8, KC=8).

The subjects were matched for age and gender. History of contact lens wear, atopy and

Changes to the tear film proteome in keratoconus 48 Chapter 2: Levels of lactoferrin, secretory immunoglobulin A and serum albumin in the tears of keratoconus patients eye rubbing of the subjects were recorded. All the contact lens wearers (C or KC) were using rigid gas permeable (RGP) type of lenses for 2 to 3 years of duration. No participant gave a history of active allergy during the study and all the people diagnosed with KC gave a history of frequent eye rubbing. Exclusion criteria included people with any kind of ocular infections, ocular pathologies, those taking topical or systemic medications or who had undergone any form of corneal surgery. Demographics of the subjects recruited are illustrated in Table 2.1.

Changes to the tear film proteome in keratoconus 49 Chapter 2: Levels of lactoferrin, secretory immunoglobulin A and serum albumin in the tears of keratoconus patients

Table 2.1 : Demographics of C and KC subjects.

Groups Number of Subjects C KC Tear proteins Total protein Lactoferrin sIgA Serum albumin Total protein Lactoferrin sIgA Serum albumin Total 28 28 26 15 26 26 20 18 Men 14 14 11 7 13 13 9 8

Women 14 14 15 8 13 13 11 10 20-30 8 8 6 3 8 8 9 8

31-40 10 10 6 4 9 9 5 5

>40 10 10 14 8 9 9 6 5 RGP lens wear 18 18 11 4 19 19 14 13

Non-contact lens wear 10 10 15 11 7 7 6 5 Atopic 5 5 9 7 20 20 15 13

Non-atopic 23 23 17 8 6 6 5 5 C-Control; KC-Keratoconus; RGP-Rigid gas permeable

Changes to the tear film proteome in keratoconus 50 Chapter 2: Levels of lactoferrin, secretory immunoglobulin A and serum albumin in the tears of keratoconus patients

2.2.3. Collection of Basal tears

Subjects were seated for the tear collection procedure. The subjects were instructed to tilt their head towards the side of tear collection and look in the opposite direction. The head tilt facilitates tear flow towards the lateral canthus of the eye. Looking in the opposite direction helps the subjects not to be aware of the tear collection procedure, which is essential for the collection of basal tears.

A 10 µL calibrated, transparent glass micro capillary tube (BLAUBRAND® intraMARK, Wertheim, Germany) was placed at the inferior cul-de-sac close to the lateral canthus of the subject (Figure 2.1a). The blunt polished end of the micro capillary tube was held by the investigator’s index finger and the thumb wearing protective gloves. The other hand of the investigator was used for immobilizing the subject’s head.

Special care was taken not to touch the ocular surface or the eye lids during tear collection. The subjects were also instructed not to blink while the glass capillary tube was resting at the lateral canthus. These precautions were taken to prevent reflex secretion of tears.

A minimum of 5-7 µL of basal tears was collected from both eyes by the same investigator for every subject (C or KC). Tears were collected only until the orange band on the tube, 1.5 mm from the collecting end, which is equivalent to a volume of

1.67 µL (Figure 2.1b). Around 6 to 7 tubes were used for tear collection from each eye to collect the minimum amount of tears. This procedure was followed to ensure collection of only basal tears with a steady tear flow rate of < 1 µL/min (Fullard &

Snyder 1990). The average time taken for collecting tears was 20-25 min for each eye.

Changes to the tear film proteome in keratoconus 51 Chapter 2: Levels of lactoferrin, secretory immunoglobulin A and serum albumin in the tears of keratoconus patients

Tear collection was carried out with contact lenses in place for contact lens wearers to avoid the possibility of reflex tearing or tear contamination during the removal of contact lenses. Collection of tears was immediately suspended at the first sign of reflex tearing. The tear fluid collected in the glass tube by the principle of capillary action was expelled into a 0.5 mL tube (Scientific Specialities Inc., CA, USA) using a small manual rubber pump (Figure 2.1c). The tear samples were centrifuged at 5000 rpm for

10 min at 4 °C to remove debris or cells (Sitaramamma et al. 1998). After centrifugation, the tear fluid was transferred into a 0.65 mL siliconized polypropylene micro centrifuge tube (Sigma-Aldrich, Steinheim, Germany). Parafilm was used to seal the lids of the tube. The samples were stored at -70 °C until used for analysis

(Sitaramamma et al. 1998).

Figure 2.1: (a) Tear sample collection using a thin polished glass micro capillary tube placed at the lateral canthus of the eye (b) Polished glass micro capillary tube (c) Rubber pump to expel the tears. Photo courtesy of Brien Holden Vision Institute, Sydney, Australia.

2.2.4. Corneal topography

The assessment of the shape of the corneal curvature was made using corneal topography maps. The simulated keratometry reading of the corneal curvature was recorded using a Medmont Corneal topographer E300 (Medmont Pty Ltd, Vermont,

Changes to the tear film proteome in keratoconus 52 Chapter 2: Levels of lactoferrin, secretory immunoglobulin A and serum albumin in the tears of keratoconus patients

Victoria 3133, Australia).

The Medmont corneal topographer E300 is an automated computerized video- keratometer (Figure 2.2). It is commonly used to assess the surface of the cornea by the principle of Placido’s disk. The Placido’s disk consists of a series of 32 regular placido rings illuminated by red light-emitting diodes. These illuminated rings are placed in front of the cornea. The image of the concentric rings which is reflected off the cornea gives an image of the surface regularity or irregularity of the corneal surface (Mejia-

Barbosa & Malacara-Hernandez 2001). These images are recorded by a video camera and, the computerised software in-built with the corneal topographer constructs the curvature maps with reference of up to 15120 measurement points. According to the manufacturer, the accuracy of the Medmont E300 is < ± 0.1 D and only maps with a score of above 95% were included in the analysis. Calibration of the instrument was checked prior to taking the measurements as recommended by the manufacturer.

The subjects were requested to rest their chin and forehead on the chin rest and the forehead strap respectively of the Medmont topographer (Figure 2.2). Instruction was given to the subjects to look straight ahead into the placido’s rings in the instrument without blinking for few seconds.

The steepest simulated keratometry reading of the corneal curvature (K2) was recorded on KC patients and normal or control (C) subjects without contact lenses in place. The difference between the mean inferior and superior (I-S) power in the cornea was used to verify the diagnosis of keratoconus (Rabinowitz 1998). KC patients were grouped into mild (<45D), moderate (45-52D) and severe (>52D) stages according to the steepest keratometry reading (K2) (Zadnik et al. 1998).

Changes to the tear film proteome in keratoconus 53 Chapter 2: Levels of lactoferrin, secretory immunoglobulin A and serum albumin in the tears of keratoconus patients

Figure 2.2: Examination of the corneal curvature using Medmont corneal topographer. Photo courtesy of Brien Holden Vision Institute, Sydney, Australia.

2.2.5. SDS-PAGE

Sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis (PAGE) is the most common and an effective laboratory technique used to analyse samples containing complex proteins. To separate tear proteins, one dimensional electrophoresis (1D) was performed with SDS-PAGE using the Laemmli system (Laemmli 1970). The complex tear proteins were separated in the gel system, based on their molecular weights.

The tears from all C and KC subjects were pooled separately for the analysis, based on similarities in the steepest keratometry reading of the corneas within each group.

Undiluted pooled tears from C (4 µL) or KC subjects (4 µL) were mixed with 5x sample buffer (10% SDS, 10 mM beta-mercapto-ethanol, 20% v/v glycerol, 0.2M Tris-HCL,

0.05% w/v bromophenol blue, ph 6.8) in the ratio 4:1. The mixture was heated at 65 oC for 10 min. Samples were loaded onto 4-12% NuPAGE® Novex 4-12% Bis-Tris gel. A lane with molecular weight standards (Precision Plus Protein™ Dual Xtra Standards,

Bio-Rad, Gladesville, New South Wales, Australia) was included. Electrophoresis was

Changes to the tear film proteome in keratoconus 54 Chapter 2: Levels of lactoferrin, secretory immunoglobulin A and serum albumin in the tears of keratoconus patients carried out at 150 V in 1x running buffer (50 mM MES, 50 mM Tris Base, 0.1% SDS, 1 mM EDTA, pH 7.3) until complete. After the run, the gel was stained with Coomassie brilliant blue R-250 solution from Biorad (0.1 % w/v in 50% v/v methanol and 10% v/v acetic acid) and destained (10% (v/v) acetic acid, 10% (v/v) methanol) to visualize the bands. The TotalLab Quant software, version 12.2 was used to analyse the peaks and densitometry of the individual bands in the gel.

2.2.6. Total protein concentration of tears

Bicinchoninic acid (BCA) protein assay (Smith et al. 1985) was used to determine the total protein concentration in 2 µL of unpooled tear samples from individual C or KC subjects. Bovine serum albumin (BSA) standard curve was plotted by preparing serial dilutions of BSA (Figure 3). The tear samples (1:10 dilution) were mixed with BCA solution (Pierce BCA kit, Thermo Scientific, Scoresby, Victoria, Australia) and the absorbance was measured at 562 nm using spectrophotometer (Tecan Spectrofluoro

Plus; Tecan Group Ltd., Männedorf, Switzerland). The levels of total protein in individual tears were estimated against the standard BSA curve (Figure 2.2).

Changes to the tear film proteome in keratoconus 55 Chapter 2: Levels of lactoferrin, secretory immunoglobulin A and serum albumin in the tears of keratoconus patients

Figure 2.3: Standard curve plot of serial dilutions of BSA.

2.2.7. Quantification of lactoferrin, sIgA and serum albumin in tears

The levels of lactoferrin, sIgA and serum albumin in unpooled individual tear samples were determined by using commercially available ELISA kits (Hycult Biotechnologies,

Uden, Netherlands; Immundiagnostik, Bensheim, Germany; Bethyl Laboratories,

Montgomery, USA respectively). The manufacturer’s recommendation was followed for all the assays. Tear samples were mixed with reagent diluents (provided in the assay kits) at 1:250,000 dilution for lactoferrin, 1:15,000 for sIgA and, 1:150 for measuring the levels of serum albumin. The sensitivity of the assay kits for lactoferrin, sIgA and serum albumin were 0.4 ng/mL, 13.4 ng/mL and 6.25 ng/mL respectively, as mentioned by the manufacturers. Samples and standards were incubated in microtitre well plates, coated with antibodies specific to lactoferrin or sIgA or serum albumin. Biotinylated secondary antibody and streptavidin-peroxidase conjugate were then sequentially added to the wells. The substrate (tetramethylbenzidine) was added. This substrate reacts with the streptavidin conjugate and the resulting coloured solution was read at 450 nm using

Changes to the tear film proteome in keratoconus 56 Chapter 2: Levels of lactoferrin, secretory immunoglobulin A and serum albumin in the tears of keratoconus patients spectrophotometer (Tecan Spectrofluoro Plus).

2.2.8. Statistical Analysis

The results obtained were expressed as mean ± standard deviation. Significant differences between the groups i.e., between C and KC, were determined by using

Student’s t-test. One-way ANOVA was used to assess the significant differences within the groups. Linear regression analysis was used to correlate the keratometry readings with the total and individual protein concentrations. SPSS predictive analytics software, version 18 was used for all the analysis and a p value of < 0.05 was considered significant.

2.3. Results

The proportion of KC subjects in each group, based on the severity of the disease is listed in Table 2.2. KC subjects recruited belonged to mild (<45D), moderate (45-52D) and severe (>52D) stages of the disease.

Table 2.2: Percentage (%) of KC subjects in each group based on disease severity.

% of KC subjects used for analysis Stages of KC Total protein Lactoferrin sIgA Serum albumin Mild (<45D) 15.4 11.6 10 11.2 Moderate (45-52D) 30.8 26.9 35 50 Severe (>52D) 53.8 61.5 55 38.8

2.3.1. SDS-PAGE

The tear proteomic patterns of normal and keratoconus tear are illustrated in Figure 2.4.

Changes to the tear film proteome in keratoconus 57 Chapter 2: Levels of lactoferrin, secretory immunoglobulin A and serum albumin in the tears of keratoconus patients

Equal volumes (4 µL) of tears from C (lane 2) and KC (lane 3) as well as molecular weight standards (lane 1) were run on 4-12 % Bis-Tris gel (Figure 2.4). Densitometry or pixel position of the individual bands in C lane were more pronounced compared to the

KC lane, indicating a decreased tear protein level in KC subjects (Figure 2.5).

Figure 2.4: 1D SDS-PAGE of tears from KC patients and C. Standard markers were loaded in Lane 1. C and KC tear samples were loaded in Lane 2 and Lane 3 respectively.

Changes to the tear film proteome in keratoconus 58 Chapter 2: Levels of lactoferrin, secretory immunoglobulin A and serum albumin in the tears of keratoconus patients

Figure 2.5: Comparison of the densitometry of the bands between C (Lane 2) and KC (Lane 3).

2.3.2. Total tear protein concentration

The total tear protein concentration in C and KC groups is shown in Table 2.3. KC patients had a significantly lower level of total tear protein (3.86 ± 1.62 mg/mL) compared with the level in the C (normal) group (7.00 ± 1.58 mg/mL) (p < 0.0001).

This was approximately a two-fold decrease in total protein concentration in the KC group compared to C group. Gender, age and atopy did not appear to influence the total protein concentration in either KC or the C group. RGP contact lens wear did not modify the protein level in either of the groups (Table 2.3).

The correlation between keratometry readings and total tear protein level in C or KC subjects were studied and the results are given in Figure 2.6. A significant (p < 0.0001) negative correlation was found between total tear protein and keratometry reading by simple regression analysis. The correlation coefficient associated with the total protein concentration and keratometry reading was, r = -0.71.

2.3.3. Tear lactoferrin, sIgA and serum albumin levels

The tear lactoferrin, sIgA and serum albumin concentration in C and KC groups are

Changes to the tear film proteome in keratoconus 59 Chapter 2: Levels of lactoferrin, secretory immunoglobulin A and serum albumin in the tears of keratoconus patients shown in Table 2.3. KC patients had a significantly lower level of lactoferrin and sIgA when compared to the control group (p < 0.0001). The decrease in serum albumin level in KC group was not significant (p > 0.05) compared to the C group. There was no influence of gender, age, atopy or contact lens (RGP) wear on lactoferrin, sIgA and serum albumin concentration in the tears of either KC or the C group (Table 2.3).

The correlation of keratometry with lactoferrin and sIgA are shown in Figure 2.7 and

Figure 2.8 respectively. A significant negative correlation was found with sIgA (r = -

0.40, p < 0.001) and lactoferrin (r = -0.46, p < 0.0001) when compared to keratometry.

There was no significant correlation (r = -0.15, p > 0.05) between keratometry and the tear concentration of albumin (Figure 2.9).

Changes to the tear film proteome in keratoconus 60 Chapter 2: Levels of lactoferrin, secretory immunoglobulin A and serum albumin in the tears of keratoconus patients

Table 2.3: Levels of total protein, lactoferrin and sIgA in the tears of C and KC subjects.

Groups Total protein (mg/mL) Lactoferrin (mg/mL) sIgA (mg/mL) Albumin (µg/mL) Subjects C KC p-value C KC p-value C KC p-value C KC p-value Total 7.00±1.58 3.86±1.62 0.0001 1.13±0.29 0.67±0.28 0.0001 1.70±0.66 0.78±0.36 0.0001 11.66±8.20 8.18±4.72 0.14 Men 7.30±1.46 3.82±1.56 0.0001 1.21±0.36 0.75±0.26 0.001 1.87±0.64 0.85±0.44 0.0001 14.18±10.50 8.91±4.62 0.22

Women 6.64±1.67 3.90±1.75 0.0003 1.07±0.21 0.60±0.29 0.0001 1.53±0.58 0.73±0.30 0.0003 10.28±7.69 6.28±2.48 0.14 20-30 yrs 7.10±1.22 3.87±1.67 0.0005 1.06±0.27 0.74±0.13 0.01 2.03±0.68 0.79±0.38 0.0005 8.51±2.68 5.95±2.63 0.18

31-40 yrs 6.80±1.91 3.50±1.88 0.001 1.28±0.28 0.67±0.35 0.004 1.50±0.50 0.57±0.30 0.005 10.18±6.17 6.36±1.70 0.22

> 40 yrs 6.65±1.40 4.02±0.08 0.0001 0.99±0.28 0.56±0.19 0.001 1.67±0.78 0.94±0.42 0.04 10.18±5.27 10.67±8.34 0.88 RGP wear 7.18±2.34 3.82±0.55 0.0001 1.13±0.21 0.72±0.10 0.0001 1.64±0.52 0.82±0.34 0.0001 10.18±2.95 8.33±5.51 0.63

Non-contact 6.93±1.43 4.10±1.01 0.0004 1.14±0.32 0.56±0.35 0.002 1.68±0.69 0.67±0.45 0.007 14.60±8.60 7.74±2.62 0.10 lens wear Atopic 7.18±2.34 3.99±1.53 0.0006 1.14±0.30 0.73±0.48 0.0003 1.61±0.69 0.80±0.39 0.001 9.24±4.38 8.56±5.64 0.48

Non-atopic 6.93±1.43 2.88±1.06 0.0001 1.14±0.26 0.48±0.03 0.00001 1.69±0.67 0.73±0.20 0.005 12.33±5.90 6.74±1.75 0.10

C-Control; KC-Keratoconus; RGP-Rigid gas permeable; Results are expressed as mean ± standard deviation.

Changes to the tear film proteome in keratoconus 61 Chapter 2: Levels of lactoferrin, secretory immunoglobulin A and serum albumin in the tears of keratoconus patients

Table 2.4: Influence of gender, age, contact lens wear and atopy on the level of tear proteins.

Groups Gender Age groups (years) Contact lens wear (RGP) Atopy Men Women p- 20-30 31-40 > 40 p- Yes No p- Yes No p- value value value value

Total C 7.30±1.46 6.64±1.67 0.27 7.10±1.22 6.80±1.91 6.65±1.40 0.82 7.18±2.34 6.93±1.43 0.73 7.18±2.34 6.93±1.43 0.76 Protein KC 3.82±1.56 3.90±1.75 0.90 3.87±1.67 3.50±1.88 4.02±0.08 0.79 3.82±0.55 4.10±1.01 0.39 3.99±1.53 2.88±1.06 0.06 (mg/mL) Lacto- C 1.21±0.36 1.07±0.21 0.22 1.06±0.27 1.28±0.28 0.99±0.28 0.08 1.13±0.21 1.14±0.32 0.92 1.14±0.30 1.14±0.26 1.00 ferrin KC 0.75±0.26 0.60±0.29 0.17 0.74±0.13 0.67±0.35 0.56±0.19 0.32 0.72±0.10 0.56±0.35 0.13 0.73±0.48 0.48±0.03 0.14 (mg/mL) sIgA C 1.87±0.64 1.53±0.58 0.16 2.03±0.68 1.50±0.50 1.67±0.78 0.20 1.64±0.52 1.68±0.69 0.86 1.61±0.69 1.69±0.67 0.76 (mg/mL) KC 0.85±0.44 0.73±0.30 0.48 0.79±0.38 0.57±0.30 0.94±0.42 0.23 0.82±0.34 0.67±0.45 0.41 0.80±0.39 0.73±0.20 0.62 Serum C 14.18±10.50 10.28±7.69 0.43 8.51±2.68 10.18±6.17 10.18±5.27 0.83 10.18±2.95 14.60±8.60 0.19 9.24±4.38 12.33±5.90 0.27 albumin KC 8.91±4.62 6.28±2.48 0.15 5.95±2.63 6.36±1.70 10.67±8.34 0.22 8.33±5.51 7.74±2.62 0.78 8.56±5.64 6.74±1.75 0.39 (µg/mL)

C-Control; KC-Keratoconus; RGP-Rigid gas permeable; Results are expressed as mean ± standard deviation.

Changes to the tear film proteome in keratoconus 62 Chapter 2: Levels of lactoferrin, secretory immunoglobulin A and serum albumin in the tears of keratoconus patients

Figure 2.6: Negative correlation between total tear protein levels and corneal curvature (n=C+KC).

Figure 2.7: Negative correlation between tear lactoferrin levels and corneal curvature (n=C+KC).

Changes to the tear film proteome in keratoconus 63 Chapter 2: Levels of lactoferrin, secretory immunoglobulin A and serum albumin in the tears of keratoconus patients

Figure 2.8: Negative correlation between tear sIgA and corneal curvature (n=C+KC).

Figure 2.9: Insignificant correlation between serum albumin and corneal curvature (n=C+KC).

Changes to the tear film proteome in keratoconus 64 Chapter 2: Levels of lactoferrin, secretory immunoglobulin A and serum albumin in the tears of keratoconus patients

2.4. Discussion

The present study demonstrates altered tear protein profile in the KC subjects. The concentration of total tear protein, lactoferrin, sIgA and albumin in normal subjects in this study agrees well with that reported in the literature (Gachon et al. 1982; Toshitani et al. 1999; Flanagan & Willcox 2009).

Our initial observation, that the tears of KC subjects appeared to have reduced protein levels upon gel electrophoresis, was confirmed using a total protein assay using BCA.

Similar results indicating a reduction in total tear protein levels have been reported recently (Acera et al. 2011).

Tear proteins are secreted either constitutively or regulated by the flow of aqueous component of tears. sIgA is the main constitutively produced tear protein i.e. its concentration decreases upon stimulation of tear flow (Fullard & Snyder 1990; Fullard

& Tucker 1991). Lactoferrin, as well as lysozyme and tear lipocalin, are regulated proteins, i.e. their concentration remains almost constant upon stimulation of tear flow

(Fullard & Snyder 1990; Fullard & Tucker 1991). Therefore, we sought to determine whether there was difference in the amounts of the regulated protein lactoferrin and the constitutive protein sIgA in the tears of KC compared to C subjects. This knowledge would help to understand whether tear flow, or production of reflex tearing, had changed in the KC subjects versus the normals (C). If the ratio of lactoferrin to sIgA remained the same there would likely be no difference in tear flow rate. The ratio of lactoferrin to sIgA was 0.66 for normals and 0.86 for KC (p = 0.25, not significant) in the present study.

We observed that the tears of KC patients were deficient in both lactoferrin and sIgA.

Changes to the tear film proteome in keratoconus 65 Chapter 2: Levels of lactoferrin, secretory immunoglobulin A and serum albumin in the tears of keratoconus patients

Importantly, the levels of lactoferrin and sIgA were not influenced by RGP type of contact lens wear or atopy of the subjects. Previous studies have confirmed that tear lactoferrin levels are independent of age and gender (Kijlstra et al. 1983) or contact lens wear (Sack et al. 1992). A recent study has also demonstrated that lactoferrin in tears was down-regulated during KC, although the two dimensional gel electrophoresis results shown in that article could only demonstrate this for an isotype of lactoferrin and the results were only semi-quantitative (Lema et al. 2010).

Lactoferrin has anti-inflammatory properties and a decrease in lactoferrin levels could increase the levels of inflammatory markers present in KC tears (Lema & Duran 2005).

Being an iron binding protein, reduction in lactoferrin levels may contribute to free iron deposition in a KC cornea presenting the classical sign Fleischer’s ring (Iwamoto &

DeVoe 1976).

Reduced IgA levels might disrupt the normal immune function of the tear film in KC and studies have reported the occurrence of infectious keratitis (Donnenfeld et al. 1996), vernal conjunctivitis (Totan et al. 2001) and Acanthamoeba keratitis (Wolf et al. 2009) in KC corneas.

Metaplastic squamous epithelial cells were reported in the conjunctiva of KC patients

(Dogru et al. 2003). This might affect the production of sIgA since it is secreted into the tears by the plasma cells in the adenoid or epithelial layer of the conjunctiva in addition to sub-epithelial cells in the lacrimal gland (Franklin et al. 1973). Lower lactoferrin and sIgA levels might indicate the contribution of inflammatory and immune mediated mechanisms in the pathophysiology of KC.

Several of the protein changes, such as decreased total protein, lactoferrin and sIgA,

Changes to the tear film proteome in keratoconus 66 Chapter 2: Levels of lactoferrin, secretory immunoglobulin A and serum albumin in the tears of keratoconus patients seen with the KC subjects have also been seen associated with subjects with dry eye

(Mackie & Seal 1984). However, dry-eye is associated with increased concentration of albumin in tears (Bron & Mengher 1989; Versura et al. 2010). In this study, there was a decrease in serum albumin concentration in KC tears, although this did not reach significance (Table 2.2). These differences might be useful in differentiating KC from dry eye conditions. Increased expression of serum albumin in KC has also been reported

(Acera et al. 2011; Joseph et al. 2011). Further studies are required to determine the effect of KC on tear film albumin that would be crucial to differentiate KC from dry eye conditions.

KC is characterised by thickening of corneal nerves resulting in decreased corneal sensation (Zabala & Archila 1988; Patel et al. 2009). The neural regulation of the lacrimal gland has recently been reviewed (Dartt 2009). Stimulation of afferent, sensory corneal (and conjunctival) nerves activates efferent nerves to the lacrimal gland, thus stimulating secretion of electrolytes, water, and proteins. At low levels of sensory nerve stimulation sufficient tears are produced to cover the ocular surface as the precorneal tear film. More intense stimulation causes increased tearing.

Our results, of decreased levels of lactoferrin, lysozyme and sIgA in tears but no significant difference in the level of albumin, are in agreement with a report that showed similar changes after photorefractive keratectomy (PRK) (Fust et al. 2003). It is therefore likely that similar mechanisms are at work to reduce the concentration of certain tear proteins, and like the previous publication (Fust et al. 2003); one of the most likely explanations is changes in corneal nerves. Corneal innervation can modulate lacrimal gland tear film secretion (Dartt 2009). With PRK, the nerves are damaged and it is also known that there is degeneration of corneal nerves in KC (Teng 1963).

Changes to the tear film proteome in keratoconus 67 Chapter 2: Levels of lactoferrin, secretory immunoglobulin A and serum albumin in the tears of keratoconus patients

The degenerated nerves in KC corneas are involved in increased expression of proteolytic enzymes (Brookes et al. 2003). Several studies have reported the over expressed proteases in the corneas and tear film of KC patients (Table 1.3, Chapter 1).

The cornea is under severe oxidative stress in KC (Kenney & Brown 2003) and this also enhances the expression of proteases such collagenases and gelatinases (Shoham et al. 2008). Proteases might have an imperative role in the reduction of tear proteins, seen in the present study. Decreased tear protein concentration seen in this study reflects the lower total protein levels in the KC corneas reported previously (Rabinowitz et al.

2005).

Although many studies have examined the changes in KC corneas, very few have analysed the tear film proteins of these patients. This study for the first time has quantified total protein, lactoferrin, sIgA and serum albumin levels in KC tears and correlated these changes to the severity of the disease. Importantly, the tear protein changes observed in this study were due to the direct effect of KC and were not due to the influence of age or contact lens wear or history of atopy.

The tears of KC subjects appear to have an altered protein profile, and one that might change with the severity of the disease. Studying the tear film proteins along with corneal topography changes is essential in understanding the course of the disease.

Having investigated some of the major tear proteins, the next step was to analyse whether there are changes in other proteins or proteases in KC. The complete tear protein profiles were investigated and compared between C and KC in Chapter 3.

Changes to the tear film proteome in keratoconus 68 Chapter 3: Identification of differentially expressed tear proteins in keratoconus

3. CHAPTER 3: IDENTIFICATION OF DIFFERENTIALLY EXPRESSED TEAR PROTEINS IN KERATOCONUS

Changes to the tear film proteome in keratoconus 69 Chapter 3: Identification of differentially expressed tear proteins in keratoconus

3.1. Introduction

The constituents of the tear film could be vulnerable to changes in ocular diseases, since it is intimately associated with the ocular surface. A balance between proteases and protease inhibitors is critical to control the cell turnover rates and barrier functions of the cornea. Changes in the ocular environment might interfere with the level of proteases or protease inhibitors, thereby reflecting changes onto tear proteins or peptides

(de Souza et al. 2006).

Modern peptidomic techniques have facilitated the examination of complex mixtures consisting of proteins or peptides. The tear fluid with a heterogeneous mixture of proteins, salts and lipids, has been subjected to separation or pre-fractionation steps to isolate the proteins, prior to analysis by mass spectrometry (MS) (Nguyen-Khuong et al.

2008).

The MicroFlow 10 (MF10) (NuSeps, French Forest, Sydney, Australia) is a device used for pre-fractionating and enriching the proteins present in complex samples of low volume (Ly & Wasinger 2008). First introduced by Horvath and co-workers in 1994,

MF10 utilizes the established principles of electrophoresis to move charged molecules in a solution (Horvath et al. 1994). In addition to this new separation technique, the development of a high-powered mass spectrometer such as Linear Ion Trap Quadrupole

Fourier Transform mass spectrometer (LTQ-FT MS) has improved the precision of exploring tear proteins (Syka et al. 2004; de Souza et al. 2006).

It has been routine to use gel electrophoresis and MS to analyse the tear protein profiles in KC. Previously, altered expressions of zinc-α2-glycoprotein (ZAG), lactoferrin, and

IGKC (immunoglobulin kappa chain) (Lema et al. 2010), several keratins, lysozyme C, lipocalin (Pannebaker et al. 2010) and cystatins (Acera et al. 2011) have been reported in KC tears using gel based techniques. The present study for the first time has

Changes to the tear film proteome in keratoconus 70 Chapter 3: Identification of differentially expressed tear proteins in keratoconus evaluated the tear proteins in KC by taking a simple gel free approach using MF10 followed by LTQ-FT MS.

3.2. Materials and Methods

The study was conducted after obtaining approval from the HREA Panel at the UNSW,

Sydney, Australia (approval no: 084081). Each subject gave written informed consent before participating in the study and all the procedures were conducted complying with the tenets of Declaration of Helsinki (Appendix A).

3.2.1. Subjects

Two groups were included in the study, consisting of control subjects (C) who had not been diagnosed with KC and subjects with KC. A total of 54 subjects (C=18, KC=36) were enrolled in the study to examine the tear protein profile. Demographics of the study are illustrated in Table 3.1. The subjects (C & KC) either gave a history of no or discontinued contact lens wear for at least one month before the study. Subjects having active allergy or history of previous ocular diseases or ocular surgery or using topical or systemic medications were excluded from the study. The participants with KC gave a history of frequent eye rubbing.

Table 3.1: Demographics of the study.

Groups Tear protein profile C KC

Total 18 36 Men 10 19

Women 8 17 Age (yrs) 36.60 ± 9.85 36.73 ± 13.12 Age is expressed as mean ± standard deviation

Changes to the tear film proteome in keratoconus 71 Chapter 3: Identification of differentially expressed tear proteins in keratoconus

3.2.2. Corneal topography

The corneal curvature was mapped using a Medmont Corneal topographer E300

(Medmont Pty Ltd, Camberwell, Victoria 3124, Australia) on KC patients and C subjects similar to the technique described in Section 2.2.4.

3.2.3. Tear collection

Collection of tear samples from the study subjects was carried out according to the method described in Section 2.2.3.

3.2.4. Evaluation of tear proteins

The outline of experimental techniques followed in the present study is shown in Figure

3.1.

Changes to the tear film proteome in keratoconus 72 Chapter 3: Identification of differentially expressed tear proteins in keratoconus

Figure 3.1: Schematic diagram showing the work flow to analyse tear proteins in the present study.

3.2.5. Total protein level

The total protein concentration of C and KC groups was quantified by Bicinchoninic acid (BCA) protein assay (Smith et al. 1985). Please refer to Section 2.2.6 for more details on BCA assay.

3.2.6. Protein fractionation

The MF10 instrument was used to fractionate the tear proteins. The tears were pooled prior to fractionation. The C group had 36 µL of tears, obtained by pooling 2 µL of tears

Changes to the tear film proteome in keratoconus 73 Chapter 3: Identification of differentially expressed tear proteins in keratoconus from 18 eyes and KC group had 72 µL of tears, obtained by pooling 2 µL of tears from

36 eyes. Total protein amounts analysed in C and KC group were equivalent before fractionation.

The MF10 instrument consists of specific pore-limiting membranes or polyacrylamide membranes (Figure 3.2). Samples containing complex peptide mixtures are separated based on their size and charge using the mass restricted polyacrylamide membranes in

MF-10 under native, denaturing or reducing conditions (Ly & Wasinger 2008).

In the present study, a 6-chamber cartridge was assembled using 5, 25, 50, 75, 150 kDa polyacrylamide membranes (NuSep, Frenchs Forest, Sydney, Australia) and a 1 kDa regenerated cellulose membrane (Millipore™, Kilsyth, Victoria, Australia) as shown in

Figure 3.2. Prior to use, the 1 kDa membrane was washed in deionised water for 1 hr with 3 changes of water. The cathode end of the cartridge was fitted with a 5 kDa membrane followed by a 150, 75, 25 and 5 kDa membrane, fitting into separate cartridges. The anode end of the cartridge was fitted with a 1 kDa membrane and a 5 kDa membrane facing the anode circulating buffer. Both ends of cathode and anode were fitted with 5 kDa membranes to remove the charged contaminants from the sample during fractionation process. The resulting assembly generated six chambers for fractionation. All cartridge assemblies had two lanes of chambers that allowed fractionation of two samples in one run. The chamber cartridge assembly was placed in the MF10 separation unit (Figure 3.2). A solution containing 80 mL of 90 mM Tris/10 mM epsilon aminocaproic acid (EACA), pH 10.2 circulated around the electrodes.

Under native conditions, 35 µL of C tears in 140 µL of sample buffer (90 mM Tris/10 mM EACA, pH 10.2) were loaded into one chamber and 70 µL of KC tears in 280 µL of sample buffer were loaded on a separate run. All the remaining chambers of both lanes were filled with an equivalent volume of sample buffer. Fractionation was

Changes to the tear film proteome in keratoconus 74 Chapter 3: Identification of differentially expressed tear proteins in keratoconus performed at 50V for 30 min, 250V for 2 h (0.5 Kv Hrs) at 15˚C. After separation, fractions were collected from the chambers using gel-loading tips (Interpath Services,

Pty Ltd., Heidelberg, Victoria, Australia).

Changes to the tear film proteome in keratoconus 75 Chapter 3: Identification of differentially expressed tear proteins in keratoconus

Figure 3.2: The Microflow 10 (MF-10) separation system, modified from (Ly & Wasinger 2011).

Changes to the tear film proteome in keratoconus 76 Chapter 3: Identification of differentially expressed tear proteins in keratoconus

3.2.7. Sample preparation for LC-MS/MS

Proteins were recovered for digestion by C18 Stage tip (Thermo Fisher Scientific,

Scoresby, Victoria, Australia) for the 1 to 5 kDa, and 5 to 25 kDa fractions and by acetone precipitation for the remaining fractions to remove salt and buffer contaminants.

The stage tips and acetone precipitations are effective in concentrating the lower and higher molecular weight proteins respectively (Polson et al. 2003; Ishihama et al. 2006).

3.2.7.1. C18 Stage tip

The C18 Stage tip was equilibrated with 25 μL of 50% methanol, 5% formic acid in distilled water (dH2O) (v/v) and washed with 25 μL 5% formic acid in dH2O. Samples were slowly passed through the Stage Tip in 25 μL aliquots. The tip was washed twice with 25 μL of 5% formic acid in dH2O (v/v). Peptides were eluted with 10 μL of 80% acetone, 5% formic acid in dH2O (v/v) into a clean 1.5 mL polypropylene tube. The samples were dried completely in a vacuum centrifuge and frozen at -20 °C until further use. Lyophilized protein samples were reconstituted with 50 μL of 50 mM ammonium bicarbonate buffer pH 8.50 before trypsin digestion.

3.2.7.2. Acetone precipitation

Ice cold acetone was added to the fractions (>25 kDa) in the ratio of 4:1. The mixture was incubated at -20 °C for 1 hr. The samples were centrifuged in a 5840R centrifuge

(Eppendorf AG, Hamburg, Germany) at 11000 rpm for 12 min at 10 °C. After centrifugation the supernatant was discarded. The pellet collected by decanting the acetone was air dried for 20 min and stored at -20 °C.

3.2.8. Trypsin digestion

The individual fractions were treated with 1 µL of trypsin (1 µg/mL) and the mixture

Changes to the tear film proteome in keratoconus 77 Chapter 3: Identification of differentially expressed tear proteins in keratoconus was incubated overnight at 37 °C. The reaction was stopped by resuspending the samples in 5 µL buffer containing 0.1% formic acid. Samples were then completely dried in a vacuum centrifuge. The lyophilized samples were treated with 5 µL of 2% acetic acid and 0.1% formic acid. Equivalent amounts of fractionated samples were analysed for each group (C or KC). Fractions were examined by liquid chromatography and mass spectrometry (LC-MS/MS). 1 to 5 kDa fractions were analysed at 1 µL from

10 µL, 5 to 25 kDa at 0.5 µL from 10 µL, 25 to 50 kDa, 0.2 µL from 10 µL, 50 to 75 kDa fractions, 0.1 µL from 10 µL and the 75 to 150 kDa fraction at 0.1 µL from 10 µL.

3.2.9. LC-MS/MS using LTQ-FT instrument

The digested MF10 fractions of the protein/peptide standard mix were evaluated using a

LTQ-FT Ultra MS (Thermo Electron, Bremen, Germany). In LTQ-FT MS, the individual peptides are converted into ions (ionized peptides) for detection. Peptides were separated by nano-LC using Ultimate 3000 High Performance Liquid

Chromatography (HPLC) and auto sampler system (Dionex, Amsterdam, Netherlands).

HPLC was similar to that described previously (Krijgsveld et al. 2006). High voltage

(1800V) was applied to low volume tee (Upchurch Scientific) and the column tip positioned ~0.5 cm from the heated capillary (T=200˚C) of a LTQ-FT. Positive ions were generated by electrospray and the instrument operated in DDA mode. A survey scan of m/z 350-1750 was acquired in the Fourier Transform Ion Cyclotron Resonance

(FT ICR) cell (Resolution = 100,000 at m/z 400). Up to six of the most abundant ions (>

2000 counts) with charge states of +2 or +3 were sequentially isolated and fragmented within the linear ion trap using collisionally induced dissociation. Mass to charge ratios selected for MS/MS were dynamically excluded for 45 s. Peak lists were generated using ‘MASCOT Daemon/ extract_msn software, version 2.2 (Matrix Science, London,

Changes to the tear film proteome in keratoconus 78 Chapter 3: Identification of differentially expressed tear proteins in keratoconus

England) using default parameters and submitted to the database search program

MASCOT.

3.2.10. Search of the database

All MS/MS spectra were searched using MASCOT search engine. A minimum

MASCOT score of 50 was taken into consideration for identification of peptides. MF10 protein/peptide fractions were searched against the SwissProt database, release 15 with the following criteria: precursor tolerance and the product ion tolerances were at 6ppm and ± 0.6 Da respectively; variable modification of methionine oxidation; and no enzyme. Analysis was carried out using Scaffold proteome software, version 3

(Proteome Software, Inc., Portland, OR, USA). Scaffold is a bioinformatics tool designed to increase the confidence in protein identification in MS based proteomic studies (Searle 2010). Peptide probabilities were capped at 95% for peptide identifications and 80% protein probability with at least two peptides required for confirmation. This provided 1.50% and 0% false positive rates for peptide and protein identification respectively.

3.2.11. Statistical methods

Significant differences in total tear protein levels between the groups were analysed using Student’s t-test. The differential expression of proteins across the samples was compared between C and KC groups based on peptide spectral counts using the

Scaffold software. The relative abundance of individual proteins across the groups was calculated by spectral counting. Spectral counting compares the MS/MS spectra assigned to each protein. It is considered sensitive for detecting the proteins that undergo changes in abundance (Old et al. 2005). The spectral count correlates with the protein abundance (Liu et al. 2008). Standard deviations were automatically calculated

Changes to the tear film proteome in keratoconus 79 Chapter 3: Identification of differentially expressed tear proteins in keratoconus across replicates and the spectral counts were normalized between the groups to remove statistical errors created from repeated measurements (Beissbarth et al. 2004). The fold change in the Scaffold analytical software was used to test the significance of the ratio of spectral counts between C and KC groups.

3.3. Results

3.3.1. Corneal topography

The mean steepest keratometry reading (K2) in C and KC group was 43.7 ± 1.2 D and

57.3 ± 10.5 D respectively. KC subjects recruited belonged to mild (<45D), moderate

(45-52D) and severe (>52D) stages of the disease. The proportion of KC subjects in mild, moderate and severe stages of KC were 13.9%, 38.8% and 47.2% respectively.

3.3.2. Total tear protein level

The differences in age or gender were not statistically significant between C and KC groups. KC patients had a significantly lower level of total tear protein (3.24 ± 0.70

µg/µL) compared to the C (non-keratoconic) group (6.35 ± 1.24 µg/µL) (p < 0.0001).

3.3.3. Tear protein profile

Pooled C (35 µL) and KC group (70 µL) tear samples had equal total protein amounts before protein fractionation and mass spectroscopy. After MS, searches to identify tear proteins were performed using the human protein database UniProtKB/SwissProt

Protein Knowledge Base. Stringent search criteria yielded a total of 75 tear proteins in C and KC (Table 3.2a). The proteins were also identified in the individual tear fractions of

C and KC i.e., 1-5 kDa (Table 3.2b), 5-25 kDa (Table 3.2c), 25-50 kDa (Table 3.2d),

50-75 kDa (Table 3.2e) and 75-150 kDa (Table 3.2f).

Changes to the tear film proteome in keratoconus 80 Chapter 3: Identification of differentially expressed tear proteins in keratoconus

Table 3.2a: List of total tear proteins identified in C and KC.

Accession Entry name Identified proteins Theoretical mass number (kDa) P63104 1433Z_HUMAN 14-3-3 protein zeta/delta 28 P63261 ACTG_HUMAN Actin-2 42 Q6UY14 ATL4_HUMAN ADAMTS-like protein 4 116 Q5JTZ9 SYAM_HUMAN Alanyl-tRNA synthetase 107 P02763 A1AG1_HUMAN Alpha-1-acid glycoprotein 1 23 P01011 AACT_HUMAN Alpha-1-antichymotrypsin 48 P01009 A1AT_HUMAN Alpha-1-antitrypsin 47 P02765 FETUA_HUMAN Alpha-2-HS-glycoprotein 39 P08758 ANXA5_HUMAN Annexin A5 36 P02647 APOA1_HUMAN Apolipoprotein A-I 31 P53367 ARFP1_HUMAN Arfaptin-1 42 P61769 B2MG_HUMAN Beta-2-microglobulin 14 O43852 CALU_HUMAN Calumenin 37 P07858 CATB_HUMAN Cathepsin B 38 P10909 CLUS_HUMAN Clusterin 52 P28325 CYTD_HUMAN Cystatin D 16 P01036 CYTS_HUMAN Cystatin S 16 P09228 CYTT_HUMAN Cystatin SA 16 P01037 CYTN_HUMAN Cystatin SN 16 Q9UGM3 DMBT1_HUMAN Deleted in malignant brain tumours 260 1 protein P81605 DCD_HUMAN Dermcidin 11 O75923 DYSF_HUMAN Dysferlin 237 Q9GZZ8 LACRT_HUMAN Extracellular glycoprotein lacritin 14 P02671 FIBA_HUMAN Fibrinogen alpha chain 95 P20930 FILA_HUMAN Filaggrin 435 P22352 GPX3_HUMAN Glutathione peroxidase 3 25

Changes to the tear film proteome in keratoconus 81 Chapter 3: Identification of differentially expressed tear proteins in keratoconus

Table 3.2a (Continued)

Accession Entry name Identified proteins Theoretical mass number (kDa) P41250 SYG_HUMAN Glycyl-tRNA synthetase beta 83 subunit P49915 GUAA_HUMAN GMP synthase [glutamine- 77 hydrolyzing] P00738 HPT_HUMAN Haptoglobin 45 P04792 HSPB1_HUMAN Heat shock protein beta 1 23 P02790 HEMO_HUMAN Hemopexin 52 P01834 IGKC_HUMAN Ig kappa chain C region 12 P02533 K1C14_HUMAN Keratin, type I cytoskeletal 14 52 P13645 K1C10_HUMAN Keratin, type I cytoskeletal 10 59 P35527 K1C9_HUMAN Keratin, type I cytoskeletal 9 62 P04264 K2C1_HUMAN Keratin, type II cytoskeletal 1 66 P35908 K22E_HUMAN Keratin, type II cytoskeletal 2 65 epidermal P13647 K2C5_HUMAN Keratin, type II cytoskeletal 5 62 Q16773 KAT1_HUMAN Kynurenine-oxoglutarate 48 transaminase P22079 PERL_HUMAN Lactoperoxidase 80 P02788 TRFL_HUMAN Lactoferrin 78 P31025 LCN1_HUMAN Lipocalin-1 19 Q86X29 LSR_HUMAN Lipolysis-stimulated lipoprotein 71 receptor P61626 LYSC_HUMAN Lysozyme C 16 O75556 SG2A1_HUMAN Mammaglobin B 11 Q13421 MSLN_HUMAN Mesothelin 69 P01033 TIMP1_HUMAN Metalloproteinase inhibitor 1 23 P16035 TIMP2_HUMAN Metalloproteinase inhibitor 2 24

Changes to the tear film proteome in keratoconus 82 Chapter 3: Identification of differentially expressed tear proteins in keratoconus

Table 3.2a (Continued)

Accession Entry name Identified proteins Theoretical mass number (kDa) P05164 PERM_HUMAN Myeloperoxidase 74 P80188 NGAL_HUMAN Neutrophil gelatinase-associated 22 lipocalin Q02818 NUCB1_HUMAN Nucleobindin-1 54 P55058 PLTP_HUMAN Phospholipid transfer protein 55 P01833 PIGR_HUMAN Polymeric immunoglobulin 83 receptor Q9ULR0 ISY1_HUMAN Pre-mRNA-splicing factor ISY1 37 P07737 PROF1_HUMAN Profilin-1 15 P12273 PIP_HUMAN Prolactin-inducible protein 16 Q99935 PROL1_HUMAN Proline-rich protein 1 27 Q16378 PROL4_HUMAN Proline-rich protein 4 15 P25815 S100P_HUMAN Protein S100-P 10 Q16740 CLPP_HUMAN Putative ATP-dependent Clp 30 protease proteolytic subunit, mitochondrial Q9HBR0 S38AA_HUMAN Putative sodium-coupled neutral 120 amino acid transporter 10 Q15293 RCN1_HUMAN Reticulocalbin-1 39 Q9BUL9 RPP25_HUMAN Ribonuclease P protein subunit p25 21 O95968 SG1D1_HUMAN Secretoglobin family 1D member 1 10 P02768 ALBU_HUMAN Serum albumin 69 Q9H299 SH3L3_HUMAN SH3 domain-binding glutamic 10 acid-rich-like protein 3 Q99954 SMR3A_HUMAN Submaxillary gland androgen- 14 regulated protein 3A P10599 THIO_HUMAN Thioredoxin 12 P20061 TCO1_HUMAN Transcobalamin-1 48

Changes to the tear film proteome in keratoconus 83 Chapter 3: Identification of differentially expressed tear proteins in keratoconus

Table 3.2a (Continued)

Accession Entry name Identified proteins Theoretical mass number (kDa) Q7Z4N2 TRPM1_HUMAN Transient receptor potential cation 33 channel subfamily O75888 TNF13_HUMAN Tumour necrosis factor ligand 26 superfamily member 13 Q9BQ24 ZFY21_HUMAN Zinc finger FYVE domain- 26 containing protein 21 Q96KM6 Z512B_HUMAN Zinc finger protein 512B 97 P25311 ZA2G_HUMAN Zinc-alpha-2-glycoprotein 34 Q96DA0 ZG16B_HUMAN Zymogen granule protein 16 23 homolog B

Changes to the tear film proteome in keratoconus 84 Chapter 3: Identification of differentially expressed tear proteins in keratoconus

Table 3.2b: Proteins in 1 to 5 kDa fractions.

Accession Entry name Identified proteins Theoretical number mass (kDa) P63261 ACTG_HUMAN Actin-2 42 P01009 A1AT_HUMAN Alpha-1-antitrypsin 47 P02765 FETUA_HUMAN Alpha-2-HS-glycoprotein 39 Q9UGM3 DMBT1_HUMAN Deleted in malignant brain tumours 1 protein 260 P02671 FIBA_HUMAN Fibrinogen alpha chain 95 P13645 K1C10_HUMAN Keratin, type I cytoskeletal 10 59 P35527 K1C9_HUMAN Keratin, type I cytoskeletal 9 62 P13647 K2C5_HUMAN Keratin, type II cytoskeletal 5 62 Q9GZZ8 LACRT_HUMAN Extracellular glycoprotein lacritin 14 P02788 TRFL_HUMAN Lactoferrin 78 P31025 LCN1_HUMAN Lipocalin-1 19 P61626 LYSC_HUMAN Lysozyme C 16 P01833 PIGR_HUMAN Polymeric immunoglobulin receptor 83 Q16378 PROL4_HUMAN Proline-rich protein 4 15 P02768 ALBU_HUMAN Serum albumin 69

Changes to the tear film proteome in keratoconus 85 Chapter 3: Identification of differentially expressed tear proteins in keratoconus

Table 3.2c: Proteins in 5 to 25 kDa fractions.

Accession Entry name Identified proteins Theoretical number mass (kDa) P63261 ACTG_HUMAN Actin-2 42 P01009 A1AT_HUMAN Alpha-1-antitrypsin 47 P02765 FETUA_HUMAN Alpha-2-HS-glycoprotein 39 P61769 B2MG_HUMAN Beta-2-microglobulin 14 P28325 CYTD_HUMAN Cystatin D 16 P01036 CYTS_HUMAN Cystatin S 16 P01037 CYTN_HUMAN Cystatin SN 16 P09228 CYTT_HUMAN Cystatin SA 16 Q9UGM3 DMBT1_HUMAN Deleted in malignant brain tumours 1 protein 260 P81605 DCD_HUMAN Dermcidin 11 Q9GZZ8 LACRT_HUMAN Extracellular glycoprotein lacritin 14 P31025 LCN1_HUMAN Lipocalin-1 19 P61626 LYSC_HUMAN Lysozyme C 16 P02671 FIBA_HUMAN Fibrinogen alpha chain 95 P49915 GUAA_HUMAN GMP synthase [glutamine-hydrolysing] 77 P13645 K1C10_HUMAN Keratin, type I cytoskeletal 10 59 P02533 K1C14_HUMAN Keratin, type I cytoskeletal 14 52 P35527 K1C9_HUMAN Keratin, type I cytoskeletal 9 62 P04264 K2C1_HUMAN Keratin, type II cytoskeletal 1 66 P35908 K22E_HUMAN Keratin, type II cytoskeletal 2 epidermal 65 P13647 K2C5_HUMAN Keratin, type II cytoskeletal 5 62 P31025 LCN1_HUMAN Lipocalin-1 19 O75556 SG2A1_HUMAN Mammaglobin B 11 Q13421 MSLN_HUMAN Mesothelin 69 P01833 PIGR_HUMAN Polymeric immunoglobulin receptor 83 Q99935 PROL1_HUMAN Proline-rich protein 1 27 Q16378 PROL4_HUMAN Proline-rich protein 4 15 P02768 ALBU_HUMAN Serum albumin 69 O75888 TNF13_HUMAN Tumour necrosis factor ligand superfamily 26 member 13

Changes to the tear film proteome in keratoconus 86 Chapter 3: Identification of differentially expressed tear proteins in keratoconus

Table 3.2d: Proteins in 25 to 50 kDa fractions.

Accession Entry name Identified proteins Theoretical number mass (kDa) P53367 ARFP1_HUMAN Arfaptin-1 42 P61769 B2MG_HUMAN Beta-2-microglobulin 14 P07858 CATB_HUMAN Cathepsin B 38 P01036 CYTS_HUMAN Cystatin S 16 P81605 DCD_HUMAN Dermcidin 11 O75923 DYSF_HUMAN Dysferlin 237 Q9GZZ8 LACRT_HUMAN Extracellular glycoprotein lacritin 14 P02671 FIBA_HUMAN Fibrinogen alpha chain 95 P13645 K1C10_HUMAN Keratin, type I cytoskeletal 10 59 P35527 K1C9_HUMAN Keratin, type I cytoskeletal 9 62 P04264 K2C1_HUMAN Keratin, type II cytoskeletal 1 66 P35908 K22E_HUMAN Keratin, type II cytoskeletal 2 epidermal 65 P31025 LCN1_HUMAN Lipocalin-1 19 O75556 SG2A1_HUMAN Mammaglobin B 11 Q16378 PROL4_HUMAN Proline-rich protein 4 15 O95968 SG1D1_HUMAN Secretoglobin family 1D member 1 10 P02768 ALBU_HUMAN Serum albumin 69

Changes to the tear film proteome in keratoconus 87 Chapter 3: Identification of differentially expressed tear proteins in keratoconus

Table 3.2e: Proteins in 50 to 75 kDa fractions.

Accession Entry name Identified proteins Theoretical number mass (kDa) P63104 1433Z_HUMAN 14-3-3 protein zeta/delta 28 P02763 A1AG1_HUMAN Alpha-1-acid glycoprotein 1 23 P01009 A1AT_HUMAN Alpha-1-antitrypsin 47 P08758 ANXA5_HUMAN Annexin A5 36 P02647 APOA1_HUMAN Apolipoprotein A-I 31 P61769 B2MG_HUMAN Beta-2-microglobulin 14 O43852 CALU_HUMAN Calumenin 37 P01036 CYTS_HUMAN Cystatin S 16 P01037 CYTN_HUMAN Cystatin SN 16 Q9GZZ8 LACRT_HUMAN Extracellular glycoprotein lacritin 14 P02671 FIBA_HUMAN Fibrinogen alpha chain 95 P20930 FILA_HUMAN Filaggrin 435 P22352 GPX3_HUMAN Glutathione peroxidase 3 25 P41250 SYG_HUMAN Glycyl-tRNA synthetase beta subunit 83 P13645 K1C10_HUMAN Keratin, type I cytoskeletal 10 59 P02533 K1C14_HUMAN Keratin, type I cytoskeletal 14 52 P04264 K2C1_HUMAN Keratin, type II cytoskeletal 1 66 P13647 K2C5_HUMAN Keratin, type II cytoskeletal 5 62 Q16773 KAT1_HUMAN Kynurenine-oxoglutarate transaminase 48 P22079 PERL_HUMAN Lactoperoxidase 80 P31025 LCN1_HUMAN Lipocalin-1 19 O75556 SG2A1_HUMAN Mammaglobin B 11 Q13421 MSLN_HUMAN Mesothelin 69 P16035 TIMP2_HUMAN Metalloproteinase inhibitor 2 24 P80188 NGAL_HUMAN Neutrophil gelatinase-associated lipocalin 22 P12273 PIP_HUMAN Prolactin-inducible protein 16 Q16378 PROL4_HUMAN Proline-rich protein 4 15 Q9HBR0 S38AA_HUMAN Putative sodium-coupled neutral amino acid 120 transporter 10 O95968 SG1D1_HUMAN Secretoglobin family 1D member 1 10 P02768 ALBU_HUMAN Serum albumin 69 Q99954 SMR3A_HUMAN Submaxillary gland androgen-regulated protein 14 3A P20061 TCO1_HUMAN Transcobalamin-1 48 P25311 ZA2G_HUMAN Zinc-alpha-2-glycoprotein 34 Q96DA0 ZG16B_HUMAN Zymogen granule protein 16 homolog B 23

Changes to the tear film proteome in keratoconus 88 Chapter 3: Identification of differentially expressed tear proteins in keratoconus

Table 3.2f: Proteins in 75 to 150 kDa fractions

Accession Entry name Identified proteins Theoretical number mass (kDa) P63261 ACTG_HUMAN Actin-2 42 P01011 AACT_HUMAN Alpha-1-antichymotrypsin 48 P01009 A1AT_HUMAN Alpha-1-antitrypsin 47 P02647 APOA1_HUMAN Apolipoprotein A-I 31 O43852 CALU_HUMAN Calumenin 37 P07858 CATB_HUMAN Cathepsin B 38 P10909 CLUS_HUMAN Clusterin 52 P01036 CYTS_HUMAN Cystatin S 16 P01037 CYTN_HUMAN Cystatin SN 16 Q9GZZ8 LACRT_HUMAN Extracellular glycoprotein lacritin 14 P02671 FIBA_HUMAN Fibrinogen alpha chain 95 P04792 HSPB1_HUMAN Heat shock protein beta-1 23 P13645 K1C10_HUMAN Keratin, type I cytoskeletal 10 59 P35527 K1C9_HUMAN Keratin, type I cytoskeletal 9 62 P04264 K2C1_HUMAN Keratin, type II cytoskeletal 1 66 P35908 K22E_HUMAN Keratin, type II cytoskeletal 2 epidermal 65 P02788 TRFL_HUMAN Lactoferrin 78 P31025 LCN1_HUMAN Lipocalin-1 19 O75556 SG2A1_HUMAN Mammaglobin B 11 Q13421 MSLN_HUMAN Mesothelin 69 P80188 NGAL_HUMAN Neutrophil gelatinase-associated lipocalin 22 P55058 PLTP_HUMAN Phospholipid transfer protein 55 P01833 PIGR_HUMAN Polymeric immunoglobulin receptor 83 P12273 PIP_HUMAN Prolactin-inducible protein 16 Q99935 PROL1_HUMAN Proline-rich protein 1 27 Q16378 PROL4_HUMAN Proline-rich protein 4 15 Q15293 RCN1_HUMAN Reticulocalbin-1 39 O95968 SG1D1_HUMAN Secretoglobin family 1D member 1 10 P02768 ALBU_HUMAN Serum albumin 69 Q99954 SMR3A_HUMAN Submaxillary gland androgen-regulated protein 14 3A P20061 TCO1_HUMAN Transcobalamin-1 48 P25311 ZA2G_HUMAN Zinc-alpha-2-glycoprotein 34 Q96DA0 ZG16B_HUMAN Zymogen granule protein 16 homolog B 23 Q6UY14 ATL4_HUMAN ADAMTS-like protein 4 116 Q5JTZ9 SYAM_HUMAN Alanyl-tRNA synthetase 107

Changes to the tear film proteome in keratoconus 89 Chapter 3: Identification of differentially expressed tear proteins in keratoconus

3.3.4. Differentially expressed proteins in KC compared to C

Based on the spectral counts, cathepsin B was over-expressed in KC compared to C

(Figure 3.3a). Polymeric immunoglobulin receptor (PIGR), fibrinogen alpha chain or α- fibrinogen, cystatin S and cystatin SN were decreased in KC compared to C (Figure

3.3b & Figure 3.3c). The tear proteins associated only with KC were keratin type-1 cytoskelatal-14 (K1C14) and keratin type-2 cytoskeletal-5 (K2C5).

Fold change is the ratio of spectral counts in KC group compared to the spectral counts in C group of a particular protein. Larger fold change values imply more differences of a particular protein between the groups. The corresponding molecular weight fractions and the fold change values of the differentially expressed proteins in KC are shown in

Table 3.3.

Changes to the tear film proteome in keratoconus 90 Chapter 3: Identification of differentially expressed tear proteins in keratoconus

Figure 3.3: Tear proteins which were relatively (a) up-regulated or (b & c) down- regulated in KC compared to C.

Table 3.3: Altered tear proteins in KC compared to C.

Accession Entry name Identified Theoretical Indentified Fold no. proteins mass (kDa) fraction (kDa) change

P07858 CATB_HUMAN Cathepsin B 38 25-50 +2.7 P01833 PIGR_HUMAN PIGR 83 75-150 -9.4 P02671 FIBA_HUMAN α-Fibrinogen 95 75-150 -8.2 P01037 CYTN_HUMAN Cystatin SN 16 5-25 -2.2 P01036 CYTS_HUMAN Cystatin S 16 5-25 -2.1 P02533 K1C14_HUMAN Keratin, type I 52 50-75 1# cytoskeletal 14 P13647 K2C5_HUMAN Keratin, type II 62 50-75 1# cytoskeletal 5

Fold change is 1 in case of missing values (K1C14 and K2C5 were absent in C group).

Changes to the tear film proteome in keratoconus 91 Chapter 3: Identification of differentially expressed tear proteins in keratoconus

3.3.5. Ontology Analysis

The proteins listed in Table 3.3 were grouped based on location (Figure 3.4), biological process (Figure 3.5) and functions performed (Figure 3.6) by using the Gene ontology

(GO) term analysis in Scaffold. Several proteins belonged to more than one category within each group. The altered proteins identified in KC are mainly located in the intracellular region (Figure 3.4), implicated in cellular or developmental processes

(Figure 3.5) and carry out important enzyme regulatory or molecular functions (Figure

3.6).

Figure 3.4: Cellular compartments of the proteins altered in KC.

Changes to the tear film proteome in keratoconus 92 Chapter 3: Identification of differentially expressed tear proteins in keratoconus

Figure 3.5: Biological processes of the proteins altered in KC.

Figure 3.6: Functions of the proteins altered in KC.

Changes to the tear film proteome in keratoconus 93 Chapter 3: Identification of differentially expressed tear proteins in keratoconus

3.4. Discussion

The use of tear proteomics to understand the pathophysiology of KC has been a topic of considerable interest in recent years. In this study, the tear proteins were examined and compared in people with or without KC for differential expression.

Electrophoresis using one-dimensional (1D) or two dimensional (2D) gels are the most common techniques used to fingerprint tear proteins. Although considered as a simple and an effective technique, many proteins go undetected or lost during the various steps involved in gel-based techniques (Zhou et al. 2005). Recent advances in the field of proteomics have improved the investigation of lower abundance proteins or peptides.

Fractionation strategies are essential to improve the isolation and enrichment of lower molecular weight proteins or peptides from complex samples. The ability of the MF10 instrument to fractionate, concentrate and desalt the samples prior to MS analysis, enhances the identification of complex tear proteins present in low volumes (Nguyen-

Khuong et al. 2008).

The present study, for the first time has used MF10 to examine the tear proteins in KC.

The MF10 coupled with LTQ-FT showed relative over-expression of cathepsin B and down-regulation of PIGR, α-fibrinogen, cystatin SN and cystatin S in the tears of people with KC. Keratins such as K1C14 and K2C5 were present only in KC tears.

The tear proteins identified were present in more than one molecular weight fractions using MF10 and this might be due to the inherent ability of proteins to form complexes with other proteins or due to the influence of protease activity. Importantly, the altered tear proteins between C and KC were identified in their native molecular weight fractions according to this study (Table 3.3), which shows the efficiency of MF10 protein separation.

Cathepsin B belongs to the group of lysosomal protease. It is localized in the corneal

Changes to the tear film proteome in keratoconus 94 Chapter 3: Identification of differentially expressed tear proteins in keratoconus epithelium (Im & Kazlauskas 2007) and known to degrade the extracellular matrix

(Berquin & Sloane 1996). Although, cathepsin B has been shown to be elevated in KC corneas (Zhou et al. 1998), its expression in KC tears was not studied previously. Over- expressed cathepsin B in KC corneas might be the reason for its increased expression in the tear film reported in this study. These proteases may play a vital role in the apoptosis of keratocytes, which is the major form of cell death in KC (Kaldawy et al.

2002).

The keratins (K1C14 & K2C5) are normally found in the outer or epidermal layer of human skin and not necessarily present in the tear film. A possible explanation for the presence of keratins in KC tears could be the habit of frequent eye rubbing seen among people with KC in the present study. This result is consistent with elevated keratin levels in the corneal epithelium and tears of KC patients reported previously

(Pannebaker et al. 2010; Joseph et al. 2011).

The epithelial cells of the lacrimal gland express PIGR (Mestecky & McGhee 1987).

These receptors are essential for the transport of secretory immunoglobulin A (sIgA) into the tear film. Binding of PIGR to sIgA protects the sIgA from proteolytic degradation (Song et al. 1994). Tear fluids are rich in sIgA and low levels of PIGR might affect the concentration of sIgA in the tears of KC, thereby disrupting the normal immune function of the tear film. Low levels of tear sIgA in KC have been reported previously in Chapter 2.

Fibrinogen is an important glycoprotein present in the epithelial basement membrane of the cornea. It is a complex protein consisting of α, β and γ chains (Millin et al. 1986).

Degradation of the epithelial basement membrane is an important feature of KC and studies have demonstrated weak expression of fibrinogen in the epithelial basement

Changes to the tear film proteome in keratoconus 95 Chapter 3: Identification of differentially expressed tear proteins in keratoconus membranes of KC corneas (Newsome et al. 1981; Millin et al. 1986). Fibrinogen is involved in corneal wound healing by assisting the adhesion and migration of epithelial cells (Kao et al. 1998). This study is the first to report the down regulation of α- fibrinogen in KC tears and the role of α component of fibrinogen in KC needs further investigation.

Cystatins are a group of lysosomal or cysteine protease inhibitors. Cystatin S and cystatin SN are extracellular proteins belonging to the family of cystatins. These levels were significantly less in the KC tears compared to C. The tear film exhibits important ocular defence functions in the presence of protease inhibitors such as cystatins. These cystatins monitor the extensive degradation of the intracellular and extracellular proteins mediated by the cysteine or lysosomal proteases (Barka et al. 1991). Our results are concurrent with the decreased levels of cystatin SN and cystatin S reported previously in KC tears (Acera et al. 2011). Low levels of protease inhibitors cystatin S and cystatin

SN might increase the level of tear proteases and its activity in KC.

MF10 has been used in identifying proteins in plasma (Wasinger et al. 2005), urine

(Wasinger et al. 2008) and tear film (Nguyen-Khuong et al. 2008), but the instrument is still too developmental to be used routinely for separating proteins. Although the low abundant proteins, proteases and protease inhibitors were identified in this study, the strategies to deplete the high abundant proteins during MF10 were not used. The masking effect of one or more high abundant tear proteins such as lactoferrin, secretory

IgA (sIgA), serum albumin, lysozyme and lipocalin might reduce the chances of detecting other tear proteins which are less abundant in tears (Green-Church et al.

2008). Tear proteins present at low concentrations might provide valuable information about the ocular environment (Azzarolo et al. 2004) and future studies are essential to further uncover the tear proteins present in low concentrations in KC by depleting high

Changes to the tear film proteome in keratoconus 96 Chapter 3: Identification of differentially expressed tear proteins in keratoconus abundant proteins.

In summary, the tear proteins differentially expressed in KC are mainly intracellular and this could be indicative of an increased rate of cell turnover in KC corneas. The tears of people with KC also show increasing evidence of increased apoptotic activity in the cornea linked to increased proteases and decreased protease inhibitors. Further comprehensive studies investigating tear proteases and protease inhibitors are vital to understand the course of the disease. The next chapter examines the proteases, protease inhibitors and inflammatory molecules in KC tears.

Changes to the tear film proteome in keratoconus 97 Chapter 4: Proteases and inflammatory molecules in the tears of people with keratoconus

4. CHAPTER 4: PROTEASES AND INFLAMMATORY MOLECULES IN THE TEARS OF PEOPLE WITH KERATOCONUS

Changes to the tear film proteome in keratoconus 98 Chapter 4: Proteases and inflammatory molecules in the tears of people with keratoconus

4.1. Introduction

KC is a poorly understood degenerative disease of the cornea. Although considered not common, KC is not rare and is often under diagnosed. Screening for KC is crucial to avoid complications such as ectasia or progressive thinning of the cornea after laser assisted in-situ keratomileusis (LASIK) (Binder 2003). Once diagnosed, vision can be partially restored using contact lenses. Corneal collagen cross-linking (CXL) using

UVA/riboflavin is a technique followed to slow or stop the progression of KC

(Wollensak et al. 2003).

Type I collagen is predominant in the corneal stroma and type IV collagen is observed in the epithelial basement membrane and Descemet’s membrane (Nakayasu et al. 1986).

These collagen fibrils are interwoven by mature elastic fibres, consisting of protein elastin (Kamma-Lorger et al. 2010).

MMPs are a family of zinc dependent endopeptidases, that include gelatinases (MMP-2,

-9), collagenases (MMP-1, -8, -13), stromelysins (MMP-3, -10) and matrilysins (MMP-

7, -26) and are known to degrade collagens (Table 1.2, Chapter 1). MMPs are synthesized by corneal epithelial cells and stromal cells (Fini et al. 1998) and, are inhibited by TIMPs which include TIMP-1, -2, -3, and -4. Raised levels of MMP-1 and

-9 have been reported in tears of VKC (Leonardi et al. 2003) and MMP-8 in the tears of

ABC (Maatta et al. 2008). Cathepsins S (CATS) exhibits elastinolytic properties

(Alexander & Werb 1989; Taleb et al. 2005), which could be important to maintain the

ECM architecture of the cornea. It has been shown that the corneas of people with KC have increased levels of proteases (Table 1.3, Chapter 1) and, these might contribute to the changes in corneal shape. Although there have been several reports of tear proteases in KC patients, for example increased levels of MMP-9 (Lema & Duran 2005; Lema et

Changes to the tear film proteome in keratoconus 99 Chapter 4: Proteases and inflammatory molecules in the tears of people with keratoconus al. 2009) or MMP-1 (Pannebaker et al. 2010), the relative activity of these proteases are unknown and have not been studied or reported previously.

Cytokines or inflammatory molecules play an important role in the regulation of ocular surface inflammation and immunological reactions. These cytokines are multipotent peptides expressed by the cells of the ocular surface (Nakamura et al. 1998). The stromal cells in KC express high levels of binding sites for IL-1 in KC (Fabre et al.

1991). Levels of IL-4 and -5 have been shown to be increased in tears of people with a proliferative type of AKC (Uchio et al. 2000). Tears of people with dry eye syndrome have elevated levels of IL-6 and TNF-α (Yoon et al. 2007). However, there has been a report of significantly decreased levels of IL-12, TNF-α, IL-13, CCL5 in the tears of people with KC compared to controls (Jun et al. 2011). Others have shown a significant increase in the level of IL-6 or TNF-α in the tears from KC (Lema & Duran 2005).

Measuring the actual concentration of cytokines can be challenging in basal tear fluid.

The data available on baseline concentrations of tear cytokines are limited and variable, and this could be due to the nature of tear fluid collected and the use of different assays to study the level of cytokines (Jun et al. 2011).

MMPs, cytokines and CATS interact with each other forming a complex network. IL-

1β, IL-6 or TNF-α can stimulate the production of several MMPs (-1, -2, -3, -7, -9, -13)

(Haro et al. 2000; Yoo et al. 2002; Li et al. 2003; Wisithphrom & Windsor 2006) and

CATS (Taleb et al. 2005). Levels of MMP-1, -2, -3, -9 have shown to inactive IL-1β

(Ito et al. 1996) and IL-10 has an inhibitory effect on CATS (Sendide et al. 2005). This interplay is illustrated in Figure 4.1.

Changes to the tear film proteome in keratoconus 100 Chapter 4: Proteases and inflammatory molecules in the tears of people with keratoconus

Figure 4.1: Illustration of the interplay between proteases and cytokines based on previous reports. IL-1β, IL-6 or TNF-α can have a stimulatory effect (+) on the production of MMPs (-1, -2, -3, -9, -13) (Haro et al. 2000; Yoo et al. 2002; Li et al. 2003; Wisithphrom & Windsor 2006) and CATS (Taleb et al. 2005). The level of MMP-2 has been enhanced by IL-1α (Kusano et al. 1998). MMPs (-1, -2, -3, -9) also been reported to have an inhibitory effect ( - ) on production of IL-1β (Ito et al. 1996) and IL-10 may have an inhibitory effect ( - ) on CATS expression (Sendide et al. 2005).

In this study we have examined the levels and activity of proteases in tears collected from normal subjects, people with KC and individuals after CXL for the management of

KC.

4.2. Materials and Methods

The Human Research Ethics Advisory Panel at the University of New South Wales approved the study (approval no: 084081). Written informed consent was obtained from each subject prior to being enrolled in the study (Appendix A). All procedures were conducted in accordance with the 2000 Declaration of Helsinki.

Changes to the tear film proteome in keratoconus 101 Chapter 4: Proteases and inflammatory molecules in the tears of people with keratoconus

4.2.1. Subjects

Three groups were included in the study, consisting of control subjects (C) who had not been diagnosed with KC, subjects with KC, and subjects who had undergone a CXL procedure to stabilize the progression of their KC. A total of 80 eyes (C=28, KC=32,

CXL=20) were used to study the total protein levels, 60 eyes (C=20, KC=25, CXL=15) were used to study the protease levels. The gelatinase and collagenase activities were studied on 49 eyes (C=17, KC=19, CXL=13) and 42 eyes (C=16, KC=15, CXL=11), respectively. These differences in subject numbers were due to the need to increase the volume of tear samples collected during the study. The demographic profile of the subjects is illustrated in Table 4.1. Tears were collected during single study visits and no subject was in more than one group. The subjects recruited with KC gave a history of frequent eye rubbing. All the subjects either gave a history of no or discontinued contact lens wear for at least one month before the study. No participant in this study gave a history of active allergy at the time of tear collection. The CXL subjects had a post- operative period ranging from 3-6 months. Subjects who had a history of any ocular surgery, or were under topical or systemic medication were excluded from the study.

Changes to the tear film proteome in keratoconus 102 Chapter 4: Proteases and inflammatory molecules in the tears of people with keratoconus

Table 4.1 : Demographics of the study.

Protease activity Total protein Protease levels Groups Gelatinase Collagenase

C KC CXL C KC CXL C KC CXL C KC CXL

Total 28 32 20 20 25 15 17 19 13 16 15 11

Men 16 17 7 8 16 6 9 8 8 9 6 5

Women 12 15 13 12 9 9 8 11 5 7 9 6

Age (years) 32.6±11.1 29.5±9.4 27.3±5.2 29.8±8.9 27.4±6.0 27±5.0 30.5±9.4 26.7±5.0 28.4±5.1 30.2±1.0 26.8±5.6 24.6±3.5

C–Controls; KC–Keratoconus; CXL–Cross-linked

Changes to the tear film proteome in keratoconus 103 Chapter 4: Proteases and inflammatory molecules in the tears of people with keratoconus

4.2.2. Tear collection

Basal tears were collected from all the study participants using the method described previously in Section 2.2.3.

4.2.3. Corneal topography

The cornea was mapped using the Allegro Oculyzer (Wavelight® GmbH, Am

Wolfsmantel 5, 91058 Erlangen, Germany) (Figure 4.2) on all the subjects. The Allegro

Oculyzer with the Pentacam technology examines the cornea three dimensionally using a built-in high resolution imaging camera. The rotating camera operates according to the

Scheimpflug principle (Koretz et al. 2004). The Belin/Ambrosio Enhanced Ectasia

Display was used for KC screening (Ambrosio et al. 2011).

Figure 4.2: The Allegro Oculyzer examination set up. Photo courtesy of Wavelight®

4.2.4. Total tear proteins

The total protein concentration of the tear samples was quantified by using the method

Changes to the tear film proteome in keratoconus 104 Chapter 4: Proteases and inflammatory molecules in the tears of people with keratoconus described previously in Section 2.2.6.

4.2.5. Proteases and cytokines in tears

The proteases or the cytokines in the tear film were measured using a RayBio® human custom G -series antibody array (Ray Biotech, Inc., Norcross, Georgia, United States).

The array on a glass chip provides a highly sensitive approach to simultaneously detect multiple active forms of proteases or cytokines from individual samples. A preliminary cross-reactivity test was performed and no cross-reactivity was found for the selected antibodies of proteases and cytokines. In brief, antibodies to the individual proteases, protease inhibitors or cytokines were immobilized onto glass chips in discrete spots.

Then, tear samples were incubated with the antibodies on the glass chips for 2 h at room temperature and washed with phosphate buffer saline. A cocktail of biotinylated antibodies was added and incubated for further 2 h. The final step involved addition of labelled streptavidin before reading the fluorescence (excitation: 532 nm) using Axon

GenePix® scanner. The signal intensities between the samples were studied using the

Ray Bio® analysis tool. The ratio of fluorescence intensity to total protein is taken as the measure of expression of individual proteases and cytokines in each sample. The layout of the entire protein profile in the custom G -series array is shown in Table. Biotinylated protein and BSA served as positive and negative controls in the array.

Changes to the tear film proteome in keratoconus 105 Chapter 4: Proteases and inflammatory molecules in the tears of people with keratoconus

Table 4.2: Layout of the custom G-series array.

A B C D E F G H

1 POS 1 POS 2 POS 3 NEG NEG MMP-1 MMP-2 MMP-3

2 POS 1 POS 2 POS 3 NEG NEG MMP-1 MMP-2 MMP-3

3 MMP-7 MMP-8 MMP-9 MMP-13 Cathepsin S TIMP-1 TIMP-2 IL-1α

4 MMP-7 MMP-8 MMP-9 MMP-13 Cathepsin S TIMP-1 TIMP-2 IL-1α

5 IL-1β IL-3 IL-4 IL-5 IL-6 IL-7 IL-8 IL-9

6 IL-1β IL-3 IL-4 IL-5 IL-6 IL-7 IL-8 IL-9

7 IL-10 TNF-α TNF-β NEG NEG NEG NEG POS 2

8 IL-10 TNF-α TNF-β NEG NEG NEG NEG POS 2

POS-Positive control; NEG-Negative control

Tear samples were pooled based on similarities in the steepest keratometry reading of

the corneas within each group. Fifty µL of pooled tear sample from each group was

used in analysis (recommended minimum volume of individual tear sample for the

array). The C group had four tear samples, each sample obtained by pooling tears from

five eyes. The KC group had five samples, each obtained by pooling tears from five

eyes. The CXL group had three samples, each obtained by pooling from five eyes. All

the samples were masked before examination. The average total protein concentrations

in KC, CXL and C groups were 3.73 µg/µL, 4.76 µg/µL and 5.84 µg/µL respectively.

4.2.6. Activity of proteases

The gelatinase and collagenase activities in individual tear samples were examined

using the Enzchek® Gelatinase/Collagenase Assay Kit (Invitrogen Australia Pty,

Victoria, Australia). The protocol was modified in our laboratory to be used with tear

samples. Individual tear samples (2 µL) were incubated with the substrate and reaction

buffer (0.5 M Tris-HCL, 1.5 M NaCl, 50 mM CaCl2, 2 mM NaN3 and pH 7.6) at room

Changes to the tear film proteome in keratoconus 106 Chapter 4: Proteases and inflammatory molecules in the tears of people with keratoconus temperature for 2 h. DQ™ gelatin (pig skin) and DQ™ collagen (Type IV from human placenta) were the substrates used to determine the active forms of gelatinases and collagenases, respectively. These substrates are heavily labeled with fluorescein and express highly fluorescent peptides when digested by gelatinases or collagenases due to release of fluorescein from self-quenching in the unprocessed substrates. The intensity of the fluorescence (excitation: 485 nm, emission: 535 nm) was measured by a fluorescence microplate reader (Tecan Spectrofluoro Plus). The ratio of fluorescence intensity to total protein was taken as the measure of tear gelatinolytic or collagenolytic activity.

4.2.7. Statistical methods

The results obtained were expressed as mean ± standard deviation. Three way comparisons of C, KC and CXL groups were made by using Kruskal-Wallis non- parametric test. Significant differences in total tear protein levels and protease activity between the three groups were determined by univariate analysis followed by post-hoc testing. Mann-Whitney U test was used to confirm the interaction of individual proteases between the three groups. The simulated keratometry reading was correlated to total protein, protease levels and protease activity using linear regression. The sensitivity and specificity of the protease activity tests was assessed by Receiver operating curve (ROC) analysis. SPSS predictive analytics software, version 18 was used for all the analysis and a p value of < 0.05 was considered significant.

4.3. Results

4.3.1. Total tear protein concentration

Neither age- nor gender- related differences were not statistically significant between

Changes to the tear film proteome in keratoconus 107 Chapter 4: Proteases and inflammatory molecules in the tears of people with keratoconus the groups. The total tear protein concentrations in C, KC and CXL groups were compared. KC patients (4.1 ± 0.9 mg/mL) had a significantly lower level of total tear protein compared to the C (6.7 ± 1.4 mg/mL) (p = 0.0001) and CXL (5.7 ± 2.3 mg/mL)

(p = 0.003) groups. The difference in total protein was not significant between C (6.7 ±

1.4 mg/mL) and CXL (5.7 ± 2.3 mg/mL) (p = 0.086). The total tear protein levels were correlated with the keratometry reading. A significant (p = 0.01) but not strong negative correlation (r = -0.28) was found between total tear protein and keratometry reading by simple regression analysis (Figure 4.3). A relatively strong negative correlation (r = -

0.55, p < 0.0001) was found when the CXL group was excluded from the analysis

(Figure 4.4).

Figure 4.3: Correlation analysis between total protein levels and keratometry in 80 eyes. X- axis represents keratometry values in dioptres (D) and the Y- axis shows the total protein levels in mg/mL. An increase in keratometry was accompanied by a decrease in level of total tear protein in tears.

Changes to the tear film proteome in keratoconus 108 Chapter 4: Proteases and inflammatory molecules in the tears of people with keratoconus

Figure 4.4: Correlation analysis between total protein levels and keratometry in 60 eyes (without CXL group). X- axis represents keratometry values in dioptres (D) and the Y- axis shows the total protein levels in mg/ml. An increase in keratometry was accompanied by a decrease in level of total tear protein in tears.

4.3.2. Tear Proteases

The proteases and their expression levels in C, KC and CXL are shown in Table 4.3.

The proteases (measured as fluorescence intensity units (FIU) per mg total protein) that were significantly increased in KC patients when compared to C group were MMP-1, -

3, -7, and -13. The difference in protease levels observed in CXL was not significant compared to KC or C.

Levels of individual proteases or protease inhibitors were correlated to the keratometry reading (Table 4.4a). Significant positive correlations were found. As the level of

MMP-13, CATS, TIMP-1 or TIMP-2 increased in the tear film so did the level of corneal steepening (increase in keratometry reading). The correlations were insignificant when CXL group was included in the analysis (Table 4.4b). The ratios of

MMPs to TIMPs were not significant (p = 0.368) between the three groups and did not correlate with keratometry (Table 4.5).

Changes to the tear film proteome in keratoconus 109 Chapter 4: Proteases and inflammatory molecules in the tears of people with keratoconus

Table 4.3: Expression of proteases and cytokines in tears of the three groups.

Proteases ; Cytokines FIU/mg total protein Mediators C KC CXL p-value

MMP-1 30.4 ± 5.4 45.6 ± 8.9 39.8 ± 13.0 0.048*

MMP-2 37.1 ± 4.8 52 ± 8.2 55.1 ± 31.1 0.091

MMP-3 57.7 ± 4.4 150.6 ± 62.0 502.5 ± 679.0 0.025*

MMP-7 17.8 ± 3.3 28.9 ± 4.4 22.7 ± 6.8 0.046*

MMP-8 356.8 ± 569.3 377.3 ± 380.7 191.9 ± 180.7 0.572

MMP-9 349.1 ± 545.3 442.1 ± 474.5 217.5 ± 211.2 0.375

MMP-13 52.8 ± 3.9 86.7 ± 6.5 76.7 ± 20.7 0.022*

TIMP-1 3272.8 ± 553.3 4533.6 ± 1015.4 3288.8 ± 1429.6 0.174

TIMP-2 3937.3 ± 2720.2 6576.5 ± 1793.0 3631.8 ± 1166.3 0.087

CATS 6524.9 ± 570.4 8321.1 ± 698.9 7072.6 ± 2295.2 0.125

IL-1α 85.7 ± 6.8 133.6 ± 9.5 119.9 ± 59.2 0.075

IL-1β 51.7 ± 16.4 67.4 ± 14.2 57.7 ± 29.9 0.471

IL -3 44.8 ± 6.9 66.3 ± 8.4 60.6 ± 14.6 0.078

IL-4 46.7 ± 3.9 72.4 ± 4.5 62.3 ± 16.1 0.022*

IL-5 47.4 ± 3.1 81.1 ± 11.5 66 ± 21.3 0.027*

IL-6 66.7 ± 8.7 313.6 ± 232.2 109.7 ± 35.2 0.012*

IL-7 3072.5 ± 881.2 5132.5 ± 3211.1 2720.3 ± 1516.8 0.424

IL-8 1168.0 ± 405.2 2893.4 ± 1758.5 1308.1 ± 694.4 0.034*

IL-9 55.1 ± 11.2 76 ± 10.1 65.1 ± 18.8 0.113

IL-10 20.5 ± 2.1 30.6 ± 3.3 29.4 ± 13.4 0.059

TNF-α 54.1 ± 7.0 92.2 ± 8.5 80.4 ± 26.5 0.014*

TNF-β 76.8 ± 5.5 130.9 ± 21.3 114.85 ± 30.3 0.014*

* Significant levels comparing all the three groups i.e., C, KC and CXL FIU = fluorescence intensity unit.

Changes to the tear film proteome in keratoconus 110 Chapter 4: Proteases and inflammatory molecules in the tears of people with keratoconus

Table 4.4a: Correlations between tear proteases/cytokines and keratometry (D) of control (C) and keratoconus (KC) group. Proteases ; Cytokines

Mediators Equation r p - value

MMP-13 y= 1.052x + 20.22 0.55 0.019*

TIMP-1 y= 168.1x - 4041 0.71 0.027*

* TIMP-2 y= 453.9x - 16231 0.79 0.009

CATS y= 201.35x + 2072.8 0.79 0.002*

IL - 1α y= 4.633x - 108.5 0.77 0.013*

* IL - 10 y= 0.977x - 20.43 0.72 0.025

TNF- α y= 3.692x - 100.6 0.76 0.015*

* Significant positive correlation showing, increase in keratometry (D) is accompanied by an increase in the tear protease/cytokine levels.

Changes to the tear film proteome in keratoconus 111 Chapter 4: Proteases and inflammatory molecules in the tears of people with keratoconus

Table 4.4b: Correlation analysis of tear proteases and keratometry (D) in control (C), keratoconus (KC) and cross-linked (CXL) group. Proteases ; Cytokines

Mediators Equation r p - value

MMP-13 y= 1.0524x+20.224 0.38 0.224#

TIMP-1 y= 35.17x+2041.2 0.21 0.518#

TIMP-2 y= 100.11x-51.99 0.28 0.375#

# CATS y= 42.307x+5292 0.20 0.531

# IL - 1α y= 2.1371x+7.1851 0.41 0.181

# IL - 10 y= 0.3936x+7.274 0.33 0.289

# TNF- α y= 1.3606x+8.4271 0.41 0.178

# Correlation not significant.

Changes to the tear film proteome in keratoconus 112 Chapter 4: Proteases and inflammatory molecules in the tears of people with keratoconus

Table 4.5: Correlation between MMPs:TIMPs and keratometry (D) in C, KC and CXL groups. Proteases

Mediators Equation r p - value

MMP-1/TIMP-1 y= -6E-05x+0.0134 0.15 0.631#

# MMP-1/TIMP-2 y= -0.0002x+0.0216 0.43 0.139

# MMP-2/TIMP-1 y= -3E-05x+0.0143 0.08 0.798

MMP-2/TIMP-2 y= -0.0002x+0.0241 0.33 0.276#

MMP-3/TIMP-1 y= -0.0019x-0.0483 0.22 0.472#

# MMP-3/TIMP-2 y= 0.0017x-0.042 0.16 0.595

# MMP-7/TIMP-1 y= -4E-05x+0.0084 0.21 0.487

MMP-7/TIMP-2 y= -0.0002x+0.0132 0.48 0.092#

MMP-8/TIMP-1 y= -0.0024x+0.2073 0.19 0.537#

# MMP-8/TIMP-2 y= -0.0026x+0.2044 0.23 0.443

# MMP-9/TIMP-1 y= -0.0026x+0.2222 0.18 0.558

MMP-9/TIMP-2 y= -0.0027x+0.2122 0.23 0.449#

MMP-13/TIMP-1 y= -4E-05x+0.0215 0.05 0.86#

MMP-13/TIMP-2 y= -0.0004x+0.0361 0.37 0.205#

# Correlation not significant.

Changes to the tear film proteome in keratoconus 113 Chapter 4: Proteases and inflammatory molecules in the tears of people with keratoconus

4.3.3. Tear Cytokines

The cytokine expression levels in C, KC and CXL groups are shown in Table 4.2. KC patients had significantly increased tear levels of IL-4, -5, -6, -8 and TNF-α, -β compared to C subjects. IL-6 was the only cytokine over expressed in KC when compared to CXL group (p = 0.025). TNF-α was the only cytokine increased in CXL compared to C (p = 0.034). There was a significant positive correlation between the levels of IL-1α, IL-10 and TNF-α and corneal topography (Table 4.3a). The positive correlations were insignificant when CXL group was included in the analysis (Table

4.3b).

4.3.4. Proteolytic activity of tears

The activity of gelatinases and collagenases in the tear film of C, KC, and CXL groups were examined and the results are shown in Table 4.5. The tear film of KC subjects had significantly (p = 0.0001) higher gelatinolytic activity compared to C subjects. The activity of gelatinases in CXL was not significantly different compared to KC (p = 0.24) or C (p = 0.076). Similar results were observed for the activity of collagenases.

Collagenases were significantly (p = 0.025) more active in KC compared to C group but were not significantly more active when the CXL group was compared to KC (p = 1.00) and C (p = 0.13) groups. The activity of gelatinases and collagenases was maximum in the KC subjects followed by the CXL. The insignificant correlation between collagenolytic (r = 0.22, p = 0.16) activity and corneal topography readings turned significant after excluding CXL group from the analysis (r = 0.41, p = 0.023) (Figure

4.5 & 4.6). There was no correlation between gelatinolytic activity and keratometry readings with or without CXL group (r = 0.16, p = 0.26) (Figure 4.7). The substrate specificity of MMPs exhibiting gelatinolytic or collagenolytic activity (Alexander &

Changes to the tear film proteome in keratoconus 114 Chapter 4: Proteases and inflammatory molecules in the tears of people with keratoconus

Werb 1989) is shown in Table 4.6.

Figure 4.5: Insignificant correlation between collagenolytic activity and keratometry reading in C, KC and CXL groups.

Figure 4.6: Significant positive correlation between collagenolytic activity and keratometry reading in C and KC groups.

Changes to the tear film proteome in keratoconus 115 Chapter 4: Proteases and inflammatory molecules in the tears of people with keratoconus

Figure 4.7: Insignificant correlation between gelatinolytic activity and keratometry reading in C, KC, CXL groups.

Table 4.6: Gelatinase and collagenase activities in tear samples. Protease activity

Groups C KC CXL p-value

Gelatinase activity 45.8 ± 24.6 87.5 ± 33.6 69.6 ± 22.2 0.0001* (FIU/mg total protein)

Collagenase activity 3.6 ± 2.0 6.1 ± 3.2 5.7 ± 3.3 0.025* (FIU/mg total protein)

* Significant levels comparing all the three groups i.e., C, KC and CXL FIU = fluorescence intensity unit.

Changes to the tear film proteome in keratoconus 116 Chapter 4: Proteases and inflammatory molecules in the tears of people with keratoconus

Table 4.7: Substrate specificity of collagenases (MMP-1,-8,-13), gelatinases (MMP-2,- 9), stromelysin (MMP-3) and matrilysin (MMP-7). Proteases

Substrate specificity

Proteases Gelatin Collagen IV

MMP-1 * (+) (–)

MMP-2 # (+) (+)

MMP-3 * (+) (+)

MMP-7 * (+) (+)

MMP-8 # (+) (–)

MMP-9 # (+) (+)

MMP-13 * (+) (–)

* Levels of protease significantly increased in KC tears compared to C.

# No significant difference in tear protease levels between C, KC and CXL.

(+) Active degradation of the substrate,

(–) No active degradation of the substrate (Alexander & Werb 1989; Taleb et al. 2005).

4.3.5. ROC analysis of proteolytic activities

The performances of gelatinolytic activity and collagenolytic activity as diagnostic tests for KC was studied using the ROC analysis (Metz 1978). Sensitivity was plotted against the specificity to generate ROC curves shown in Figure 4.8. The sensitivity and specificity of gelatinolytic activity test were 97.4 % and 96.3 % respectively.

Changes to the tear film proteome in keratoconus 117 Chapter 4: Proteases and inflammatory molecules in the tears of people with keratoconus

Collagenolytic activity test had sensitivity and specificity rates of 73.3%. The accuracy of these tests was measured by the area under the curve in ROC (AUROC) with 95% confidence intervals (Bewick et al. 2004). Gelatinolytic activity had an AUC score of

0.85 (0.726-0.983, p = 0.001) and collagenolytic activity had an AUC of 0.74 (0.557-

0.918, p = 0.026).

(a) Gelatinase activity

(b) Collagenase activity

Figure 4.8: ROC analysis of the protease activities in C and KC groups (a) Gelatinase activity (b) Collagenase activity.

Changes to the tear film proteome in keratoconus 118 Chapter 4: Proteases and inflammatory molecules in the tears of people with keratoconus

4.4. Discussion

In KC, the cornea is known to have increased gelatinolytic and collagenolytic activity

(Kao et al. 1982; Rehany et al. 1982; Brown et al. 1993) and increased tear levels of pro-MMP-9 (Lema and Duran 2005) or active MMP1 (Pannebaker et al. 2010).

However, there have been no reports on the activity of proteases in the tear film from subjects with KC. This study, for the first time, has examined the activities of gelatinases and collagenases in the tears of people with or without KC, and in people after CXL for the treatment of KC.

In this study we have found increased expression of collagenases (MMP-1, MMP-13), stromelysin (MMP-3) and matrilysin (MMP-7) in tears of KC patients using the antibody array. The finding of increased levels of active MMP-1 concurs with the results of a previous study (Pannebaker et al. 2010), as does the lack of a significant increase in levels of TIMP-1 or TIMP-2. TIMP levels have been demonstrated to increase in KC corneas only after scarring (Kenney et al. 1998). The subjects (C, KC or

CXL) recruited in this study did not have scarred corneas and this might be the reason for the insignificant levels of TIMPs between the groups. The levels of MMP-13,

TIMP-1, -2 and CATS were positively correlated to the keratometry values (Table

4.3a). CATS had higher expression in KC patients compared to normal subjects but this expression was not statistically significant. The proteolytic activities of MMP-1, -3, -7 and CATS might affect the stability of architecture of the cornea, due to their ability to degrade elastin (Shi et al. 1992; Li et al. 2000; Heinz et al. 2011) which is interwoven with collagen fibres in the cornea (Kamma-Lorger et al. 2010).

Our study indicates no difference in the level of active MMP-2 and -9 between the three groups. Whilst increased levels of pro-MMP-9 have been reported (Lema and Duran,

Changes to the tear film proteome in keratoconus 119 Chapter 4: Proteases and inflammatory molecules in the tears of people with keratoconus

2005), in the present study MMP-9 levels were detected using antibodies, which might explain the difference. These gelatinases are known for their ability to degrade denatured collagen. The collagenases are responsible for the “first insult”, causing the collagen to denature (Alexander & Werb 1989). KC corneas have a weak arrangement of collagen fibrils, and normal levels of gelatinases would still have a greater gelatinolytic activity in KC compared to a normal cornea with a healthy collagen. As

KC is a slowly progressive disease, even minute levels and activity of proteases might add to the steepening effect on the cornea (Balasubramanian et al. 2010).

The tear proteolysis, i.e. gelatinolytic and collagenolytic activities, were significantly increased in KC patients compared to the controls. Based on the AUC scores in ROC analysis, the quality of a diagnostic test can be classified as excellent (0.90-1), good

(0.80-0.90), fair (0.70-0.80), poor (0.60-0.70) and fail (0.50-0.60) (Metz 1978). In the present study, gelatinolytic activity with an AUC score of 0.85 (0.726-0.983, p = 0.001) is considered a good test and collagenolytic activity with an AUC of 0.74 (0.557-0.918, p = 0.026), a fair test for the diagnosis of KC. Further studies are required to establish the use of protease activity tests as a non-invasive method for early diagnosis of KC.

The protease expression or activities are not always continuous and this could be the reason for certain protease levels (MMP-1, -2, -3, -7, -9) and the gelatinolytic activities being inconsistent with the keratometry readings in KC.

There have been various studies with conflicting reports on the expression of various

MMPs in KC corneas. Studies have shown increased levels of MMP-1 (Seppala et al.

2006) and MMP-13 (Mackiewicz et al. 2006) in KC corneas. Others have shown that normal and KC corneas showed no difference in the levels of MMP-2 and -9 (Fini et al.

1992) and MMP-3 and -10 (Saghizadeh et al. 2001). Increased levels of tear MMP-1, -

Changes to the tear film proteome in keratoconus 120 Chapter 4: Proteases and inflammatory molecules in the tears of people with keratoconus

13 shown in the current study supports the over-expression of MMP-1, -13 observed in

KC corneas, reported previously (Mackiewicz et al. 2006; Seppala et al. 2006). Our data also support the fact that there are no differences in the levels of MMP-1 or MMP-9 during KC, although we did see an increase in the level of MMP-3 in tears during KC.

CXL has a “freezing effect” on the KC cornea (Wollensak et al. 2003). Wolf et al. have reported decrease in corneal steepening in KC patients after CXL but cases of keratopathy (Rodriguez-Ausin et al. 2011) and corneal melting (Labiris et al. 2011) after the procedure have also been reported. The complications and failure rates of CXL were

2.9 % and 7.6 % respectively (Koller et al. 2009). There have been no reports on the behaviour of tear proteases after CXL. In this study, the tears were collected from CXL group 3-6 months after CXL procedure. At this post-operative period, the epithelium and stroma are fully regenerated with a regular corneal surface in KC (Mazzotta et al.

2008). The levels of MMPs in CXL were intermediate between the KC and normal subjects. The positive correlations between keratometry and proteases/cytokines (Table

4.3b) or collagenase activity (Figure 4.4a) became insignificant when CXL group was included in the analysis. No difference was observed in the tear protease activity of

CXL when compared to C or KC groups. Reduced protease levels and protease activity were observed in the CXL group compared to KC patients. Thus, CXL might positively affect protease levels and activities in the KC cornea. Since tears from the subjects were not analysed before CXL procedure in this study, future studies may need to confirm the direct effect of cross linking on tear proteases.

KC is defined as a non-inflammatory disease of the cornea but inflammatory molecules such as IL and TNF have been shown to be over-expressed in KC corneas (Fabre et al.

1991) and tear film of KC patients (Lema & Duran 2005). McMonnies suggested

Changes to the tear film proteome in keratoconus 121 Chapter 4: Proteases and inflammatory molecules in the tears of people with keratoconus classifying KC as a non-inflammatory condition (McMonnies 2007). However, the present study found increased expression of IL-4,-5,-6 and TNF-α,-β in the tears of KC patients and the levels of IL-1α,-10, TNF-α were positively correlated to keratometry.

IL-6 was elevated in KC compared to CXL subjects. The expression of TNF-α, in particular, was elevated in CXL compared to the control or C group.

As mentioned earlier, there is an active interplay between MMPs and ILs (Figure 4.1).

MMP-1, -2, -3, -9 and IL-10 has an inhibitory effect on IL-1β (Ito et al. 1996) and

CATS (Sendide et al. 2005) respectively (Figure 1). This interaction could contribute to the insignificant levels of IL-1β and CATS in the present study. The increased expression of TNF-α in CXL compared to C might trigger the levels of MMPs and

CATS leading to similar levels observed in a KC tear film as shown in Figure 4.9.

Figure 4.9: Diagram showing the relative expression of MMPs and cytokines in the tears of controls (C), keratoconus (KC) and cross-linked (CXL) groups. The amount of MMPs (-1, -3, -7, -13), ILs (-4, -5, -6, -8) and TNFs (-α, -β) were significantly elevated in tears of KC compared to C. TNF-α was the only cytokine over-expressed in tears of CXL compared to C, and IL-6 was the only cytokine elevated in tears of KC compared to CXL.

Unwinding this interplay would be crucial to establish the role of proteases and

Changes to the tear film proteome in keratoconus 122 Chapter 4: Proteases and inflammatory molecules in the tears of people with keratoconus cytokines in KC.

In conclusion, the expression and activity of proteases in the KC tear film appears to be significantly altered. An in-depth analysis is essential to determine the use of tear proteases in early diagnosis of KC, monitoring the disease progression before and after

CXL.

Throughout the course of this study, the most prominent feature noted among people suffering with KC was the positive history of frequent eye rubbing. Evaluating the role of eye rubbing in the development and progression of KC is fundamental to understand the nature of the disease. The next chapter examines the possible causal links between eye rubbing and KC.

Changes to the tear film proteome in keratoconus 123 Chapter 5: Effects of eye rubbing on the concentration of proteases and cytokines in the tears

5. CHAPTER 5: EFFECTS OF EYE RUBBING ON THE CONCENTRATION OF PROTEASES AND CYTOKINES IN THE TEARS

Changes to the tear film proteome in keratoconus 124 Chapter 5: Effects of eye rubbing on the concentration of proteases and cytokines in the tears

5.1. Introduction

Eye rubbing is a physiological response to uncomfortable eyes brought about by such things as fatigue, emotional stress or exposure to dust particles or allergens. It is also common before or after sleep or during contact lens fittings. Finger tips or knuckles or palms of the hand are mostly used for rubbing one or both eyes. The average duration of an episode of eye rubbing for most people is a few seconds (McMonnies & Boneham

2003).

KC is a progressive, debilitating disease of the eye extensively linked to the habits of abnormal or forceful eye rubbing (Coyle 1984; Krachmer 2004). A multivariate analysis for determining the risk factors for KC indicated that eye rubbing was the only significant predictor for the disease (Bawazeer et al. 2000). Increased frequency of eye rubbing is also seen in other ocular conditions such as dry eye syndrome (Sullivan et al.

2002), allergic (Raizman et al. 2000) or bacterial or viral conjunctivitis (Senaratne &

Gilbert 2005), misdirected eye lashes or trichiasis and blepharitis (Sihota & Tandon

2011). Frequent eye rubbing is also common in dermatological conditions such as eczema or atopic dermatitis involving the eye lids (De Benedetto et al. 2009).

Ocular rubbing is considered abnormal when there is a combination of amplified frequency, intensity and duration of rubbing episodes over an extended period of time.

The technique used by most KC patients to rub their eyes is distinct or different to that used by other people (Carlson 2009). Vigorous ocular rubbing in KC involves the use of finger tips or knuckles. The use of finger tips exerts a pointed or localized pressure on the cornea. The characteristic feature of KC eye rubbing is stroking the closed eyes with pointed pressure using finger tips or middle knuckles in a circular motion restricted to

Changes to the tear film proteome in keratoconus 125 Chapter 5: Effects of eye rubbing on the concentration of proteases and cytokines in the tears the cornea (Figure 5.1a & 5.1b) (Carlson 2009).

Figure 5.1: KC patients use their (a) finger tips or (b) knuckle to generate pressure localized to the cornea in a circular motion. Images obtained with kind permission from Dr. Alan Carlson (Carlson 2009) and Review of Ophthalmology®.

On the other hand, people with allergic or infective ocular diseases tend to use the back of their hand or palm or finger pad to rub their eyes (Figure 5.2a, 5.2b & 5.2c) (Carlson

2009). The rubbing movement in these conditions is usually horizontal involving the eye lids and caruncle, with minimal pressure spreading to the cornea (Figure 5.2a &

5.2c). This type of eye rubbing often lasts less than 15 s but the duration of KC rub is much longer, usually extending from 10 to 180 s (Carlson 2010). McMonnies and

Boneham have shown that people without allergy or KC have a decreased frequency per day and duration (less than 5 s) of eye rubbing compared to atopic or KC subjects

(McMonnies & Boneham 2003). The method of eye rubbing may be used as an indicator for differentiating between normal or allergic subjects and those with KC.

Changes to the tear film proteome in keratoconus 126 Chapter 5: Effects of eye rubbing on the concentration of proteases and cytokines in the tears

Figure 5.2: Different methods adopted by allergic patients for eye rubbing using (a) back of the hand, (b) palm and (c) finger pad involving the caruncle. Images obtained with kind permission from Dr. Alan Carlson (Carlson 2009) and Review of Ophthalmology®.

Several studies have investigated the role of proteolytic enzymes such as matrix metalloproteinases (MMPs) in KC (Table 1.2). MMPs are involved in the degradation of ECM or activation of cellular apoptosis (Ollivier et al. 2007). In the human cornea,

MMPs are secreted by epithelial cells, stromal cells and neutrophils (Fini et al. 1998).

In KC, the cornea is known to express elevated levels of MMP-1 (Seppala et al. 2006) and MMP-13 (Mackiewicz et al. 2006). The tear analysis in KC has shown increased levels of MMP-1, MMP-3, MMP-7 and MMP-13 (Table 4.2). Elevated gelatinolytic and collagenolytic activities have been also reported in the corneas and (Kao et al. 1982;

Changes to the tear film proteome in keratoconus 127 Chapter 5: Effects of eye rubbing on the concentration of proteases and cytokines in the tears

Rehany et al. 1982; Brown et al. 1993) tear film of KC (Table 4.5).

MMP-13 or collagenase 3 is expressed by human corneal epithelial cells and is essential for the regulation of corneal wound healing and remodelling (Li et al. 2003). In addition to the strong collagenolytic activity, MMP-13 also has gelatinolytic properties (Knauper et al. 1996). Increased levels of MMP-13 have also been reported in VKC (Leonardi et al. 2007) and systemic immune disorders such as rheumatoid arthritis (Lindy et al.

1997) and osteoarthritis (Takaishi et al. 2008).

KC is defined as a non-inflammatory disease of the cornea. However, inflammatory molecules such as ILs and TNFs have been shown to be over-expressed in the corneas

(Fabre et al. 1991; Becker et al. 1995; Ha et al. 2004; Lema & Duran 2005) and tear film of KC patients (Lema & Duran 2005). The increased expressions of IL-4, -5, -6 and TNF-α, -β in the tears of KC patients have been reported previously (Table 4.2).

Cytokines such as IL-6 and TNF-α regulate immune responses and inflammation. The cells of the ocular surface express IL - 6 and TNF- α (Nakamura et al. 1998; Sugaya et al. 2011) and, the levels of these inflammatory mediators are particularly increased in response to corneal wound healing (Wilson et al. 2001; Ebihara et al. 2011). Increased levels of IL-6 and TNF-α have also been reported in dry eye syndromes (Yoon et al.

2007), allergic keratoconjunctivitis (Leonardi et al. 2003; Shoji et al. 2007) and VKC

(Leonardi et al. 1998).

Studies have investigated the impact of experimental eye rubbing on biomechanical properties of the cornea (Liu et al. 2011), thickness of the layers of the cornea

(McMonnies et al. 2010; Prakasam et al. 2012), the corneal curvature (Mansour &

Haddad 2002), tear levels of IL-8 and epidermal growth factor (EGF) (Kallinikos &

Changes to the tear film proteome in keratoconus 128 Chapter 5: Effects of eye rubbing on the concentration of proteases and cytokines in the tears

Efron 2004) during contact lens wear. However, there have been no reports in the literature examining the influence of eye rubbing on tear protease, proteolytic activities and inflammatory markers. To the knowledge of this candidate, the present study is the first to report the tear levels of MMP-13, IL- 6, TNF- α and collagenase activity before and after experimental eye rubbing.

5.2. Materials and Methods

Ethics was granted by the Human Research Ethics Advisory Panel at the University of

New South Wales (approval no: 11039). Written informed consent was obtained from each subject after briefly outlining the nature and possible consequences of the study

(Appendix A). All the procedures were conducted in accordance with the 2000

Declaration of Helsinki.

5.2.1. Subjects

Normal volunteers were recruited for the study. A group consisting of 17 subjects was used to study the levels of MMP-13, IL-6 and TNF-α. The total protein levels and collagenase activities in the tears were examined in 14 subjects and 11 subjects respectively, from the same group. Subjects with KC were not included in the study for ethical reasons. Contact lens wearers were excluded from the study. Other exclusion criteria included subjects with a history of dry eye disorders, ocular or systemic allergic conditions, ocular surgery and use of topical or systemic medications. The demographics of the subjects recruited are illustrated in Table 1.

Changes to the tear film proteome in keratoconus 129 Chapter 5: Effects of eye rubbing on the concentration of proteases and cytokines in the tears

Table 5.1: Demographics of the study

Groups Total protein Mediator levels Collagenase activity

Total 14 17 11

Men 6 7 6

Women 8 10 5

Mean age 32.36 ± 7.08 32.58 ± 7.95 33.18 ± 7.70

Age is expressed as mean ± standard deviation

5.2.2. Corneal topography The corneal curvature was mapped using a Medmont Corneal topographer E300

(Medmont Pty Ltd, Camberwell, Victoria 3124, Australia) on all the subjects similar to the technique described in Section 2.2.4.

5.2.3. Eye rubbing technique

The typical KC eye rub was demonstrated by the investigator to all the subjects prior to the study. Subjects were then encouraged to follow the KC eye rubbing technique for exactly 60 s without interruption on both eyes, one eye at a time. The technique and duration of the eye rubbing was supervised and monitored by the investigator. Using three finger tips (index, middle and ring), all the subjects performed the KC eye rub with their eyes closed. The right hand was used to rub the right eye and vice versa. The amount of force applied for rubbing was limited so as not to cause any discomfort to the eyes. Subjects were advised to exert equal pressure on both eyes. Further care was taken by the subjects to adapt to the circular eye rubbing movement, applying constant pressure towards the centre of the cornea. Instructions were also given to the subjects to maintain a steady central fixed gaze position with the contra lateral eye opened during

Changes to the tear film proteome in keratoconus 130 Chapter 5: Effects of eye rubbing on the concentration of proteases and cytokines in the tears eye rubbing to assist central corneal rubbing (McMonnies et al. 2010).

5.2.4. Tear collection

Basal tears were collected, using glass capillary tubes before and immediately after 60 seconds of rubbing the right eye followed by the other eye. The method followed to collect basal tears has been explained previously in Section 2.2.3. A minimum of 10 µL of basal tears was collected before and after eye rubbing. Before rubbing (BR) tears from both eyes and after rubbing (AR) tears from both eyes were pooled separately for every subject in order to increase the sample volume for the analysis.

5.2.5. Total tear protein concentration

The total protein concentration of BR and AR tear samples were quantified by using

BCA protein assay described previously in Section 2.2.6.

5.2.6. Quantification of MMP-13, IL-6 and TNF-α

The levels of MMP-13 in BR and AR tear samples of individual subjects were determined by using commercially available ELISA kit (Ray Biotech, Inc., Norcross,

Georgia, United States). Similarly, the tear samples were assayed for IL-6 and TNF-α with commercially available ELISA kits (Quantikine® HS, R&D Systems, Inc.,

Minneapolis, USA. The minimum detection limit of the assays was 6 pg/mL for MMP-

13, 0.016 pg/mL for IL-6, and 0.038 pg/mL for TNF-α. Tear samples were diluted in

1:50 with the assay diluents and the analysis was performed according to the manufacturer’s protocols. The limited volume of tear samples precluded the inclusion of other mediators involved in KC for analysis.

Changes to the tear film proteome in keratoconus 131 Chapter 5: Effects of eye rubbing on the concentration of proteases and cytokines in the tears

5.2.7. Protease activities

The collagenase and gelatinase activities in BR and AR tear samples of individual subjects were examined using the Enzchek® Gelatinase/Collagenase Assay Kit

(Invitrogen Australia Pty, Victoria, Australia). Please refer Section 4.2.6.

5.2.8. Statistical methods

A sample size with a minimum of 10 patients in each group was required to determine a statistically significant difference with 80% power at 95% confidence, based on previous reports (Lema & Duran 2005; Balasubramanian et al. 2012). Paired Student t- test was performed to analyse for significant differences in the levels of total tear protein, MMP-13, IL-6, TNF-α and collagenase activities in BR and AR tear samples.

SPSS predictive analytics software, version 18 was used for all the analysis and a p value of < 0.05 was considered significant.

5.3. Results

5.3.1. Total tear protein concentration

The total protein level in tear samples BR and AR is shown in Table 5.2. BR tear samples had a total protein concentration not significantly (p = 0.68) different compared to AR samples.

5.3.2. Tear levels of MMP-13, IL-6 and TNF-α

The concentration of MMP-13, IL-6 and TNF-α in BR and AR tears are shown in Table

5.2. The concentration of all these mediators increased significantly (p < 0.05) in AR when compared to BR tears (Table 5.2).

Changes to the tear film proteome in keratoconus 132 Chapter 5: Effects of eye rubbing on the concentration of proteases and cytokines in the tears

5.3.3. Tear proteolytic activities

The activity of collagenases in the tear samples of BR and AR of individual subjects were examined and the results are shown in Table 5.2. Collagenolytic activities in BR tear samples were not significantly (p > 0.05) different compared to the AR samples

(Table 5.2).

Table 5.2: Levels of total protein, inflammatory mediators, proteolytic activities in the tear samples.

Groups BR AR p-value Total protein 7.74 ± 1.52 8.03 ± 2.15 0.68 (mg/mL) MMP-13 (pg/mL) 51.86 ± 34.29 63 ± 36.80 0.006

Collagenase activity 5.02 ± 3.00 7.50 ± 3.90 0.14 (FIU/mg total protein) IL-6 (pg/mL) 1.25 ± 0.98 2.02 ± 1.52 0.004

TNF-α (pg/mL) 1.16 ± 0.74 1.45 ± 0.66 0.003

BR-Before eye rubbing; AR-After eye rubbing; Results are expressed as mean ± standard deviation.

5.4. Discussion

The expression of MMP-13 (Mackiewicz et al. 2006), protease activities (Kao et al.

1982; Rehany et al. 1982; Brown et al. 1993), IL-6 and TNF-α (Lema & Duran 2005;

Jun et al. 2011) have been previously examined to establish their involvement in of KC.

In Chapter 4, the tear film of KC showed increased levels of MMP-13, IL-6 and TNF-α when compared to normal controls. To establish a causal link between KC and eye rubbing, this study was designed to investigate the influence of eye rubbing on the tear

Changes to the tear film proteome in keratoconus 133 Chapter 5: Effects of eye rubbing on the concentration of proteases and cytokines in the tears levels of MMP-13, IL-6 and TNF-α and protease activities. Despite studies indicating a strong association between KC and eye rubbing (McMonnies & Boneham 2003;

McMonnies 2009; McMonnies et al. 2010), a cause and an effect relationship has not been recognized. The present study is the first experimental evidence to demonstrate significantly elevated levels of MMP-13, IL-6 and TNF-α in the tear film after eye rubbing.

Various case reports have explained the involvement of eye rubbing in KC (Koenig &

Smith 1993; Jafri et al. 2004; Ioannidis et al. 2005; Koenig 2008). Other case studies have linked eye rubbing in KC to nasolacrimal duct obstruction (Diniz et al. 2005), punctual agenesis (Lindsay et al. 2000) and Tourette syndrome (Kandarakis et al. 2011).

Although there is no experimental evidence to explain why eye rubbing might influence the progression of or susceptibility to KC, the close association between eye rubbing and KC reported in many studies (Copeman 1965; Karseras & Ruben 1976; Tretter et al. 1995) have meant clinicians often advise KC patients about the risk of aggravating the condition if they indulge in vigorous eye rubbing.

The ocular environment changes dynamically during eye rubbing. There is increased friction between palpebral conjunctiva of the under surface of upper eye lid and the ocular surface during closed eye rubbing. Animal model studies on eye rubbing have illustrated significant infiltration of inflammatory cells, degranulation of mast cells, surface irregularity of upper tarsal conjunctiva (Greiner et al. 1997).

The mechanical force exerted on the corneal surface might result in rubbing related epithelial trauma (McMonnies et al. 2010). The flattening or displacement of the cells during ocular rubbing might have a profound effect on the corneal expression of proteases or interleukins which could be reflected onto the tear film.

The increased levels of tear MMP-13, IL-6 and TNF- α after 60 s of closed eye,

Changes to the tear film proteome in keratoconus 134 Chapter 5: Effects of eye rubbing on the concentration of proteases and cytokines in the tears experimental eye rubbing on normal subjects seen in the present study could be due to the mechanical effects of ocular rubbing on the surface of the cornea. These proteases and inflammatory molecules have the potential to induce keratocyte apoptosis (Balkwill

& Burke 1989; Ollivier et al. 2007), which is the major form of cell death of keratocytes in KC cornea (Kim et al. 1999; Kaldawy et al. 2002).

Levels of MMP-13, IL-6 and TNF-α are known to be also elevated in AKC or VKC

(Leonardi et al. 1998; Leonardi et al. 2003; Leonardi et al. 2007; Shoji et al. 2007).

Immune mediated mechanisms triggered by environmental allergens are actively involved in AKC or VKC-related ocular rubbing. Mast cells of the conjunctiva play a central role in causing the signs and symptoms of ocular itching. The allergens bind to immunoglobin E (IgE) molecules on the surface of mast cells stimulating the release of histamine tempting to rub the eyes (Raizman et al. 2000). Along with the release of histamine, the mast cells also liberate cytokines leading to inflammatory changes in the cornea. Increased levels of serum IgE has been also reported in KC (Rahi et al. 1977;

Kemp & Lewis 1982). However, the type of itch-rub cycle seen in AKC or VKC is absent in KC (Krachmer 2005).

The temptation to rub the eyes in AKC or VKC is probably due to ocular itching unlike in KC where the motivating factors have been shown to be relief from burning or a gritty sensation (Carlson 2009). Nevertheless, AKC or VKC aggravate the progression of KC (Totan et al. 2001; Kaya et al. 2007), where a combination of both atopic-type and KC-type rubbing might have a significant role in these conditions.

Keratocytes produce IL-6 when exposed to TNF-α (Planck et al. 1994) and MMP-13 levels are increased in human epithelial cell cultures when treated with TNF-α (Li et al.

2003). As previously explained in Figure 4.1 the active interplay between proteases and cytokines might be aggravated during persistent eye rubbing seen in KC. McMonnies

Changes to the tear film proteome in keratoconus 135 Chapter 5: Effects of eye rubbing on the concentration of proteases and cytokines in the tears hypothesised that rubbing can cause spikes in corneal temperature that might increase collagenase activity (McMonnies 2009). Whilst, the activity of tear collagenases was not statistically different, an increase in the trend for collagenase activity related to eye rubbing was observed.

In summary, the levels of MMP-13, IL-6 and TNF-α were over-expressed after experimental eye rubbing. In ectatic corneal conditions such as KC, persistent eye rubbing might cause additional increase in the levels and activity of these mediators to many folds, contributing to the development or progression of the disease. Up-regulated proteases and inflammatory molecules could be the possible causal link between eye rubbing and KC. Elucidating the factors responsible for the habit of chronic central eye rubbing in KC is critical to identify the aetiology of the disease, which could be crucial in arresting its progression.

Changes to the tear film proteome in keratoconus 136 Chapter 6: Summary, conclusions and future directions

6. CHAPTER 6: SUMMARY, CONCLUSIONS AND FUTURE DIRECTIONS

Changes to the tear film proteome in keratoconus 137 Chapter 6: Summary, conclusions and future directions

6.1. Significance of research

The thinning of the cornea in KC has been well studied and has been documented to occur as a result of degradation of corneal collagen. However, the reason for this tissue degradation is not certain but has been hypothesized to be linked with proteases.

Early studies demonstrated elevated levels of collagenolytic and gelatinolytic activities in laboratory cultures of KC corneas (Table 1.3). Although, thought to be a non- inflammatory disease, inflammatory molecules are elevated in KC, and these inflammatory molecules might mediate the production and activation of proteases.

The role of proteases and cytokines in KC has been a subject of extensive discussion and speculation for many years. The thinning of the cornea during KC is likely to cause changes in proteins, proteolytic enzymes and cytokines to manifest in the tears of KC patients and not be present in people without KC. The aim of this thesis was, therefore, to understand the role and interactions of proteases and inflammatory molecules in the pathogenesis of KC using tear proteomics.

Due to the close proximity of the tear film to the ocular surface, analysing the tear proteome is indispensable in monitoring the health status of the cornea. Therefore, tears were used in this study as a non-invasive method to understand the thinning of the cornea in KC.

6.2. Novel findings

Chapter 2

 The levels of total protein, lactoferrin, sIgA and serum albumin levels were

quantified for the first time in the tears of KC.

Changes to the tear film proteome in keratoconus 138 Chapter 6: Summary, conclusions and future directions

 The levels of total protein, lactoferrin and sIgA were decreased in KC and this

decrease correlated with the severity of the disease.

Chapter 3

 The MF10 coupled with LTQ-FT showed relative over-expression of cathepsin

B and down-regulation of cystatin S, cystatin SN, PIGR and α-fibrinogen in the

tears of KC.

Chapter 4

 The levels of tear MMP- 1, -3, -7, and 13 were increased in KC patients.

 Tear proteolytic activity was examined for the first time in KC.

 Elevated collagenolytic and gelatinolytic activities were observed in the tears of

KC.

 The collagenolytic activity increased according to the severity of the disease.

 Cytokines such as IL- 4, -5, -6, -8, TNF-α, -β were over-expressed in KC when

compared to controls.

 First study to investigate the MMPs and cytokines in KC patients who had

undergone CXL procedure.

 Reduced protease levels and protease activity was observed after CXL.

 The level of TNF- α was significantly elevated in CXL patients compared to

controls.

Chapter 5

 This study, for the first time has established a causal link between eye rubbing

and KC through tear proteases, protease activity and cytokines.

 The levels of MMP-13, IL-6 and TNF-α were elevated after eye rubbing.

Changes to the tear film proteome in keratoconus 139 Chapter 6: Summary, conclusions and future directions

6.3. Summary

Open-eye, basal tears were collected using glass capillary tubes from normal and KC subjects for investigating the tear proteome in this thesis. All the tear samples were collected by the same experienced investigator and, the type of tears collected, tear collection time (between 10 am to 4 pm) and the tear collection technique were kept constant throughout the study, to avoid possible influence of these factors in the tear analysis.

During the early stages of this study, the most intriguing finding observed in the tear analysis of KC was the global reduction of the concentration of total proteins. Further examination of KC tear proteins showed significant decrease in the amount of lactoferrin and sIgA compared to normals. Neither age, gender, contact lens wear or history of atopy appeared to influence the tear protein changes observed in KC.

Lactoferrin and sIgA have anti-inflammatory and immune defence functions respectively and, lower levels of these proteins might indicate the contribution of inflammatory and immune mediated mechanisms in the pathophysiology of KC. The degenerated corneal nerves along with increased expression of proteolytic enzymes reported in KC (Brookes et al. 2003) might be the reason for tear protein changes seen in this study. Indeed, a follow up study from this research should investigate the association between changes in corneal sensitivity during KC and changes in tear film proteins.

The protein changes detected in the tears is crucial to understand the pathological events involved in KC corneas. Cathepsin B, a lysosomal cysteine protease was up-regulated and protease inhibitors such as cystatin S and cystatin SN were decreased in KC tears.

This imbalance between proteases and protease inhibitors might add to the increased

Changes to the tear film proteome in keratoconus 140 Chapter 6: Summary, conclusions and future directions apoptotic activity and increased cell turnover rates seen in the stromal keratocytes of

KC (Chwieralski et al. 2006).

The substantial increase of tear MMPs, ILs and TNFs confirms the important role of proteases and inflammatory molecules in the development of KC. Elevated levels of tear MMP-1, -3, -7, 13 reflects the over-expression of these proteases in KC corneas.

Higher levels of these proteases and its activities might have a significant impact in the arrangement of collagen fibrils in KC corneas.

Cytokines promote wound healing and the increased corneal tissue degradation in KC corneas might stimulate the expression of ILs and TNFs. This mechanism is evident with elevated levels of IL-4, -5, -6, -8 and TNF-α, -β in KC tears seen in the present study.

The CXL procedure is becoming a widely used technique for the management of KC.

Although, this treatment with UV-A/riboflavin reduces the tear protease expression levels and its activities in KC, the level of TNF-α was still elevated compared to normal tears. Since TNF-α triggers the expression of MMPs, CXL corneas may possibly be prone to recurrences.

The mechanical force exerted on the corneal surface during eye rubbing augments the level of MMP-13, IL- 6 and TNF-α in the tear film. Therefore, persistent eye rubbing seen in people with KC might increase the concentration and activities of these mediators many fold, aggravating the disease.

6.4. Implication of research

The changes in tear proteins or protease activity or cytokines identified in this study might form the basis for the development of non-invasive diagnostic or prognostic tests, specifically for KC. Such clinical tests could help in identifying the onset of the disease

Changes to the tear film proteome in keratoconus 141 Chapter 6: Summary, conclusions and future directions prior to its maturation and people at the risk of rapid progression. These tests could also be used in assessing people most likely to benefit from therapies such as CXL or determine the success of CXL. In addition to diagnostic or prognostic tests, this work might lead the way exploring the use of medical or conservative treatments to reduce or stop the progression of KC using potential protease inhibitors.

6.5. Limitation of the research and recommendations for future studies

In general, the small volume of tears collected (~5-10 µL), arduous tear collection method and the search for reliable tear testing techniques to study the large dynamic range of tear proteins are the main challenges faced by researchers in the tear laboratory today. Overcoming these obstacles is essential to use tear proteins as a routine diagnostic or a prognostic tool for ocular diseases in clinical practice.

Recruitment of large number of KC patients was a difficult task faced in this thesis. In the present study, the proportion of subjects recruited in the early stages of KC was less than 20%. Future work would look forward to include more subjects either in the early stage or subclinical (forme-fruste) KC. This would enable the use of tear proteomics to understand the early manifestations of the disease or in the early diagnosis of KC before the onset of clinical signs.

Basal tear samples were collected throughout the study to analyse the tear proteome.

Collection of basal tears using capillary tubes was tedious. The investigator took approximately 20-25 min to collect <10 µL of basal tears with a minimal tear flow rate from each eye. When collecting basal tears, it is also challenging to avoid accidental contact with the lids or lashes or ocular surface, which might induce reflex tearing and alter the tear proteome. An improvement in the basal tear collection technique is

Changes to the tear film proteome in keratoconus 142 Chapter 6: Summary, conclusions and future directions necessary to make it less demanding for the investigators and the subjects.

Due to the nature of the low volume of basal tears, the tear samples were pooled to generate adequate tear volume to examine the tear proteins using mass spectrometry and tear protease or cytokine levels using antibody array. Pooling the tear samples minimizes the chance of interpreting laboratory results to one patient or one subject.

The low sample volume of tears also restricted the study from determining the effect of

KC or CXL on the level of proteins such as lipocalin, lysozyme, plasmin and protease inhibitors such as alpha 1-antiprotease and alpha 2-macroglobin levels.

Although the investigator did not experience paucity of tear flow while collecting tears from KC or CXL or normal subjects, a test for dry eye was not included in this study.

Several of the protein changes, such as decreased total protein, lactoferrin, sIgA and increased level of cytokines seen with the KC subjects have also been seen associated with subjects with dry eye (Mackie & Seal 1984). However, dry-eye is associated with increased concentration of albumin in tears (Bron & Mengher 1989; Versura et al.

2010). In this study, there was a decrease in serum albumin concentration in KC tears, although this did not reach significance. These differences might be useful in differentiating KC from dry-eye conditions. Further studies into tear proteome are required to differentiate KC from dry-eye conditions.

KC is characterised by thickening of corneal nerves resulting in decreased corneal sensation (Zabala & Archila 1988; Patel et al. 2009). The degenerated nerves in KC corneas are involved in increased expression of proteolytic enzymes (Brookes et al.

2003) which might contribute to the changes in the tear protein level in KC. Studies investigating the association between changes in corneal nerves or corneal sensitivity and tear protein changes in KC are essential.

Changes to the tear film proteome in keratoconus 143 Chapter 6: Summary, conclusions and future directions

In the present study, the high abundant proteins were not depleted during MF-10. The presence of high abundant tear proteins decreases the chances of detecting other proteins that are less abundant in tears, which could provide valuable information about the ocular environment (Azzarolo et al. 2004). Future studies are essential to uncover the tear proteins present in low concentrations in KC by depleting high abundant proteins. Studies are also required to quantify, validate and verify the candidate tear peptide biomarkers reported in this study.

CXL procedure has been shown to reduce the protease levels and its activities in KC tears. However, the tears or severity of KC were not examined in subjects before CXL procedure in this study and hence, future studies may need to confirm the direct effect of cross linking on tear proteases.

A cause-and-effect relationship could not be established from this cross-sectional study and only a prospective study would allow such a determination, whereby tear samples should be analysed prior to disease onset, especially in people at the risk of developing

KC.

6.6. Conclusions

The work performed in this doctoral dissertation, involved isolating the tear proteins, proteases and cytokines that are significantly altered during KC, thereby contributing to the knowledge of the underlying molecular mechanisms in KC. The decreased levels of tear proteins, elevated levels of tear proteases, and proteolysis signify the extensive corneal tissue degenerative process in KC. The increased levels of tear cytokines reveals the chronic inflammatory events at work in the pathogenesis of the disease, disputing the definition of KC as a non-inflammatory disease. The habitual eye rubbing seen in

Changes to the tear film proteome in keratoconus 144 Chapter 6: Summary, conclusions and future directions people with KC might have deleterious effects on the stability of the collagen architecture by stimulating the expression of proteolytic enzymes and inflammatory mediators.

Changes to the tear film proteome in keratoconus 145 References

REFERENCES

7. Abalain JH, H Dossou, J Colin & HH Floch (2000): Levels of collagen degradation products (telopeptides) in the tear film of patients with keratoconus. Cornea 19: 474-476.

8. Abelson MB, AA Leonardi, LM Smith, IA Fregona, MA George & AG Secchi (1995): Histaminase activity in patients with vernal keratoconjunctivitis. Ophthalmology 102: 1958-1963.

9. Acera A, T Suarez, I Rodriguez-Agirretxe, E Vecino & JA Duran (2011): Changes in tear protein profile in patients with conjunctivochalasis. Cornea 30: 42-49.

10. Acera A, E Vecino, I Rodriguez-Agirretxe, K Aloria, JM Arizmendi, C Morales & JA Duran (2011): Changes in tear protein profile in keratoconus disease. Eye (Lond) 25: 1225-1233.

11. Alexander CM & Z Werb (1989): Proteinases and extracellular matrix remodeling. Curr Opin Cell Biol 1: 974-982.

12. Ambrosio R, Jr., LP Nogueira, DL Caldas, BM Fontes, A Luz, JO Cazal, MR Alves & MW Belin (2011): Evaluation of corneal shape and biomechanics before LASIK. Int Ophthalmol Clin 51: 11-38.

13. Ananthi S, T Chitra, R Bini, NV Prajna, P Lalitha & K Dharmalingam (2008): Comparative analysis of the tear protein profile in mycotic keratitis patients. Mol Vis 14: 500-507.

14. Applebaum A (1936): Keratoconus. Arch Ophthalmol.

15. Assouti M, DH Vynios, ST Anagnostides, G Papadopoulos, CD Georgakopoulos & SP Gartaganis (2006): Collagen type IX and HNK-1 epitope in tears of patients with pseudoexfoliation syndrome. Biochim Biophys Acta 1762: 54-58.

16. Avisar R, R Menache, P Shaked, J Rubinstein, I Machtey & H Savir (1979): Lysozyme content of tears in patients with Sjogren's syndrome and rheumatoid arthritis. Am J Ophthalmol 87: 148-151.

17. Azzarolo AM, K Brew, S Kota, O Ponomareva, J Schwartz & C Zylberberg (2004): Presence of tear lipocalin and other major proteins in lacrimal fluid of rabbits. Comp Biochem Physiol B Biochem Mol Biol 138: 111-117.

18. Baird PN, M Schache & M Dirani (2010): The GEnes in Myopia (GEM) study in understanding the aetiology of refractive errors. Progress in retinal and eye research 29: 520-542.

19. Balasubramanian SA, S Mohan, DC Pye & MD Willcox (2012): Proteases, proteolysis and inflammatory molecules in the tears of people with keratoconus. Acta Ophthalmol.

Changes to the tear film proteome in keratoconus 146 References

20. Balasubramanian SA, DC Pye & MD Willcox (2010): Are proteinases the reason for keratoconus? Curr Eye Res 35: 185-191.

21. Balasubramanian SA, DC Pye & MD Willcox (2011): Levels of lactoferrin, secretory IgA and serum albumin in the tear film of people with keratoconus. Exp Eye Res.

22. Balkwill FR (1989): Tumour necrosis factor. Br Med Bull 45: 389-400.

23. Balkwill FR & F Burke (1989): The cytokine network. Immunol Today 10: 299- 304.

24. Ballow M, PC Donshik, P Rapacz & L Samartino (1987): Tear lactoferrin levels in patients with external inflammatory ocular disease. Invest Ophthalmol Vis Sci 28: 543-545.

25. Barka T, PA Asbell, H van der Noen & A Prasad (1991): Cystatins in human tear fluid. Curr Eye Res 10: 25-34.

26. Barksby HE, JM Milner, AM Patterson, NJ Peake, W Hui, T Robson, R Lakey, J Middleton, TE Cawston, CD Richards & AD Rowan (2006): Matrix metalloproteinase 10 promotion of collagenolysis via procollagenase activation: implications for cartilage degradation in arthritis. Arthritis Rheum 54: 3244- 3253.

27. Bawazeer AM, WG Hodge & B Lorimer (2000): Atopy and keratoconus: a multivariate analysis. Br J Ophthalmol 84: 834-836.

28. Bechara SJ, GO Waring, 3rd & MS Insler (1996): Keratoconus in two pairs of identical twins. Cornea 15: 90-93.

29. Bechrakis N, ML Blom, WJ Stark & WR Green (1994): Recurrent keratoconus. Cornea 13: 73-77.

30. Becker J, S Salla, U Dohmen, C Redbrake & M Reim (1995): Explorative study of interleukin levels in the human cornea. Graefes Arch Clin Exp Ophthalmol 233: 766-771.

31. Beissbarth T, L Hyde, GK Smyth, C Job, WM Boon, SS Tan, HS Scott & TP Speed (2004): Statistical modeling of sequencing errors in SAGE libraries. Bioinformatics 20 Suppl 1: i31-39.

32. Benedetto DA, DO Shah & HE Kaufman (1975): The instilled fluid dynamics and surface chemistry of polymers in the preocular tear film. Invest Ophthalmol 14: 887-902.

33. Berquin IM & BF Sloane (1996): Cathepsin B expression in human tumors. Adv Exp Med Biol 389: 281-294.

34. Berta A, J Tozser & FJ Holly (1990): Determination of plasminogen activator activities in normal and pathological human tears. The significance of tear plasminogen activators in the inflammatory and traumatic lesions of the cornea

Changes to the tear film proteome in keratoconus 147 References

and the conjunctiva. Acta Ophthalmol (Copenh) 68: 508-514.

35. Beuerman RW & L Pedroza (1996): Ultrastructure of the human cornea. Microsc Res Tech 33: 320-335.

36. Bewick V, L Cheek & J Ball (2004): Statistics review 13: receiver operating characteristic curves. Crit Care 8: 508-512.

37. Bhuyan KC & DK Bhuyan (1970): Catalase in ocular tissue and its intracellular distribution in corneal epithelium. Am J Ophthalmol 69: 147-153.

38. Binder PS (2003): Ectasia after laser in situ keratomileusis. J Cataract Refract Surg 29: 2419-2429.

39. Bisceglia L, M Ciaschetti, P De Bonis, PA Campo, C Pizzicoli, C Scala, M Grifa, P Ciavarella, N Delle Noci, F Vaira, C Macaluso & L Zelante (2005): VSX1 mutational analysis in a series of Italian patients affected by keratoconus: detection of a novel mutation. Invest Ophthalmol Vis Sci 46: 39-45.

40. Bisceglia L, P De Bonis, C Pizzicoli, L Fischetti, A Laborante, M Di Perna, F Giuliani, N Delle Noci, L Buzzonetti & L Zelante (2009): Linkage analysis in keratoconus: replication of locus 5q21.2 and identification of other suggestive Loci. Invest Ophthalmol Vis Sci 50: 1081-1086.

41. Bjerrum KB & JU Prause (1994): Collection and concentration of tear proteins studied by SDS gel electrophoresis. Presentation of a new method with special reference to dry eye patients. Graefes Arch Clin Exp Ophthalmol 232: 402-405.

42. Bode W, C Fernandez-Catalan, F Grams, FX Gomis-Ruth, H Nagase, H Tschesche & K Maskos (1999): Insights into MMP-TIMP interactions. Ann N Y Acad Sci 878: 73-91.

43. Bonavida B, AT Sapse & EE Sercarz (1969): Specific tear prealbumin: a unique lachrymal protein absent from serum and other secretions. Nature 221: 375-376.

44. Brightbill FS, CNJ McGhee & PJ McDonnell (2009): Corneal surgery: theory, technique and tissue. Mosby.

45. Bromberg BB (1981): Autonomic control of lacrimal protein secretion. Invest Ophthalmol Vis Sci 20: 110-116.

46. Bron AJ (2001): The architecture of the corneal stroma. Br J Ophthalmol 85: 379-381.

47. Bron AJ & LS Mengher (1989): The ocular surface in keratoconjunctivitis sicca. Eye (Lond) 3 ( Pt 4): 428-437.

48. Bron AJ & JM Tiffany (1998): The meibomian glands and tear film lipids. Structure, function, and control. Adv Exp Med Biol 438: 281-295.

49. Brookes NH, IP Loh, GM Clover, CA Poole & T Sherwin (2003): Involvement of corneal nerves in the progression of keratoconus. Exp Eye Res 77: 515-524.

Changes to the tear film proteome in keratoconus 148 References

50. Brown D, MM Chwa, A Opbroek & MC Kenney (1993): Keratoconus corneas: increased gelatinolytic activity appears after modification of inhibitors. Curr Eye Res 12: 571-581.

51. Buck MR, DG Karustis, NA Day, KV Honn & BF Sloane (1992): Degradation of extracellular-matrix proteins by human cathepsin B from normal and tumour tissues. Biochem J 282 ( Pt 1): 273-278.

52. Burdon KP, DJ Coster, JC Charlesworth, RA Mills, KJ Laurie, C Giunta, AW Hewitt, P Latimer & JE Craig (2008): Apparent autosomal dominant keratoconus in a large Australian pedigree accounted for by digenic inheritance of two novel loci. Hum Genet 124: 379-386.

53. Bureau J, Y Pouliquen & G Lorans (1993): [Fibrocyte reaction to interleukin 1 stimulation in keratoconus. Original title: Fibrocyte response to interleukin 1 stimulation in keratoconus]. Klin Monbl Augenheilkd 203: 269-274.

54. Burgess TL & RB Kelly (1987): Constitutive and regulated secretion of proteins. Annu Rev Cell Biol 3: 243-293.

55. Carlson AN (2009): Keratoconus: Time to Rewrite the Textbooks. Review of Ophthalmology.

56. Carlson AN (2010): Expanding our understanding of eye rubbing and keratoconus. Cornea 29: 245.

57. Choy CK, P Cho, WY Chung & IF Benzie (2001): Water-soluble antioxidants in human tears: effect of the collection method. Invest Ophthalmol Vis Sci 42: 3130-3134.

58. Chwa M, SR Atilano, D Hertzog, H Zheng, J Langberg, DW Kim & MC Kenney (2008): Hypersensitive response to oxidative stress in keratoconus corneal fibroblasts. Invest Ophthalmol Vis Sci 49: 4361-4369.

59. Chwieralski CE, T Welte & F Buhling (2006): Cathepsin-regulated apoptosis. Apoptosis : an international journal on programmed cell death 11: 143-149.

60. Colin J, B Cochener, G Savary & F Malet (2000): Correcting keratoconus with intracorneal rings. J Cataract Refract Surg 26: 1117-1122.

61. Collier SA (2001): Is the corneal degradation in keratoconus caused by matrix- metalloproteinases? Clin Experiment Ophthalmol 29: 340-344.

62. Collier SA, MC Madigan & PL Penfold (2000): Expression of membrane-type 1 matrix metalloproteinase (MT1-MMP) and MMP-2 in normal and keratoconus corneas. Curr Eye Res 21: 662-668.

63. Copeman PW (1965): Eczema and keratoconus. Br Med J 2: 977-979.

64. Cox SW, BM Eley, M Kiili, A Asikainen, T Tervahartiala & T Sorsa (2006): Collagen degradation by interleukin-1beta-stimulated gingival fibroblasts is accompanied by release and activation of multiple matrix metalloproteinases and

Changes to the tear film proteome in keratoconus 149 References

cysteine proteinases. Oral diseases 12: 34-40.

65. Coyle JT (1984): Keratoconus and eye rubbing. Am J Ophthalmol 97: 527-528.

66. Craig JP & A Tomlinson (1997): Importance of the lipid layer in human tear film stability and evaporation. Optom Vis Sci 74: 8-13.

67. Cristina Kenney M & DJ Brown (2003): The cascade hypothesis of keratoconus. Cont Lens Anterior Eye 26: 139-146.

68. Cullen JF & HG Butler (1963): Mongolism (Down's Syndrome) and Keratoconus. Br J Ophthalmol 47: 321-330.

69. Dartt DA (1989): Signal transduction and control of lacrimal gland protein secretion: a review. Curr Eye Res 8: 619-636.

70. Dartt DA (2009): Neural regulation of lacrimal gland secretory processes: relevance in dry eye diseases. Progress in retinal and eye research 28: 155-177.

71. Dartt DA, P Bex, P D'Amore, R Dana, L Mcloon & J Niederkorn (2011): Ocular Periphery and Disorders. Elsevier Science.

72. Dastjerdi MH & H Hashemi (1998): A quantitative corneal topography index for detection of keratoconus. J Refract Surg 14: 427-436.

73. De Benedetto A, R Agnihothri, LY McGirt, LG Bankova & LA Beck (2009): Atopic dermatitis: a disease caused by innate immune defects? J Invest Dermatol 129: 14-30.

74. de Souza GA, LM Godoy & M Mann (2006): Identification of 491 proteins in the tear fluid proteome reveals a large number of proteases and protease inhibitors. Genome biology 7: R72.

75. Diniz CM, PF Tzelikis, A Rodrigues Junior, S Alvim Hda, RR Dantas & AR Figueredo (2005): [Unilateral keratoconus associated with continual eye rubbing due to nasolacrimal obstruction--case report]. Arq Bras Oftalmol 68: 122-125.

76. Dogru M, H Karakaya, H Ozcetin, H Erturk, A Yucel, A Ozmen, M Baykara & K Tsubota (2003): Tear function and ocular surface changes in keratoconus. Ophthalmology 110: 1110-1118.

77. Donnenfeld ED, A Schrier, HD Perry, HJ Ingraham, R Lasonde, A Epstein & B Farber (1996): Infectious keratitis with corneal perforation associated with corneal hydrops and contact lens wear in keratoconus. Br J Ophthalmol 80: 409- 412.

78. Ebihara N, A Matsuda, S Nakamura, H Matsuda & A Murakami (2011): Role of the IL-6 classic- and trans-signaling pathways in corneal sterile inflammation and wound healing. Invest Ophthalmol Vis Sci 52: 8549-8557.

79. Edrington TB, K Zadnik & JT Barr (1995): Keratoconus. Optometry clinics : the official publication of the Prentice Society 4: 65-73.

Changes to the tear film proteome in keratoconus 150 References

80. Edwards M, CN McGhee & S Dean (2001): The genetics of keratoconus. Clin Experiment Ophthalmol 29: 345-351.

81. Edwards M, CN McGhee & S Dean (2001): The genetics of keratoconus. Clin Experiment Ophthalmol 29: 345-351.

82. Ehlers N, S Heegaard, J Hjortdal, A Ivarsen, K Nielsen & JU Prause (2010): Morphological evaluation of normal human corneal epithelium. Acta Ophthalmol 88: 858-861.

83. Eperon S, A Sauty, R Lanz, A Leimgruber, F Lurati & Y Guex-Crosier (2004): Eotaxin-1 (CCL11) up-regulation in tears during seasonal allergic conjunctivitis. Graefes Arch Clin Exp Ophthalmol 242: 966-970.

84. Epstein AJ, TN de Castro, PR Laibson, EJ Cohen & CJ Rapuano (2006): Risk factors for the first episode of corneal graft rejection in keratoconus. Cornea 25: 1005-1011.

85. Ertan A & O Muftuoglu (2008): Keratoconus clinical findings according to different age and gender groups. Cornea 27: 1109-1113.

86. Evans V, C Vockler, M Friedlander, B Walsh & MD Willcox (2001): Lacryglobin in human tears, a potential marker for cancer. Clin Experiment Ophthalmol 29: 161-163.

87. Fabre EJ, J Bureau, Y Pouliquen & G Lorans (1991): Binding sites for human interleukin 1 alpha, gamma interferon and tumor necrosis factor on cultured fibroblasts of normal cornea and keratoconus. Curr Eye Res 10: 585-592.

88. Farber JM (1993): HuMig: a new human member of the chemokine family of cytokines. Biochem Biophys Res Commun 192: 223-230.

89. Ferrero-Miliani L, OH Nielsen, PS Andersen & SE Girardin (2007): Chronic inflammation: importance of NOD2 and NALP3 in interleukin-1beta generation. Clin Exp Immunol 147: 227-235.

90. Fini ME, JR Cook & R Mohan (1998): Proteolytic mechanisms in corneal ulceration and repair. Archives of dermatological research 290 Suppl: S12-23.

91. Fini ME, MT Girard & M Matsubara (1992): Collagenolytic/gelatinolytic enzymes in corneal wound healing. Acta Ophthalmol Suppl: 26-33.

92. Fini ME, BY Yue & J Sugar (1992): Collagenolytic/gelatinolytic metalloproteinases in normal and keratoconus corneas. Curr Eye Res 11: 849- 862.

93. Fink BA, H Wagner, K Steger-May, C Rosenstiel, T Roediger, TT McMahon, MO Gordon & K Zadnik (2005): Differences in keratoconus as a function of gender. Am J Ophthalmol 140: 459-468.

94. Flanagan JL & MD Willcox (2009): Role of lactoferrin in the tear film. Biochimie 91: 35-43.

Changes to the tear film proteome in keratoconus 151 References

95. Foronjy R, K Imai, T Shiomi, B Mercer, P Sklepkiewicz, J Thankachen, P Bodine & J D'Armiento (2010): The divergent roles of secreted frizzled related protein-1 (SFRP1) in lung morphogenesis and emphysema. Am J Pathol 177: 598-607.

96. Foulks GN & AJ Bron (2003): Meibomian gland dysfunction: a clinical scheme for description, diagnosis, classification, and grading. Ocul Surf 1: 107-126.

97. Franceschetti A (1965): Keratoconus. In (ed). In: King JH and McTigue JW (eds.) Book Keratoconus. In (ed). City. Butterworths: 152-168.

98. Franklin RM, KR Kenyon & TB Tomasi, Jr. (1973): Immunohistologic studies of human lacrimal gland: Localization of immunoglobulins, secretory component and lactoferrin. The Journal of Immunology 110: 984-992.

99. Fukuda M, RJ Fullard, MD Willcox, C Baleriola-Lucas, F Bestawros, D Sweeney & BA Holden (1996): Fibronectin in the tear film. Invest Ophthalmol Vis Sci 37: 459-467.

Fullard RJ & LG Carney (1984): Diurnal variation in human tear enzymes. Exp Eye Res 38: 15-26.

100. Fullard RJ & C Snyder (1990): Protein levels in nonstimulated and stimulated tears of normal human subjects. Invest Ophthalmol Vis Sci 31: 1119-1126.

101. Fullard RJ & D Tucker (1994): Tear protein composition and the effects of stimulus. Adv Exp Med Biol 350: 309-314.

102. Fullard RJ & DL Tucker (1991): Changes in human tear protein levels with progressively increasing stimulus. Invest Ophthalmol Vis Sci 32: 2290-2301.

103. Fullerton J, P Paprocki, S Foote, DA Mackey, R Williamson & S Forrest (2002): Identity-by-descent approach to gene localisation in eight individuals affected by keratoconus from north-west Tasmania, Australia. Hum Genet 110: 462-470.

104. Funnell CL, J Ball & BA Noble (2006): Comparative cohort study of the outcomes of deep lamellar keratoplasty and penetrating keratoplasty for keratoconus. Eye (Lond) 20: 527-532.

105. Fust A, A Veres, P Kiszel, ZZ Nagy, L Cervenak, B Csakany, E Maka, I Suveges & FH Grus (2003): Changes in tear protein pattern after photorefractive keratectomy. Eur J Ophthalmol 13: 525-531.

106. Gachon AM, J Richard & B Dastugue (1982): Human tears: normal protein pattern and individual protein determinations in adults. Curr Eye Res 2: 301- 308.

107. Gachon AM, P Verrelle, G Betail & B Dastugue (1979): Immunological and electrophoretic studies of human tear proteins. Exp Eye Res 29: 539-553.

108. Gasset AR, WL Houde & M Garcia-Bengochea (1978): Hard contact lens wear

Changes to the tear film proteome in keratoconus 152 References

as an environmental risk in keratoconus. Am J Ophthalmol 85: 339-341.

109. Gasymov OK, AR Abduragimov, P Prasher, TN Yusifov & BJ Glasgow (2005): Tear lipocalin: evidence for a scavenging function to remove lipids from the human corneal surface. Invest Ophthalmol Vis Sci 46: 3589-3596.

110. Georgiou T, CL Funnell, A Cassels-Brown & R O'Conor (2004): Influence of ethnic origin on the incidence of keratoconus and associated atopic disease in Asians and white patients. Eye (Lond) 18: 379-383.

111. Gillette TE & MR Allansmith (1980): Lactoferrin in human ocular tissues. Am J Ophthalmol 90: 30-37.

112. Giraud JP, Y Pouliquen, G Offret & P Payrau (1975): Statistical morphometric studies in normal human and rabbit corneal stroma. Exp Eye Res 21: 221-229.

113. Glasgow BJ, AR Abduragimov, TN Yusifov, OK Gasymov, J Horwitz, WL Hubbell & KF Faull (1998): A conserved disulfide motif in human tear lipocalins influences ligand binding. Biochemistry 37: 2215-2225.

114. Glasson MJ, F Stapleton, L Keay, D Sweeney & MD Willcox (2003): Differences in clinical parameters and tear film of tolerant and intolerant contact lens wearers. Invest Ophthalmol Vis Sci 44: 5116-5124.

115. Gocheva V, W Zeng, D Ke, D Klimstra, T Reinheckel, C Peters, D Hanahan & JA Joyce (2006): Distinct roles for cysteine cathepsin genes in multistage tumorigenesis. Genes Dev 20: 543-556.

116. Gonzalez V & PJ McDonnell (1992): Computer-assisted corneal topography in parents of patients with keratoconus. Arch Ophthalmol 110: 1413-1414.

117. Green-Church KB, KK Nichols, NM Kleinholz, L Zhang & JJ Nichols (2008): Investigation of the human tear film proteome using multiple proteomic approaches. Mol Vis 14: 456-470.

118. Greiner JV, CD Leahy, DA Welter, SL Hearn, TA Weidman & DR Korb (1997): Histopathology of the ocular surface after eye rubbing. Cornea 16: 327- 332.

119. Gross J & CM Lapiere (1962): Collagenolytic activity in amphibian tissues: a tissue culture assay. Proc Natl Acad Sci U S A 48: 1014-1022.

120. Grus FH, AJ Augustin, NG Evangelou & K Toth-Sagi (1998): Analysis of tear- protein patterns as a diagnostic tool for the detection of dry eyes. Eur J Ophthalmol 8: 90-97.

121. Guo Q & H Zhang (1995): Tear malate dehydrogenase, lactate dehydrogenase and their isoenzymes in normal Chinese subjects and patients of ocular surface disorders. Yan Ke Xue Bao 11: 61-64.

122. Gupta AK, GS Sarin, PA Lamba & P D'Souza (1986): Immunoassay of tear lysozyme in acute adenovirus conjunctivitis. Br J Ophthalmol 70: 439-441.

Changes to the tear film proteome in keratoconus 153 References

123. Gupta AK, GS Sarin, MD Mathur & B Ghosh (1988): Alpha 1-antitrypsin and serum albumin in tear fluids in acute adenovirus conjunctivitis. Br J Ophthalmol 72: 390-393.

124. Gupta AK, GS Sarin, MD Mathur & B Ghosh (1988): Alpha 1-antitrypsin and serum albumin in tear fluids in acute adenovirus conjunctivitis. Br J Ophthalmol 72: 390-393.

125. Ha NT, K Nakayasu, A Murakami, K Ishidoh & A Kanai (2004): Microarray analysis identified differentially expressed genes in keratocytes from keratoconus patients. Curr Eye Res 28: 373-379.

126. Haeckel C, S Krueger, F Buehling, D Broemme, K Franke, A Schuetze, I Roese & A Roessner (1999): Expression of cathepsin K in the human embryo and fetus. Dev Dyn 216: 89-95.

127. Hammerstein W (1972): [Keratoconus concurrent in identical twins]. Ophthalmologica 165: 449-452.

128. Hanemaaijer R, H Visser, P Koolwijk, T Sorsa, T Salo, LM Golub & VW van Hinsbergh (1998): Inhibition of MMP synthesis by doxycycline and chemically modified tetracyclines (CMTs) in human endothelial cells. Advances in dental research 12: 114-118.

129. Hanna C, DS Bicknell & JE O'Brien (1961): Cell turnover in the adult human eye. Arch Ophthalmol 65: 695-698.

130. Haro H, HC Crawford, B Fingleton, K Shinomiya, DM Spengler & LM Matrisian (2000): Matrix metalloproteinase-7-dependent release of tumor necrosis factor-alpha in a model of herniated disc resorption. The Journal of clinical investigation 105: 143-150.

131. Harper E, KJ Bloch & J Gross (1971): The zymogen of tadpole collagenase. Biochemistry 10: 3035-3041.

132. Heinz A, S Taddese, W Sippl, RH Neubert & CE Schmelzer (2011): Insights into the degradation of human elastin by matrilysin-1. Biochimie 93: 187-194.

133. Herber S, FH Grus, P Sabuncuo & AJ Augustin (2001): Two-dimensional analysis of tear protein patterns of diabetic patients. Electrophoresis 22: 1838- 1844.

134. Herber S, P Kalden, KF Kreitner, C Riedel, JD Rompe & M Thelen (2001): [MRI in chronic epicondylitis humeri radialis using 1.0 T equipment--contrast medium administration necessary?]. Rofo 173: 454-459.

135. Hettlich HJ, K Lucke & CF Kreiner (1992): Light-induced endocapsular polymerization of injectable lens refilling materials. German journal of ophthalmology 1: 346-349.

136. Heusel JW, RD Hanson, GA Silverman & TJ Ley (1991): Structure and expression of a cluster of human hematopoietic serine protease genes found on

Changes to the tear film proteome in keratoconus 154 References

chromosome 14q11.2. J Biol Chem 266: 6152-6158.

137. Hofstetter HW (1959): A keratoscopic survey of 13,395 eyes. Am J Optom Arch Am Acad Optom 36: 3-11.

138. Hogan MJ, JA Alvarado & JE Wedell (1971): Histology of the Human Eye. Philadelphia. W B Saunders.

139. Hollingsworth JG & N Efron (2005): Observations of banding patterns (Vogt striae) in keratoconus: a confocal microscopy study. Cornea 24: 162-166.

140. Holly FJ (1973): Formation and rupture of the tear film. Exp Eye Res 15: 515- 525.

141. Holly FJ & MA Lemp (1977): Tear physiology and dry eyes. Surv Ophthalmol 22: 69-87.

142. Horner JF (1869): Zur Behandlung des Keratoconus. Klinische Monatsblätter für Augenheilkunde.

143. Horvath ZS, GL Corthals, CW Wrigley & J Margolis (1994): Multifunctional apparatus for electrokinetic processing of proteins. Electrophoresis 15: 968-971.

144. Ihalainen A (1986): Clinical and epidemiological features of keratoconus genetic and external factors in the pathogenesis of the disease. Acta Ophthalmol Suppl 178: 1-64.

145. Ihalainen A, T Salo, H Forsius & L Peltonen (1986): Increase in type I and type IV collagenolytic activity in primary cultures of keratoconus cornea. Eur J Clin Invest 16: 78-84.

146. Im E & A Kazlauskas (2007): The role of cathepsins in ocular physiology and pathology. Exp Eye Res 84: 383-388.

147. Im E & A Kazlauskas (2007): The role of cathepsins in ocular physiology and pathology. Exp Eye Res 84: 383-388.

148. Imanishi J, K Kamiyama, I Iguchi, M Kita, C Sotozono & S Kinoshita (2000): Growth factors: importance in wound healing and maintenance of transparency of the cornea. Progress in retinal and eye research 19: 113-129.

149. Ingraham HJ, ED Donnenfeld & HD Perry (1991): Keratoconus with spontaneous perforation of the cornea. Arch Ophthalmol 109: 1651-1652.

150. Ioannidis AS, L Speedwell & KK Nischal (2005): Unilateral keratoconus in a child with chronic and persistent eye rubbing. Am J Ophthalmol 139: 356-357.

151. Ishidoh K & E Kominami (1995): Procathepsin L degrades extracellular matrix proteins in the presence of glycosaminoglycans in vitro. Biochem Biophys Res Commun 217: 624-631.

152. Ishihama Y, J Rappsilber & M Mann (2006): Modular stop and go extraction

Changes to the tear film proteome in keratoconus 155 References

tips with stacked disks for parallel and multidimensional Peptide fractionation in proteomics. Journal of proteome research 5: 988-994.

153. Ito A, A Mukaiyama, Y Itoh, H Nagase, IB Thogersen, JJ Enghild, Y Sasaguri & Y Mori (1996): Degradation of interleukin 1beta by matrix metalloproteinases. J Biol Chem 271: 14657-14660.

154. Iwamoto T & AG DeVoe (1976): Electron microscopical study of the Fleisher ring. Arch Ophthalmol 94: 1579-1584.

155. Jacob JT & B Ham (2008): Compositional Profiling and Biomarker Identification of the Tear Film. Ocul Surf 6: 175-185.

156. Jafri B, H Lichter & RD Stulting (2004): Asymmetric keratoconus attributed to eye rubbing. Cornea 23: 560-564.

157. James P (1997): Protein identification in the post-genome era: the rapid rise of proteomics. Q Rev Biophys 30: 279-331.

158. Jensen OL, BS Gluud & HS Birgens (1985): The concentration of lactoferrin in tears during post-operative ocular inflammation. Acta Ophthalmol (Copenh) 63: 341-345.

159. Jester JV, T Moller-Pedersen, J Huang, CM Sax, WT Kays, HD Cavangh, WM Petroll & J Piatigorsky (1999): The cellular basis of corneal transparency: evidence for 'corneal crystallins'. J Cell Sci 112 ( Pt 5): 613-622.

160. Jones DT, D Monroy & SC Pflugfelder (1997): A novel method of tear collection: comparison of glass capillary micropipettes with porous polyester rods. Cornea 16: 450-458.

161. Joseph R, OP Srivastava & RR Pfister (2011): Differential epithelial and stromal protein profiles in keratoconus and normal human corneas. Exp Eye Res 92: 282-298.

162. Jun AS, L Cope, C Speck, X Feng, S Lee, H Meng, A Hamad & S Chakravarti (2011): Subnormal cytokine profile in the tear fluid of keratoconus patients. PLoS One 6: e16437.

163. Kaldawy RM, J Wagner, S Ching & GM Seigel (2002): Evidence of apoptotic cell death in keratoconus. Cornea 21: 206-209.

164. Kallinikos P & N Efron (2004): On the etiology of keratocyte loss during contact lens wear. Invest Ophthalmol Vis Sci 45: 3011-3020.

165. Kamma-Lorger CS, C Boote, S Hayes, J Moger, M Burghammer, C Knupp, AJ Quantock, T Sorensen, E Di Cola, N White, RD Young & KM Meek (2010): Collagen and mature elastic fibre organisation as a function of depth in the human cornea and limbus. Journal of structural biology 169: 424-430.

166. Kandarakis A, M Karampelas, V Soumplis, C Panos, N Makris, S Kandarakis & D Karagiannis (2011): A case of bilateral self-induced keratoconus in a patient

Changes to the tear film proteome in keratoconus 156 References

with tourette syndrome associated with compulsive eye rubbing: case report. BMC Ophthalmol 11: 28.

167. Kao WW, CW Kao, AH Kaufman, KW Kombrinck, RL Converse, WV Good, TH Bugge & JL Degen (1998): Healing of corneal epithelial defects in plasminogen- and fibrinogen-deficient mice. Invest Ophthalmol Vis Sci 39: 502- 508.

168. Kao WW, JP Vergnes, J Ebert, CV Sundar-Raj & SI Brown (1982): Increased collagenase and gelatinase activities in keratoconus. Biochem Biophys Res Commun 107: 929-936.

169. Karseras AG & M Ruben (1976): Aetiology of keratoconus. Br J Ophthalmol 60: 522-525.

170. Kaufman PL, A Alm & FH Adler (2003): Adler's physiology of the eye: clinical application. Mosby.

171. Kawai S, T Nakajima, S Hokari, T Komoda & K Kawai (2002): Apolipoprotein A-I concentration in tears in diabetic retinopathy. Ann Clin Biochem 39: 56-61.

172. Kaya V, M Karakaya, CA Utine, S Albayrak, OF Oge & OF Yilmaz (2007): Evaluation of the corneal topographic characteristics of keratoconus with orbscan II in patients with and without atopy. Cornea 26: 945-948.

173. Kemp EG & CJ Lewis (1982): Immunoglobulin patterns in keratoconus with particular reference to total and specific IgE levels. Br J Ophthalmol 66: 717- 720.

174. Kennedy RH, WM Bourne & JA Dyer (1986): A 48-year clinical and epidemiologic study of keratoconus. Am J Ophthalmol 101: 267-273.

175. Kennedy RH, WM Bourne & JA Dyer (1986): A 48-year clinical and epidemiologic study of keratoconus. Am J Ophthalmol 101: 267-273.

176. Kenney CM & DJ Brown (2003): The cascade hypothesis of keratoconus. Cont Lens Anterior Eye 26: 139-146.

177. Kenney MC, M Chwa, A Alba, M Saghizadeh, ZS Huang & DJ Brown (1998): Localization of TIMP-1, TIMP-2, TIMP-3, gelatinase A and gelatinase B in pathological human corneas. Curr Eye Res 17: 238-246.

178. Kenney MC, M Chwa, M Escobar & D Brown (1989): Altered gelatinolytic activity by keratoconus corneal cells. Biochem Biophys Res Commun 161: 353- 357.

179. Kenney MC, M Chwa, AJ Opbroek & DJ Brown (1994): Increased gelatinolytic activity in keratoconus keratocyte cultures. A correlation to an altered matrix metalloproteinase-2/tissue inhibitor of metalloproteinase ratio. Cornea 13: 114- 124.

180. Kijlstra A (1990): The role of lactoferrin in the nonspecific immune response on

Changes to the tear film proteome in keratoconus 157 References

the ocular surface. Reg Immunol 3: 193-197.

181. Kijlstra A, SH Jeurissen & KM Koning (1983): Lactoferrin levels in normal human tears. Br J Ophthalmol 67: 199-202.

182. Kijlstra A & A Kuizenga (1994): Analysis and function of the human tear proteins. Adv Exp Med Biol 350: 299-308.

183. Kim HS, T Shang, Z Chen, SC Pflugfelder & DQ Li (2004): TGF-beta1 stimulates production of gelatinase (MMP-9), collagenases (MMP-1, -13) and stromelysins (MMP-3, -10, -11) by human corneal epithelial cells. Exp Eye Res 79: 263-274.

184. Kim SH, JW Mok, HS Kim & CK Joo (2008): Association of -31T>C and -511 C>T polymorphisms in the interleukin 1 beta (IL1B) promoter in Korean keratoconus patients. Mol Vis 14: 2109-2116.

185. Kim WJ, YS Rabinowitz, DM Meisler & SE Wilson (1999): Keratocyte apoptosis associated with keratoconus. Exp Eye Res 69: 475-481.

186. King-Smith PE, BA Fink, RM Hill, KW Koelling & JM Tiffany (2004): The thickness of the tear film. Curr Eye Res 29: 357-368.

187. Knauper V, H Will, C Lopez-Otin, B Smith, SJ Atkinson, H Stanton, RM Hembry & G Murphy (1996): Cellular mechanisms for human procollagenase-3 (MMP-13) activation. Evidence that MT1-MMP (MMP-14) and gelatinase a (MMP-2) are able to generate active enzyme. J Biol Chem 271: 17124-17131.

188. Koenig SB (2008): Bilateral recurrent self-induced keratoconus. Eye Contact Lens 34: 343-344.

189. Koenig SB & RW Smith (1993): Keratoconus and corneal hydrops associated with compulsive eye rubbing. Refract Corneal Surg 9: 383-384.

190. Kohlhaas M, E Spoerl, T Schilde, G Unger, C Wittig & LE Pillunat (2006): Biomechanical evidence of the distribution of cross-links in corneas treated with riboflavin and ultraviolet A light. J Cataract Refract Surg 32: 279-283.

191. Koller T, M Mrochen & T Seiler (2009): Complication and failure rates after corneal crosslinking. J Cataract Refract Surg 35: 1358-1362.

192. Komai Y & T Ushiki (1991): The three-dimensional organization of collagen fibrils in the human cornea and sclera. Invest Ophthalmol Vis Sci 32: 2244- 2258.

193. Koo BS, DY Lee, HS Ha, JC Kim & CW Kim (2005): Comparative analysis of the tear protein expression in blepharitis patients using two-dimensional electrophoresis. Journal of proteome research 4: 719-724.

194. Koretz JE, SA Strenk, LM Strenk & JL Semmlow (2004): Scheimpflug and high-resolution magnetic resonance imaging of the anterior segment: a comparative study. J Opt Soc Am A Opt Image Sci Vis 21: 346-354.

Changes to the tear film proteome in keratoconus 158 References

195. Krachmer JH (2004): Eye rubbing can cause keratoconus. Cornea 23: 539-540.

196. Krachmer JH, RS Feder & MW Belin (1984): Keratoconus and related noninflammatory corneal thinning disorders. Surv Ophthalmol 28: 293-322.

197. Krachmer JH, MJ Mannis & EJ Holland (2005): Cornea. Elsevier Mosby.

198. Kremer I, RC Eagle, CJ Rapuano & PR Laibson (1995): Histologic evidence of recurrent keratoconus seven years after keratoplasty. Am J Ophthalmol 119: 511-512.

199. Krijgsveld J, S Gauci, W Dormeyer & AJ Heck (2006): In-gel isoelectric focusing of peptides as a tool for improved protein identification. Journal of proteome research 5: 1721-1730.

200. Kusano K, C Miyaura, M Inada, T Tamura, A Ito, H Nagase, K Kamoi & T Suda (1998): Regulation of matrix metalloproteinases (MMP-2, -3, -9, and -13) by interleukin-1 and interleukin-6 in mouse calvaria: association of MMP induction with bone resorption. Endocrinology 139: 1338-1345.

201. Kymes SM, JJ Walline, K Zadnik & MO Gordon (2004): Quality of life in keratoconus. Am J Ophthalmol 138: 527-535.

202. Labiris G, E Kaloghianni, S Koukoula, A Zissimopoulos & VP Kozobolis (2011): Corneal melting after collagen cross-linking for keratoconus: a case report. J Med Case Reports 5: 152.

203. Laemmli UK (1970): Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685.

204. Lass JH, A Sugar, BA Benetz, RW Beck, M Dontchev, RL Gal, C Kollman, R Gross, E Heck, EJ Holland, MJ Mannis, I Raber, W Stark & RD Stulting (2010): Endothelial cell density to predict endothelial graft failure after penetrating keratoplasty. Arch Ophthalmol 128: 63-69.

205. Lee BS, WJ Stark & AS Jun (2011): Descemet-stripping automated endothelial keratoplasty: a successful alternative to repeat penetrating keratoplasty. Clin Experiment Ophthalmol 39: 195-200.

206. Lee LR, LW Hirst & G Readshaw (1995): Clinical detection of unilateral keratoconus. Aust N Z J Ophthalmol 23: 129-133.

207. Lema I, D Brea, R Rodriguez-Gonzalez, E Diez-Feijoo & T Sobrino (2010): Proteomic analysis of the tear film in patients with keratoconus. Mol Vis 16: 2055-2061.

208. Lema I & JA Duran (2005): Inflammatory molecules in the tears of patients with keratoconus. Ophthalmology 112: 654-659.

209. Lema I, T Sobrino, JA Duran, D Brea & E Diez-Feijoo (2009): Subclinical keratoconus and inflammatory molecules from tears. Br J Ophthalmol 93: 820- 824.

Changes to the tear film proteome in keratoconus 159 References

210. Leonardi A, F Borghesan, M DePaoli, M Plebani & AG Secchi (1998): Procollagens and inflammatory cytokine concentrations in tarsal and limbal vernal keratoconjunctivitis. Exp Eye Res 67: 105-112.

211. Leonardi A, P Brun, G Abatangelo, M Plebani & AG Secchi (2003): Tear levels and activity of matrix metalloproteinase (MMP)-1 and MMP-9 in vernal keratoconjunctivitis. Invest Ophthalmol Vis Sci 44: 3052-3058.

212. Leonardi A, P Brun, A Di Stefano, L Motterle & G Abatangelo (2007): Matrix metalloproteases in vernal keratoconjunctivitis, nasal polyps and allergic asthma. Clin Exp Allergy 37: 872-879.

213. Leonardi A, P Brun, M Tavolato, M Plebani, G Abatangelo & AG Secchi (2003): Tumor necrosis factor-alpha (TNF-alpha) in seasonal allergic conjunctivitis and vernal keratoconjunctivitis. Eur J Ophthalmol 13: 606-610.

214. Leonardi A, S Sathe, M Bortolotti, A Beaton & R Sack (2009): Cytokines, matrix metalloproteases, angiogenic and growth factors in tears of normal subjects and vernal keratoconjunctivitis patients. Allergy 64: 710-717.

215. Li DQ, BL Lokeshwar, A Solomon, D Monroy, Z Ji & SC Pflugfelder (2001): Regulation of MMP-9 production by human corneal epithelial cells. Exp Eye Res 73: 449-459.

216. Li DQ, D Meller, Y Liu & SC Tseng (2000): Overexpression of MMP-1 and MMP-3 by cultured conjunctivochalasis fibroblasts. Invest Ophthalmol Vis Sci 41: 404-410.

217. Li DQ, TY Shang, HS Kim, A Solomon, BL Lokeshwar & SC Pflugfelder (2003): Regulated expression of collagenases MMP-1, -8, and -13 and stromelysins MMP-3, -10, and -11 by human corneal epithelial cells. Invest Ophthalmol Vis Sci 44: 2928-2936.

218. Li N, N Wang, J Zheng, XM Liu, OW Lever, PM Erickson & L Li (2005): Characterization of human tear proteome using multiple proteomic analysis techniques. Journal of proteome research 4: 2052-2061.

219. Lim L, K Pesudovs & DJ Coster (2000): Penetrating keratoplasty for keratoconus: visual outcome and success. Ophthalmology 107: 1125-1131.

220. Lindsay RG, AS Bruce & IF Gutteridge (2000): Keratoconus associated with continual eye rubbing due to punctal agenesis. Cornea 19: 567-569.

221. Lindy O, YT Konttinen, T Sorsa, Y Ding, S Santavirta, A Ceponis & C Lopez- Otin (1997): Matrix metalloproteinase 13 (collagenase 3) in human rheumatoid synovium. Arthritis Rheum 40: 1391-1399.

222. Little JM, YM Centifanto & HE Kaufman (1969): Immunoglobulins in human tears. Am J Ophthalmol 68: 898-905.

223. Liu WC, SM Lee, AD Graham & MC Lin (2011): Effects of eye rubbing and breath holding on corneal biomechanical properties and intraocular pressure.

Changes to the tear film proteome in keratoconus 160 References

Cornea 30: 855-860.

224. Liu Y, X Peng, J Tan, DS Darling, HJ Kaplan & DC Dean (2008): Zeb1 mutant mice as a model of posterior corneal dystrophy. Invest Ophthalmol Vis Sci 49: 1843-1849.

225. Liu Z, AJ Huang & SC Pflugfelder (1999): Evaluation of corneal thickness and topography in normal eyes using the Orbscan corneal topography system. Br J Ophthalmol 83: 774-778.

226. Lowell FC & JM Carroll (1970): A study of the occurrence of atopic traits in patients with keratoconus. The Journal of allergy 46: 32-39.

227. Ly L & VC Wasinger (2008): Peptide enrichment and protein fractionation using selective electrophoresis. Proteomics 8: 4197-4208.

228. Ly L & VC Wasinger (2011): Protein and peptide fractionation, enrichment and depletion: tools for the complex proteome. Proteomics 11: 513-534.

229. Maatta M, O Kari, T Tervahartiala, J Wahlgren, S Peltonen, M Kari, P Rytila, M Saari & T Sorsa (2008): Elevated expression and activation of matrix metalloproteinase 8 in tear fluid in atopic blepharoconjunctivitis. Cornea 27: 297-301.

230. Mackie IA & DV Seal (1984): Diagnostic implications of tear protein profiles. Br J Ophthalmol 68: 321-324.

231. Mackiewicz Z, M Maatta, M Stenman, L Konttinen, T Tervo & YT Konttinen (2006): Collagenolytic proteinases in keratoconus. Cornea 25: 603-610.

232. Madlener M, WC Parks & S Werner (1998): Matrix metalloproteinases (MMPs) and their physiological inhibitors (TIMPs) are differentially expressed during excisional skin wound repair. Exp Cell Res 242: 201-210.

233. Maguire LJ & WM Bourne (1989): Corneal topography of early keratoconus. Am J Ophthalmol 108: 107-112.

234. Mai J, M Sameni, T Mikkelsen & BF Sloane (2002): Degradation of extracellular matrix protein tenascin-C by cathepsin B: an interaction involved in the progression of gliomas. Biological chemistry 383: 1407-1413.

235. Maier P, A Broszinski, U Heizmann, D Bohringer & T Reinhardau (2007): Active transforming growth factor-beta2 is increased in the aqueous humor of keratoconus patients. Mol Vis 13: 1198-1202.

236. Mansour AM & RS Haddad (2002): Corneal topography after ocular rubbing. Cornea 21: 756-758.

237. Markoulli M, E Papas, A Petznick & B Holden (2011): Validation of the flush method as an alternative to basal or reflex tear collection. Curr Eye Res 36: 198- 207.

Changes to the tear film proteome in keratoconus 161 References

238. Matthews FJ, SD Cook, MA Majid, AD Dick & VA Smith (2007): Changes in the balance of the tissue inhibitor of matrix metalloproteinases (TIMPs)-1 and -3 may promote keratocyte apoptosis in keratoconus. Exp Eye Res 84: 1125-1134.

239. Maumenee IH (1981): The eye in the Marfan syndrome. Trans Am Ophthalmol Soc 79: 684-733.

240. Maurice DM (1957): The structure and transparency of the cornea. J Physiol 136: 263-286.

241. Mazzotta C, C Traversi, S Baiocchi, O Caporossi, C Bovone, MC Sparano, A Balestrazzi & A Caporossi (2008): Corneal healing after riboflavin ultraviolet-A collagen cross-linking determined by confocal laser scanning microscopy in vivo: early and late modifications. Am J Ophthalmol 146: 527-533.

242. McCall AS, S Kraft, HF Edelhauser, GW Kidder, RR Lundquist, HE Bradshaw, Z Dedeic, MJ Dionne, EM Clement & GW Conrad (2010): Mechanisms of corneal tissue cross-linking in response to treatment with topical riboflavin and long-wavelength ultraviolet radiation (UVA). Invest Ophthalmol Vis Sci 51: 129-138.

243. McMahon TT, LS Kim, GA Fishman, EM Stone, XC Zhao, RW Yee & J Malicki (2009): CRB1 gene mutations are associated with keratoconus in patients with leber congenital amaurosis. Invest Ophthalmol Vis Sci 50: 3185- 3187.

244. McMahon TT, JA Shin, A Newlin, TB Edrington, J Sugar & K Zadnik (1999): Discordance for keratoconus in two pairs of monozygotic twins. Cornea 18: 444- 451.

245. McMonnies CW (2007): Abnormal rubbing and keratectasia. Eye Contact Lens 33: 265-271.

246. McMonnies CW (2008): Management of chronic habits of abnormal eye rubbing. Cont Lens Anterior Eye 31: 95-102.

247. McMonnies CW (2009): Mechanisms of rubbing-related corneal trauma in keratoconus. Cornea 28: 607-615.

248. McMonnies CW, A Alharbi & GC Boneham (2010): Epithelial responses to rubbing-related mechanical forces. Cornea 29: 1223-1231.

249. McMonnies CW & GC Boneham (2003): Keratoconus, allergy, itch, eye- rubbing and hand-dominance. Clin Exp Optom 86: 376-384.

250. McPherson SD, Jr. & GT Kiffney, Jr. (1968): Some histologic findings in keratoconus. Arch Ophthalmol 79: 669-673.

251. Meek KM, S Dennis & S Khan (2003): Changes in the refractive index of the stroma and its extrafibrillar matrix when the cornea swells. Biophys J 85: 2205- 2212.

Changes to the tear film proteome in keratoconus 162 References

252. Mejia-Barbosa Y & D Malacara-Hernandez (2001): A review of methods for measuring corneal topography. Optom Vis Sci 78: 240-253.

253. Mencucci R, M Marini, I Paladini, E Sarchielli, E Sgambati, U Menchini & GB Vannelli (2010): Effects of riboflavin/UVA corneal cross-linking on keratocytes and collagen fibres in human cornea. Clin Experiment Ophthalmol 38: 49-56.

254. Mestecky J & JR McGhee (1987): Immunoglobulin A (IgA): molecular and cellular interactions involved in IgA biosynthesis and immune response. Advances in immunology 40: 153-245.

255. Metz CE (1978): Basic principles of ROC analysis. Semin Nucl Med 8: 283- 298.

256. Millin JA, BM Golub & CS Foster (1986): Human basement membrane components of keratoconus and normal corneas. Invest Ophthalmol Vis Sci 27: 604-607.

257. Mishima S (1965): Some Physiological Aspects of the Precorneal Tear Film. Arch Ophthalmol 73: 233-241.

258. Mohamed MM & BF Sloane (2006): Cysteine cathepsins: multifunctional enzymes in cancer. Nature reviews. Cancer 6: 764-775.

259. Montes-Mico R (2007): Role of the tear film in the optical quality of the human eye. J Cataract Refract Surg 33: 1631-1635.

260. Murphy G & V Knauper (1997): Relating matrix metalloproteinase structure to function: why the "hemopexin" domain? Matrix Biol 15: 511-518.

261. Murphy G & H Nagase (2008): Progress in matrix metalloproteinase research. Molecular aspects of medicine 29: 290-308.

262. Murphy G, H Stanton, S Cowell, G Butler, V Knauper, S Atkinson & J Gavrilovic (1999): Mechanisms for pro matrix metalloproteinase activation. APMIS : acta pathologica, microbiologica, et immunologica Scandinavica 107: 38-44.

263. Murphy G, F Willenbrock, T Crabbe, M O'Shea, R Ward, S Atkinson, J O'Connell & A Docherty (1994): Regulation of matrix metalloproteinase activity. Ann N Y Acad Sci 732: 31-41.

264. Murphy GJ, G Murphy & JJ Reynolds (1991): The origin of matrix metalloproteinases and their familial relationships. FEBS letters 289: 4-7.

265. Murube J (2009): Basal, reflex, and psycho-emotional tears. Ocul Surf 7: 60-66.

266. Nagase H, R Visse & G Murphy (2006): Structure and function of matrix metalloproteinases and TIMPs. Cardiovascular research 69: 562-573.

267. Nagase H & JF Woessner, Jr. (1999): Matrix metalloproteinases. J Biol Chem 274: 21491-21494.

Changes to the tear film proteome in keratoconus 163 References

268. Nakamura Y, C Sotozono & S Kinoshita (1998): Inflammatory cytokines in normal human tears. Curr Eye Res 17: 673-676.

269. Nakayasu K, M Tanaka, H Konomi & T Hayashi (1986): Distribution of types I, II, III, IV and V collagen in normal and keratoconus corneas. Ophthalmic research 18: 1-10.

270. Neurath H & KA Walsh (1976): Role of proteolytic enzymes in biological regulation (a review). Proc Natl Acad Sci U S A 73: 3825-3832.

271. Newsome DA, JM Foidart, JR Hassell, JH Krachmer, MM Rodrigues & SI Katz (1981): Detection of specific collagen types in normal and keratoconus corneas. Invest Ophthalmol Vis Sci 20: 738-750.

272. Ng V, P Cho, F Wong & Y Chan (2001): Variability of tear protein levels in normal young adults: diurnal (daytime) variation. Graefes Arch Clin Exp Ophthalmol 239: 257-263.

273. Nguyen-Khuong T, A Fitzgerald, Z Zhao, M Willcox & BJ Walsh (2008): Improvements for the visualization of low-molecular weight protein and peptides of human tears using MALDI. Proteomics 8: 3424-3432.

274. Nichols JJ & KB Green-Church (2009): Mass spectrometry-based proteomic analyses in contact lens-related dry eye. Cornea 28: 1109-1117.

275. Nichols JJ & PE King-Smith (2003): Thickness of the pre- and post-contact lens tear film measured in vivo by interferometry. Invest Ophthalmol Vis Sci 44: 68- 77.

276. Nichols JJ, K Steger-May, TB Edrington & K Zadnik (2004): The relation between disease asymmetry and severity in keratoconus. Br J Ophthalmol 88: 788-791.

277. Nielsen K, K Birkenkamp-Demtroder, N Ehlers & TF Orntoft (2003): Identification of differentially expressed genes in keratoconus epithelium analyzed on microarrays. Invest Ophthalmol Vis Sci 44: 2466-2476.

278. Nottingham J (1854): Practical Observations on Conical Cornea: And on the Short Sight, and Other Defects of Vision Connected with it. London. J. Churchill.

279. Ohashi Y, R Ishida, T Kojima, E Goto, Y Matsumoto, K Watanabe, N Ishida, K Nakata, T Takeuchi & K Tsubota (2003): Abnormal protein profiles in tears with dry eye syndrome. Am J Ophthalmol 136: 291-299.

280. Ohuchi E, K Imai, Y Fujii, H Sato, M Seiki & Y Okada (1997): Membrane type 1 matrix metalloproteinase digests interstitial collagens and other extracellular matrix macromolecules. J Biol Chem 272: 2446-2451.

281. Old WM, K Meyer-Arendt, L Aveline-Wolf, KG Pierce, A Mendoza, JR Sevinsky, KA Resing & NG Ahn (2005): Comparison of label-free methods for quantifying human proteins by shotgun proteomics. Mol Cell Proteomics 4:

Changes to the tear film proteome in keratoconus 164 References

1487-1502.

282. Ollivier FJ, BC Gilger, KP Barrie, ME Kallberg, CE Plummer, S O'Reilly, KN Gelatt & DE Brooks (2007): Proteinases of the cornea and preocular tear film. Veterinary ophthalmology 10: 199-206.

283. Oshika T & SD Klyce (1998): Corneal topography in LASIK. Semin Ophthalmol 13: 64-70.

284. Palimeris D, D Droutsas & E Chimonidou (1981): Some observations on the pathogenesis and management of keratoconus Book Some observations on the pathogenesis and management of keratoconus. City: 927-931.

285. Pannebaker C, HL Chandler & JJ Nichols (2010): Tear proteomics in keratoconus. Mol Vis 16: 1949-1957.

286. Parker J, WW Ko, G Pavlopoulos, PJ Wolfe, YS Rabinowitz & ST Feldman (1996): Videokeratography of keratoconus in monozygotic twins. J Refract Surg 12: 180-183.

287. Pataa C, L Joyon & F Roucher (1970): [Ultrastructure of keratoconus]. Arch Ophtalmol Rev Gen Ophtalmol 30: 403-417.

288. Patel DV, JY Ku, R Johnson & CN McGhee (2009): Laser scanning in vivo confocal microscopy and quantitative aesthesiometry reveal decreased corneal innervation and sensation in keratoconus. Eye (Lond) 23: 586-592.

289. Pearson AR, B Soneji, N Sarvananthan & JH Sandford-Smith (2000): Does ethnic origin influence the incidence or severity of keratoconus? Eye (Lond) 14 ( Pt 4): 625-628.

290. Pflugfelder SC, D Jones, Z Ji, A Afonso & D Monroy (1999): Altered cytokine balance in the tear fluid and conjunctiva of patients with Sjogren's syndrome keratoconjunctivitis sicca. Curr Eye Res 19: 201-211.

291. Planck SR, XN Huang, JE Robertson & JT Rosenbaum (1994): Cytokine mRNA levels in rat ocular tissues after systemic endotoxin treatment. Invest Ophthalmol Vis Sci 35: 924-930.

292. Polson C, P Sarkar, B Incledon, V Raguvaran & R Grant (2003): Optimization of protein precipitation based upon effectiveness of protein removal and ionization effect in liquid chromatography-tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 785: 263-275.

293. Prakasam RK, M Schwiede, WW Hutz, RF Guthoff & O Stachs (2012): Corneal responses to eye rubbing with spectral domain optical coherence tomography. Curr Eye Res 37: 25-32.

294. Pramod NP, E Dhevahi, K Sudhamathi, K Kannan & SP Thyagarajan (1999): Tear secretory IgA: evaluation of usefulness as a diagnostic marker in herpetic keratitis. Ocular immunology and inflammation 7: 61-67.

Changes to the tear film proteome in keratoconus 165 References

295. Prause JU (1983): Serum antiproteases and polymorphonuclear leucocyte neutral collagenolytic protease in the tear fluid of patients with corneal ulcers treated with n-butylcyano acrylate glue. Acta Ophthalmol (Copenh) 61: 283-291.

296. Prydal JI, P Artal, H Woon & FW Campbell (1992): Study of human precorneal tear film thickness and structure using laser interferometry. Invest Ophthalmol Vis Sci 33: 2006-2011.

297. Pullum KW, MA Whiting & RJ Buckley (2005): Scleral contact lenses: the expanding role. Cornea 24: 269-277.

298. Rabinowitz YS (1998): Keratoconus. Surv Ophthalmol 42: 297-319.

299. Rabinowitz YS, L Dong & G Wistow (2005): Gene expression profile studies of human keratoconus cornea for NEIBank: a novel cornea-expressed gene and the absence of transcripts for aquaporin 5. Invest Ophthalmol Vis Sci 46: 1239- 1246.

300. Rabinowitz YS, JJ Garbus, C Garbus & PJ McDonnell (1991): Contact lens selection for keratoconus using a computer-assisted videophotokeratoscope. Clao J 17: 88-93.

301. Rabinowitz YS, IH Maumenee, MK Lundergan, E Puffenberger, D Zhu, S Antonarakis & CA Francomano (1992): Molecular genetic analysis in autosomal dominant keratoconus. Cornea 11: 302-308.

302. Rahi A, P Davies, M Ruben, D Lobascher & J Menon (1977): Keratoconus and coexisting atopic disease. Br J Ophthalmol 61: 761-764.

303. Raizman MB, JS Rothman, F Maroun & WM Rand (2000): Effect of eye rubbing on signs and symptoms of allergic conjunctivitis in cat-sensitive individuals. Ophthalmology 107: 2158-2161.

304. Rao GN, T John, N Ishida & JV Aquavella (1985): Recovery of corneal sensitivity in grafts following penetrating keratoplasty. Ophthalmology 92: 1408-1411.

305. Rapacz P, J Tedesco, PC Donshik & M Ballow (1988): Tear lysozyme and lactoferrin levels in giant papillary conjunctivitis and vernal conjunctivitis. Clao J 14: 207-209.

306. Rebenitsch RL, SM Kymes, JJ Walline & MO Gordon (2011): The lifetime economic burden of keratoconus: a decision analysis using a markov model. Am J Ophthalmol 151: 768-773 e762.

307. Redl B (2000): Human tear lipocalin. Biochim Biophys Acta 1482: 241-248.

308. Rehany U, M Lahav & S Shoshan (1982): Collagenolytic activity in keratoconus. Ann Ophthalmol 14: 751-754.

309. Robertson I (1975): Keratoconus and the Ehlers-Danlos syndrome: a new aspect of keratoconus. The Medical journal of Australia 1: 571-573.

Changes to the tear film proteome in keratoconus 166 References

310. Rodriguez-Ausin P, R Gutierrez-Ortega, A Arance-Gil, M Romero-Jimenez & G Fuentes-Paez (2011): Keratopathy after cross-linking for keratoconus. Cornea 30: 1051-1053.

311. Rodriguez I, I Lema, M Blanco, M Rodriguez-Yanez, R Leira & J Castillo (2010): Vascular retinal, neuroimaging and ultrasonographic markers of lacunar infarcts. International journal of stroke : official journal of the International Stroke Society 5: 360-366.

312. Roedl JB, S Bleich, U Schlotzer-Schrehardt, N von Ahsen, J Kornhuber, GO Naumann, FE Kruse & AG Junemann (2008): Increased homocysteine levels in tear fluid of patients with primary open-angle glaucoma. Ophthalmic Res 40: 249-256.

313. Romero-Jimenez M, J Santodomingo-Rubido & JS Wolffsohn (2010): Keratoconus: a review. Cont Lens Anterior Eye 33: 157-166; quiz 205.

314. Rubsamen PE & WM McLeish (1991): Keratoconus with acute hydrops and perforation. Brief case report. Cornea 10: 83-84.

315. Saari KM, E Aine, A Posz & M Klockars (1983): Lysozyme content of tears in normal subjects and in patients with external eye infections. Graefes Arch Clin Exp Ophthalmol 221: 86-88.

316. Sack RA, A Beaton, S Sathe, C Morris, M Willcox & B Bogart (2000): Towards a closed eye model of the pre-ocular tear layer. Progress in retinal and eye research 19: 649-668.

317. Sack RA, L Conradi, D Krumholz, A Beaton, S Sathe & C Morris (2005): Membrane array characterization of 80 chemokines, cytokines, and growth factors in open- and closed-eye tears: angiogenin and other defense system constituents. Invest Ophthalmol Vis Sci 46: 1228-1238.

318. Sack RA, KO Tan & A Tan (1992): Diurnal tear cycle: evidence for a nocturnal inflammatory constitutive tear fluid. Invest Ophthalmol Vis Sci 33: 626-640.

319. Saghizadeh M, DJ Brown, R Castellon, M Chwa, GH Huang, JY Ljubimova, S Rosenberg, KS Spirin, RB Stolitenko, W Adachi, S Kinoshita, G Murphy, LJ Windsor, MC Kenney & AV Ljubimov (2001): Overexpression of matrix metalloproteinase-10 and matrix metalloproteinase-3 in human diabetic corneas: a possible mechanism of basement membrane and integrin alterations. Am J Pathol 158: 723-734.

320. Salonen EM, T Tervo, E Torma, A Tarkkanen & A Vaheri (1987): Plasmin in tear fluid of patients with corneal ulcers: basis for new therapy. Acta Ophthalmol (Copenh) 65: 3-12.

321. Sathe S, M Sakata, AR Beaton & RA Sack (1998): Identification, origins and the diurnal role of the principal serine protease inhibitors in human tear fluid. Curr Eye Res 17: 348-362.

322. Sato H, T Takino, Y Okada, J Cao, A Shinagawa, E Yamamoto & M Seiki

Changes to the tear film proteome in keratoconus 167 References

(1994): A matrix metalloproteinase expressed on the surface of invasive tumour cells. Nature 370: 61-65.

323. Sawaguchi S, T Fukuchi, H Abe, T Kaiya, J Sugar & BY Yue (1998): Three- dimensional scanning electron microscopic study of keratoconus corneas. Arch Ophthalmol 116: 62-68.

324. Schirra F, SM Richards, M Liu, T Suzuki, H Yamagami & DA Sullivan (2006): Androgen regulation of lipogenic pathways in the mouse meibomian gland. Exp Eye Res 83: 291-296.

325. Schultz CL & KS Kunert (2000): Interleukin-6 levels in tears of contact lens wearers. Journal of interferon & cytokine research : the official journal of the International Society for Interferon and Cytokine Research 20: 309-310.

326. Schultz GS, JB Davis & RA Eiferman (1988): Growth factors and corneal epithelium. Cornea 7: 96-101.

327. Searle BC (2010): Scaffold: a bioinformatic tool for validating MS/MS-based proteomic studies. Proteomics 10: 1265-1269.

328. Sen DK, GS Sarin & K Saha (1977): Immunoglobulins in tear in trachoma patients. Br J Ophthalmol 61: 218-220.

329. Senaratne T & C Gilbert (2005): Conjunctivitis. Community Eye Health 18: 73- 75.

330. Sendide K, AE Deghmane, D Pechkovsky, Y Av-Gay, A Talal & Z Hmama (2005): Mycobacterium bovis BCG attenuates surface expression of mature class II molecules through IL-10-dependent inhibition of cathepsin S. Journal of immunology 175: 5324-5332.

331. Seppala HP, M Maatta, M Rautia, Z Mackiewicz, I Tuisku, T Tervo & YT Konttinen (2006): EMMPRIN and MMP-1 in keratoconus. Cornea 25: 325-330.

332. Shapiro MB, MM Rodrigues, MR Mandel & JH Krachmer (1986): Anterior clear spaces in keratoconus. Ophthalmology 93: 1316-1319.

333. Shi GP, JS Munger, JP Meara, DH Rich & HA Chapman (1992): Molecular cloning and expression of human alveolar macrophage cathepsin S, an elastinolytic cysteine protease. J Biol Chem 267: 7258-7262.

334. Shiomi T, V Lemaitre, J D'Armiento & Y Okada (2010): Matrix metalloproteinases, a disintegrin and metalloproteinases, and a disintegrin and metalloproteinases with thrombospondin motifs in non-neoplastic diseases. Pathol Int 60: 477-496.

335. Shipley JM, RL Wesselschmidt, DK Kobayashi, TJ Ley & SD Shapiro (1996): Metalloelastase is required for macrophage-mediated proteolysis and matrix invasion in mice. Proc Natl Acad Sci U S A 93: 3942-3946.

336. Shoham A, M Hadziahmetovic, JL Dunaief, MB Mydlarski & HM Schipper

Changes to the tear film proteome in keratoconus 168 References

(2008): Oxidative stress in diseases of the human cornea. Free Radic Biol Med 45: 1047-1055.

337. Shoji J, A Kawaguchi, A Gotoh, N Inada & M Sawa (2007): Concentration of soluble interleukin-6 receptors in tears of allergic conjunctival disease patients. Jpn J Ophthalmol 51: 332-337.

338. Sihota R & R Tandon (2011): Parsons' Diseases of the eye. India. Elsevier.

339. Sitaramamma T, S Shivaji & GN Rao (1998): HPLC analysis of closed, open, and reflex eye tear proteins. Indian J Ophthalmol 46: 239-245.

340. Skrzydlewska E, M Sulkowska, M Koda & S Sulkowski (2005): Proteolytic- antiproteolytic balance and its regulation in carcinogenesis. World J Gastroenterol 11: 1251-1266.

341. Smith PK, RI Krohn, GT Hermanson, AK Mallia, FH Gartner, MD Provenzano, EK Fujimoto, NM Goeke, BJ Olson & DC Klenk (1985): Measurement of protein using bicinchoninic acid. Anal Biochem 150: 76-85.

342. Smith VA, FJ Matthews, MA Majid & SD Cook (2006): Keratoconus: matrix metalloproteinase-2 activation and TIMP modulation. Biochim Biophys Acta 1762: 431-439.

343. Smith VA, H Rishmawi, H Hussein & DL Easty (2001): Tear film MMP accumulation and corneal disease. Br J Ophthalmol 85: 147-153.

344. Snell RS & MA Lemp (1998): Clinical anatomy of the eye. Blackwell Science.

345. Snoek-van Beurden PA & JW Von den Hoff (2005): Zymographic techniques for the analysis of matrix metalloproteinases and their inhibitors. Biotechniques 38: 73-83.

346. Song W, G Apodaca & K Mostov (1994): Transcytosis of the polymeric immunoglobulin receptor is regulated in multiple intracellular compartments. J Biol Chem 269: 29474-29480.

347. Sonoda S, E Uchino, K Nakao & T Sakamoto (2006): Inflammatory cytokine of basal and reflex tears analysed by multicytokine assay. Br J Ophthalmol 90: 120-122.

348. Spandau UH, A Toksoy, S Verhaart, R Gillitzer & FE Kruse (2003): High expression of chemokines Gro-alpha (CXCL-1), IL-8 (CXCL-8), and MCP-1 (CCL-2) in inflamed human corneas in vivo. Arch Ophthalmol 121: 825-831.

349. Spoerl E, G Wollensak & T Seiler (2004): Increased resistance of crosslinked cornea against enzymatic digestion. Curr Eye Res 29: 35-40.

350. Stabuc-Silih M, M Ravnik-Glavac, D Glavac, M Hawlina & M Strazisar (2009): Polymorphisms in COL4A3 and COL4A4 genes associated with keratoconus. Mol Vis 15: 2848-2860.

Changes to the tear film proteome in keratoconus 169 References

351. Strasser G & G Grabner (1982): The influence of long-term treatment with timolol on human tear lysozyme albumin content. Graefes Arch Clin Exp Ophthalmol 218: 93-95.

352. Strongin AY, I Collier, G Bannikov, BL Marmer, GA Grant & GI Goldberg (1995): Mechanism of cell surface activation of 72-kDa type IV collagenase. Isolation of the activated form of the membrane metalloprotease. J Biol Chem 270: 5331-5338.

353. Stuchell RN, JJ Feldman, RL Farris & ID Mandel (1984): The effect of collection technique on tear composition. Invest Ophthalmol Vis Sci 25: 374- 377.

354. Sugaya S, T Sakimoto, J Shoji & M Sawa (2011): Regulation of soluble interleukin-6 (IL-6) receptor release from corneal epithelial cells and its role in the ocular surface. Jpn J Ophthalmol 55: 277-282.

355. Sullivan DA, ME Stern, K Tsubota, DA Dart, RM Sullivan & B Britt Bromberg (2002): Lacrimal gland, Tear film, and dry eye syndromes 3: Basic science and Clinical relevance. New York. Kluwer Academic/Plenum Publishers.

356. Sutton G, C Hodge & CN McGhee (2008): Rapid visual recovery after penetrating keratoplasty for keratoconus. Clin Experiment Ophthalmol 36: 725- 730.

357. Sutton G, M Madigan, A Roufas & J McAvoy (2010): Secreted frizzled-related protein 1 (SFRP1) is highly upregulated in keratoconus epithelium: a novel finding highlighting a new potential focus for keratoconus research and treatment. Clin Experiment Ophthalmol 38: 43-48.

358. Suzuki M, S Amano, N Honda, T Usui, S Yamagami & T Oshika (2007): Longitudinal changes in corneal irregular astigmatism and visual acuity in eyes with keratoconus. Jpn J Ophthalmol 51: 265-269.

359. Syka JE, JA Marto, DL Bai, S Horning, MW Senko, JC Schwartz, B Ueberheide, B Garcia, S Busby, T Muratore, J Shabanowitz & DF Hunt (2004): Novel linear quadrupole ion trap/FT mass spectrometer: performance characterization and use in the comparative analysis of histone H3 post- translational modifications. Journal of proteome research 3: 621-626.

360. Takaishi H, T Kimura, S Dalal, Y Okada & J D'Armiento (2008): Joint diseases and matrix metalloproteinases: a role for MMP-13. Curr Pharm Biotechnol 9: 47-54.

361. Taleb S, D Lacasa, JP Bastard, C Poitou, R Cancello, V Pelloux, N Viguerie, A Benis, JD Zucker, JL Bouillot, C Coussieu, A Basdevant, D Langin & K Clement (2005): Cathepsin S, a novel biomarker of adiposity: relevance to atherogenesis. The FASEB journal : official publication of the Federation of American Societies for Experimental Biology 19: 1540-1542.

362. Temel A, H Kazokoglu & Y Taga (1991): Tear lysozyme levels in contact lens wearers. Ann Ophthalmol 23: 191-194.

Changes to the tear film proteome in keratoconus 170 References

363. Teng CC (1963): Electron microscope study of the pathology of keratoconus: I. Am J Ophthalmol 55: 18-47.

364. Thakur A & MD Willcox (2000): Contact lens wear alters the production of certain inflammatory mediators in tears. Exp Eye Res 70: 255-259.

365. Thakur A, MD Willcox & F Stapleton (1998): The proinflammatory cytokines and arachidonic acid metabolites in human overnight tears: homeostatic mechanisms. J Clin Immunol 18: 61-70.

366. Tomosugi N, K Kitagawa, N Takahashi, S Sugai & I Ishikawa (2005): Diagnostic potential of tear proteomic patterns in Sjogren's syndrome. Journal of proteome research 4: 820-825.

367. Toshitani A, S Imayama, Y Shimozono, T Yoshinaga, M Furue & Y Hori (1999): Reduced amount of secretory component of IgA secretion in tears of patients with atopic dermatitis. Journal of dermatological science 19: 134-138.

368. Totan Y, IF Hepsen, O Cekic, A Gunduz & E Aydin (2001): Incidence of keratoconus in subjects with vernal keratoconjunctivitis: a videokeratographic study. Ophthalmology 108: 824-827.

369. Tran MT, M Tellaetxe-Isusi, V Elner, RM Strieter, RN Lausch & JE Oakes (1996): Proinflammatory cytokines induce RANTES and MCP-1 synthesis in human corneal keratocytes but not in corneal epithelial cells. Beta-chemokine synthesis in corneal cells. Invest Ophthalmol Vis Sci 37: 987-996.

370. Tretter TM, YS Rabinowitz, H Yang & JI Rotter (1995): Etiological factors in keratoconus. Ophthalmology (Suppl) 102: 156.

371. Turk B, D Turk & V Turk (2000): Lysosomal cysteine proteases: more than scavengers. Biochim Biophys Acta 1477: 98-111.

372. Uchio E, SY Ono, Z Ikezawa & S Ohno (2000): Tear levels of interferon- gamma, interleukin (IL) -2, IL-4 and IL-5 in patients with vernal keratoconjunctivitis, atopic keratoconjunctivitis and allergic conjunctivitis. Clinical and experimental allergy : journal of the British Society for Allergy and Clinical Immunology 30: 103-109.

373. Udar N, SR Atilano, DJ Brown, B Holguin, K Small, AB Nesburn & MC Kenney (2006): SOD1: a candidate gene for keratoconus. Invest Ophthalmol Vis Sci 47: 3345-3351.

374. Van den Steen PE, B Dubois, I Nelissen, PM Rudd, RA Dwek & G Opdenakker (2002): Biochemistry and molecular biology of gelatinase B or matrix metalloproteinase-9 (MMP-9). Crit Rev Biochem Mol Biol 37: 375-536.

375. Van Haeringen NJ (1981): Clinical biochemistry of tears. Surv Ophthalmol 26: 84-96.

376. van Haeringen NJ & E Glasius (1976): The origin of some enzymes in tear fluid, determined by comparative investigation with two collection methods.

Changes to the tear film proteome in keratoconus 171 References

Exp Eye Res 22: 267-272.

377. van Setten GB, R Stephens, T Tervo, EM Salonen, A Tarkkanen & A Vaheri (1990): Effects of the Schirmer test on the fibrinolytic system in the tear fluid. Exp Eye Res 50: 135-141.

378. van Setten GB, T Tervo, L Viinikka, K Pesonen, J Perheentupa & A Tarkkanen (1991): Ocular disease leads to decreased concentrations of epidermal growth factor in the tear fluid. Curr Eye Res 10: 523-527.

379. Versura P, P Nanni, A Bavelloni, WL Blalock, M Piazzi, A Roda & EC Campos (2010): Tear proteomics in evaporative dry eye disease. Eye (Lond) 24: 1396-1402.

380. Versura P, V Profazio & EC Campos (2010): Performance of tear osmolarity compared to previous diagnostic tests for dry eye diseases. Curr Eye Res 35: 553-564.

381. Vogt A (1919): Reflexlinien durch faltung spielgelnder der grenzflachen im bereiche von corneo, linsenkapsel und netzhaut. Albrecht von Graefes Arch Ophthalmol: 296-338.

382. Wagoner MD & R Ba-Abbad (2009): Penetrating keratoplasty for keratoconus with or without vernal keratoconjunctivitis. Cornea 28: 14-18.

383. Wagoner MD, SD Smith, WJ Rademaker & MA Mahmood (2001): Penetrating keratoplasty vs. epikeratoplasty for the surgical treatment of keratoconus. J Refract Surg 17: 138-146.

384. Wang M & TS Swartz (2009): Keratoconus and Keratoectasia: Prevention, Diagnosis, and Treatment. SLACK.

385. Wang Y, YS Rabinowitz, JI Rotter & H Yang (2000): Genetic epidemiological study of keratoconus: evidence for major gene determination. Am J Med Genet 93: 403-409.

386. Wasinger V, L Ly, A Fitzgerald & B Walsh (2008): Prefractionation, enrichment, desalting and depleting of low volume and low abundance proteins and peptides using the MF10. Methods Mol Biol 424: 257-275.

387. Wasinger VC, VL Locke, MJ Raftery, M Larance, D Rothemund, A Liew, I Bate & M Guilhaus (2005): Two-dimensional liquid chromatography/tandem mass spectrometry analysis of Gradiflow fractionated native human plasma. Proteomics 5: 3397-3401.

388. Weed KH, CJ MacEwen & CN McGhee (2006): The variable expression of keratoconus within monozygotic twins: dundee University Scottish Keratoconus Study (DUSKS). Cont Lens Anterior Eye 29: 123-126.

389. Weed KH & CN McGhee (1998): Referral patterns, treatment management and visual outcome in keratoconus. Eye (Lond) 12 ( Pt 4): 663-668.

Changes to the tear film proteome in keratoconus 172 References

390. Whitelock RB, T Fukuchi, L Zhou, SS Twining, J Sugar, RS Feder & BY Yue (1997): Cathepsin G, acid phosphatase, and alpha 1-proteinase inhibitor messenger RNA levels in keratoconus corneas. Invest Ophthalmol Vis Sci 38: 529-534.

391. Whitelock RB, Y Li, LL Zhou, J Sugar & BY Yue (1997): Expression of transcription factors in keratoconus, a cornea-thinning disease. Biochem Biophys Res Commun 235: 253-258.

392. Wilkesman J & L Kurz (2009): Protease analysis by zymography: a review on techniques and patents. Recent Pat Biotechnol 3: 175-184.

393. Williams KA, MT Lowe, MC Keane, VJ Jones, RS Loh & DJ Coster (2012): The Australian Corneal Graft Registry 2012 Report.

394. Wilson MJ (1970): Structure of the corneal stroma. Vision Res 10: 519-520.

395. Wilson SE, YG He, J Weng, Q Li, AW McDowall, M Vital & EL Chwang (1996): Epithelial injury induces keratocyte apoptosis: hypothesized role for the interleukin-1 system in the modulation of corneal tissue organization and wound healing. Exp Eye Res 62: 325-327.

396. Wilson SE, RR Mohan, R Ambrosio, Jr., J Hong & J Lee (2001): The corneal wound healing response: cytokine-mediated interaction of the epithelium, stroma, and inflammatory cells. Progress in retinal and eye research 20: 625- 637.

397. Wisithphrom K & LJ Windsor (2006): The effects of tumor necrosis factor- alpha, interleukin-1beta, interleukin-6, and transforming growth factor-beta1 on pulp fibroblast mediated collagen degradation. J Endod 32: 853-861.

398. Woessner JF, Jr. (2002): MMPs and TIMPs--an historical perspective. Molecular biotechnology 22: 33-49.

399. Wolf EJ, KJ Wolf & LZ Kleiman (2009): An atypical presentation of acanthamoeba keratitis in a patient with keratoconus treated briefly with topical anesthetic. Eye Contact Lens 35: 38-40.

400. Wollensak G, E Spoerl & T Seiler (2003): Riboflavin/ultraviolet-a-induced collagen crosslinking for the treatment of keratoconus. Am J Ophthalmol 135: 620-627.

401. Wollensak G, M Wilsch, E Spoerl & T Seiler (2004): Collagen fiber diameter in the rabbit cornea after collagen crosslinking by riboflavin/UVA. Cornea 23: 503-507.

402. Wong H, II Fatt & CJ Radke (1996): Deposition and Thinning of the Human Tear Film. Journal of colloid and interface science 184: 44-51.

403. Wu K & Y Zhang (2007): Clinical application of tear proteomics: Present and future prospects. Proteomics Clin Appl 1: 972-982.

Changes to the tear film proteome in keratoconus 173 References

404. Xue ML, D Wakefield, MD Willcox, AR Lloyd, N Di Girolamo, N Cole & A Thakur (2003): Regulation of MMPs and TIMPs by IL-1beta during corneal ulceration and infection. Invest Ophthalmol Vis Sci 44: 2020-2025.

405. Yamada M, H Mochizuki, M Kawai, K Tsubota & TJ Bryce (2005): Decreased tear lipocalin concentration in patients with meibomian gland dysfunction. Br J Ophthalmol 89: 803-805.

406. Yamada M, M Ogata, M Kawai & Y Mashima (2000): Decreased substance P concentrations in tears from patients with corneal hypesthesia. Am J Ophthalmol 129: 671-672.

407. Yeung K, F Eghbali & BA Weissman (1995): Clinical experience with piggyback contact lens systems on keratoconic eyes. J Am Optom Assoc 66: 539-543.

408. Yoo HG, BA Shin, JS Park, KH Lee, KO Chay, SY Yang, BW Ahn & YD Jung (2002): IL-1beta induces MMP-9 via reactive oxygen species and NF-kappaB in murine macrophage RAW 264.7 cells. Biochem Biophys Res Commun 298: 251-256.

409. Yoon KC, IY Jeong, YG Park & SY Yang (2007): Interleukin-6 and tumor necrosis factor-alpha levels in tears of patients with dry eye syndrome. Cornea 26: 431-437.

410. Zabala M & EA Archila (1988): Corneal sensitivity and topogometry in keratoconus. Clao J 14: 210-212.

411. Zadnik K, JT Barr, TB Edrington, DF Everett, M Jameson, TT McMahon, JA Shin, JL Sterling, H Wagner & MO Gordon (1998): Baseline findings in the Collaborative Longitudinal Evaluation of Keratoconus (CLEK) Study. Invest Ophthalmol Vis Sci 39: 2537-2546.

412. Zhao H, JE Jumblatt, TO Wood & MM Jumblatt (2001): Quantification of MUC5AC protein in human tears. Cornea 20: 873-877.

413. Zhou L, RW Beuerman, LP Ang, CM Chan, SF Li, FT Chew & DT Tan (2009): Elevation of human alpha-defensins and S100 calcium-binding proteins A8 and A9 in tear fluid of patients with pterygium. Invest Ophthalmol Vis Sci 50: 2077- 2086.

414. Zhou L, RW Beuerman, A Barathi & D Tan (2003): Analysis of rabbit tear proteins by high-pressure liquid chromatography/electrospray ionization mass spectrometry. Rapid Commun Mass Spectrom 17: 401-412.

415. Zhou L, RW Beuerman, Y Foo, S Liu, LP Ang & DT Tan (2006): Characterisation of human tear proteins using high-resolution mass spectrometry. Ann Acad Med Singapore 35: 400-407.

416. Zhou L, S Sawaguchi, SS Twining, J Sugar, RS Feder & BY Yue (1998): Expression of degradative enzymes and protease inhibitors in corneas with keratoconus. Invest Ophthalmol Vis Sci 39: 1117-1124.

Changes to the tear film proteome in keratoconus 174 References

417. Zhou L, RB Whitelock, B Yue & J Sugar (1996): Cytokine and receptor expression in keratoconus corneas. Invest Ophthalmol Vis Sci 37.

418. Zhou L, BY Yue, SS Twining, J Sugar & RS Feder (1996): Expression of wound healing and stress-related proteins in keratoconus corneas. Curr Eye Res 15: 1124-1131.

419. Zhou S, MJ Bailey, MJ Dunn, VR Preedy & PW Emery (2005): A quantitative investigation into the losses of proteins at different stages of a two-dimensional gel electrophoresis procedure. Proteomics 5: 2739-2747.

420.

Changes to the tear film proteome in keratoconus 175 Appendix

APPENDIX A: PARTICIPANT INFORMATION AND CONSENT FORMS

Approval No: 084081

Participant Information Statement and Consent Form

Severity and Progression of Keratoconus: Tear Film Analysis

Participant selection and Purpose of study

You ------are invited to participate in a study of Tear Film Analysis of Keratoconus. You have been selected as a possible participant in this study because you have been diagnosed with keratoconus/as a normal subject.

Description of Study and Risks

If you decide to participate in the study, we will collect 3-5 drops of tears by using a polished, blunt glass tube placed at the corner of your eye. This will take maximum 30 minutes of your time.

You may have redness or may feel slight irritation in the eye that may last for a few minutes.

Confidentiality and disclosure of information

Any information that is obtained in connection with this study and that can identify you, will remain confidential and will be disclosed with your permission, except as required by law. If you give us your permission by signing this document, we plan to discuss/publish the results in a Master’s Thesis, at conferences and in journals. In any publication, information will be provided in such a way that you cannot be identified.

Complaints may be directed to the Ethics Secretariat, The University of New South Wales, Sydney 2052, Australia (Phone:9385 4234, Fax: 9385 6648, Email: [email protected]) . Any complaint you make will be investigated promptly and you will be informed of the outcome.

Your Consent

Your decision whether or not to participate will not prejudice your future relations with the University of New South Wales. If you decide to participate you are free to withdraw your

Changes to the tear film proteome in keratoconus 176 Appendix consent and discontinue participation at any time.

If you have any questions, please feel free to ask us. If you have any additional quests later, Dr. Mark Willcox available on 9385 7412 or [email protected] will be happy to answer them.

You will be given a copy of this form. You are making a decision whether or not to participate. Your signature indicates that, having read the information provided above, you have decided to participate.

……………………………. ……………………………...

Signature of Research participant Signature of Witness

…………………………………… ……………………………………

Please PRINT name Please PRINT name

…………………………………….. ……………………………………

Date Nature of Witness

Revocation of Consent form

(Severity and Progression of Keratoconus: Tear Film Analysis)

I hereby wish to Withdraw my consent to participate in the research described above and understand that such a withdrawal Will Not jeopardise any treatment or relationship with the University of New South Wales.

……………………………… …………………………….

Signature Date

………………………………

Please PRINT name

This section of revocation of consent should be forwarded to:

Dr. Sivaraman Balasubramanian PhD Candidate, School of Optometry & Vision Science University of New South Wales, NSW 2052, Sydney, Australia. Ph: 93854536, Mobile: 98411 95381/0423738697

Changes to the tear film proteome in keratoconus 177 Appendix

HREA approval number: 11039

Patient Information Statement and Consent Form Effects of eye rubbing on tears Participant selection and Purpose of study

You ------are invited to participate in a study examining the effects of eye rubbing on proteins present in tears. You have been selected as a possible participant in this study because you have not been diagnosed with keratoconus, a condition in which the shape of the front surface of the eye is altered.

Description of Study and Risks

If you decide to participate in the study, you will be encouraged to rub your eyes for more than 15 seconds. We will collect 1 or 2 drops of tears by using a sterile, polished, blunt glass tube placed at the corner of your eye before and after your eye rubbing. This will take maximum 30 minutes of your time. You may feel slight irritation in the eye that may last for a few minutes.

Confidentiality and disclosure of information

Any information that is obtained in connection with this study and that can identify you, will remain confidential and will be disclosed with your permission, except as required by law. If you give us your permission by signing this document, we plan to discuss/publish the results in a PhD Thesis, at conferences and in journals. In any publication, information will be provided in such a way that you cannot be identified.

Complaints may be directed to the Ethics Secretariat, The University of New South Wales, Sydney 2052, Australia (Phone:9385 4234, Fax: 9385 6648, Email: [email protected]). Any complaint you make will be investigated promptly and you will be informed of the outcome.

Your Consent

Your decision whether or not to participate will not prejudice your future relations with the University of New South Wales. If you decide to participate you are free to withdraw your consent and discontinue participation at any time.

If you have any questions, please feel free to ask us. If you have any additional quests later, Prof. Mark Willcox available on 9385 7412 or [email protected] will be happy to answer them.

You are making a decision whether or not to participate. Your signature indicates that, having

Changes to the tear film proteome in keratoconus 178 Appendix read the information provided above, you have decided to participate.

……………………………. ……………………………..

Signature of Research participant Signature of Witness

…………………………………. …………………………………..

Please PRINT name Please PRINT name

…………………………………. ………………………………….

Date Nature of Witness

Revocation of Consent form

(Effects of eye rubbing on tears)

…………………………….. ……………………………..

Signature Date

………………………………….

Please PRINT name

This section of revocation of consent should be forwarded to:

Dr. Sivaraman Balasubramanian

PhD Candidate, School of Optometry & Vision Science

University of New South Wales, NSW 2052, Sydney, Australia.

Ph: (+61-2) 93857603 Mobile: 0423738697

Changes to the tear film proteome in keratoconus 179 Appendix

APPENDIX B: PUBLICATIONS AND PRESENTATIONS

PUBLISHED

 Balasubramanian S.A, Mohan S, Pye D, Willcox M.D. Proteases, proteolysis and inflammatory molecules in the tears of people with keratoconus. Acta Ophthalmologica (2012).

 Balasubramanian SA, Pye DC, Willcox MD. Levels of lactoferrin, secretory IgA and serum albumin in the tear film of people with keratoconus. Experimental Eye Research 96 (2012) 132-137.

 Balasubramanian S.A, Pye D, Willcox M.D. Are Proteinases the Reason for Keratoconus? Current Eye Research 35(3), 185–191, 2010.

 Balasubramanian S.A et al. Neuroretinitis with White Dot Chorioretinopathy. Journal of Tamil Nadu Ophthalmic Association 46(1):77-79, 2008.

SUBMITTED

 Balasubramanian SA, Pye DC, Willcox MD. Effect of eye rubbing on the concentration of proteases and cytokines in the tear film. Invited article written for the special issue on keratoconus in Clinical and Experimental Optometry.

 Balasubramanian SA, Valerie VW, Pye DC, Willcox MD. Differential expression of tear proteins in keratoconus. Molecular Vision.

PAPER PRESENTATIONS

 Proteases and Proteolysis in the tear film of keratoconus. European Association of Vision and Eye Research (EVER) congress, Crete, Greece. October 5-8, 2011.

 Are tear protein levels essential for good vision? One minute thesis competition at University of New South Wales, Sydney, Australia, August 4, 2011.

POSTER PRESENTATIONS

 Proteases and Proteolysis in the tear film of keratoconus. Royal Australian and New Zealand College of Ophthalmologist (RANZCO) congress, Canberra, Australia. November 19-22, 2011.

 Are tear protein levels essential for good vision? One minute thesis competition

Changes to the tear film proteome in keratoconus 180 Appendix

at University of New South Wales, Sydney, Australia, August 4, 2011.

 Tear protein levels in Keratoconus. Tear film and ocular surface society (TFOS), Florence. September 22-25, 2010.

Changes to the tear film proteome in keratoconus 181