C L I N I C A L A N D E X P E R I M E N T A L

OPTOMETRY
REVIEW

A new perspective on the pathobiology of keratoconus: interplay of

stromal wound healing and reactive species-associated processes

Clin Exp Optom 2013; 96: 188196 DOI:10.1111/cxo.12025

Isabella MY Cheung MSc PGDipSc
Charles NJ McGhee PhD FRCS FRCOphth
Trevor Sherwin PhD BSc(Hons)
Department of Ophthalmology, New Zealand
National Eye Centre, Faculty of Medical and Health
Sciences, University of Auckland, Auckland,
New Zealand
E-mail: i.cheung@auckland.ac.nz
Submitted: 31 July 2012
Revised: 3 October 2012
Accepted for publication:
30 October 2012
Severe loss of vision manifests from the corneal protrusion, thinning and distortion that
characterises keratoconus, which in its most severe form is still treated primarily by lamellar
or penetrating keratoplasty. Unfortunately, alternative therapeutic options targeting the
underlying pathobiology remain limited, attributable to an incomplete understanding of
the biological mechanisms instigating stromal deterioration and other disease processes. We
postulate that underlying abnormalities in stromal repair and reactive species-linked activi-
ties and the interaction between these phenomena are implicated in the development of
keratoconus. This revised interpretation of the pathophysiology may, with further investiga-
tion, advance our knowledge and the clinical management of this prevalent ectatic disorder.

Key words: cellular biology, cornea, disease mechanisms, keratoconus, molecular biology, reactive species, wound healing

Manifesting as a characteristic conical pro- resident keratocytes and collagen lamellae,

trusion of the cornea,1 globally keratoconus
is one of the leading indications for corneal
transplantation.2,3 Debilitating high myopia
and irregular astigmatism present con-
sequent upon localised, most commonly
inferior-temporal, stromal thinning.1 Most
currently implemented therapeutic inter-
ventions aim to restore visual function.
Fortunately, in approximately 90 per cent of
keratoconic patients, the visual impairment
which develops is successfully alleviated
with spectacles or specialised contact lenses.1
Cross-linking of the stromal collagen fibrils
is also increasingly employed to improve
the biomechanical strength of the weakened
cornea.4 Surgical options are often imple-
mented in advanced disease to reshape
the cornea or replace the degenerated
tissue.5 Corneal transplantation may be nec-
essary for 10 to 20 per cent of keratoconic
patients over their lifetime.1
Histopathologically, the triad of corneal
stromal thinning, rupture and filling of
Bowmans layer with basal epithelial cells
and deposition of ferritin in the degener-
ated basal epithelium classically character-
ises keratoconus.1 Also appreciable is the
quantitative and structural decline of the
of which the stroma is largely comprised.1,6
Despite extensive investigation, the precise
aetiology and mechanisms of disease remain
elusive; thus, no treatments targeting the
causative processes are currently available.
Many pathological features at the mole-
cular, cellular and histological levels have
been identified in the keratoconic cornea.
We propose that much of the published
biochemical changes in keratoconus cumu-
latively reflect defects at various points in
the corneal wound healing cascade. Addi-
tionally, oxidative stress has previously been
hypothesised as the origin of the pathophysi-
ology in keratoconus.7 To expand on this
contention, we also propose that repair and
oxidative pathways in the normal cornea
are linked and data supporting anomalous
reparative and oxidative processes and their
interaction in the development of kerato-
conus will be re-evaluated. We believe this
new perspective may lend credence to the
involvement of pro-inflammatory events
and enrich our comprehension of the
myriad disease mechanisms in this ectatic
disorder and consequently, with further
investigation, advance its management in
the clinic.
STROMAL REPAIR AND
THE KERATOCONIC CORNEA
Following a lesion, normal tissue integrity
is reinstated by clearing, repairing and
replacing damaged constituents. Complex
and intricately synchronised, key phases of
the wound healing cascade in the healthy
corneal stroma include apoptosis of kerato-
cytes, inflammation and extracellular matrix
(ECM) remodelling by repair fibroblasts.
Deviations in these bio-actions have been
reported in keratoconus and overall may be
the basis of the excessive and continual
repair perceptible in disease.
Apoptosis of keratocytes
Following a full thickness injury to a normal
cornea, wounded keratocytes undergo
controlled cell death, which subsequently
spreads to involve unaffected keratocytes,
forming an acellular zone in the stroma bor-
dering the site of injury.8 Wounded epithe-
lial cells induce apoptosis of keratocytes in
the stroma by releasing cytokines such as
tumour necrosis factor-alpha (TNF-a) and
interleukin-1 (IL-1).9,10 Positive feedback in
keratocytes also stimulates IL-1 synthesis in
the stroma.10 Later in the healing response,

Clinical and Experimental Optometry 96.2 March 2013 2013 The Authors
188 Clinical and Experimental Optometry 2013 Optometrists Association Australia

platelet-derived growth factor (PDGF),
epidermal growth factor (EGF) and insulin-
like growth factor originating from the
epithelium inhibit further keratocyte
death.10,11
Apoptosis is typically negligible in a heal-
thy unwounded cornea; however, signifi-
cant apoptotic death of keratocytes in
keratoconus has been substantiated by
numerous studies. Histologically, DNA
fragmentationa hallmark of programmed
cell deathis evident,12,13 as well as distinct
morphological characteristics, including
cell shrinkage, budding of apoptotic bodies
and chromatin condensation and frag-
mentation.12 The elevated transcription of
several factors in keratoconus, which facili-
tates various apoptotic processes has been
elucidated by gene expression analyses.
Demonstrated to play a part in the apopto-
sis of keratocytes in the cornea,14 possibly
through activation of the transcription
factor NF-KB (nuclear factor kappa-light-
chain-enhancer of activated B-cells),14 bone
morphogenic protein-4 also exhibits
chemo-attractive properties towards kerato-
cytes.15,16 Proteolysis by caspases, including
caspases 3, 4 and 6,17 activates endonucle-
ases and proteases to respectively mediate
the DNA and protein degradation necessary
for the structural alterations associated with
controlled cell death.18
Cofilin-1 is also involved in cellular re-
modelling19,20 and induces cell membrane
reorganisation by depolymerising the actin
cytoskeleton, which is required for initiation
of apoptotic death.15,21 The trans-membrane
protein PERP (p53 apoptosis effector re-
lated to PMP-22) may have a signalling
function in the promotion of DNA damage-
induced p53-mediated programmed cell
death,17,22 while the related factors epithelial
membrane proteins 1 and 2 facilitate apop-
totic membrane blebbing.17,23 Produced in
response to inflammatory mediators and
demonstrating anti-inflammatory effects in
the cornea,24,25 tumour necrosis factor alpha-
induced protein-6 is also implicated in p53-
independent apoptosis.24,26 Keratocytes from
corneas affected by keratoconus express a
higher number of interleukin-1 receptors.27
The induction of keratocyte apoptosis by
interleukin-1 in the cornea has been docu-
mented28 and this may occur through the
activation of Fas ligand.29 Mutation screen-
ing of keratoconic patients revealed an asso-
ciation of interleukin-1b polymorphisms
with the development of disease.30 Addition-
ally, interleukin-1 has a repulsive chemo-
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Clinical and Experimental Optometry 2013 Optometrists Association Australia
Pathobiology of keratoconus Cheung, McGhee and Sherwin
tactic effect on corneal keratocytes and imparting mechanical strength to the
modulates other physiological functions in cornea to maintain its correct curvature.44,45
these cells.16 A genome-wide linkage scan of Also abundant are the collagen-bound pro-
families affected by keratoconus identified teoglycans, decorin, lumican and keratocan,
the locus 14q11.2 to be associated with which regulate collagen fibril size, spacing
disease development; interestingly, encoded and hydration and thereby are imperative to
here is cell death-inducing DNA fragmenta- corneal transparency. During lesion repair,
tion factor A-like effector B.31 a provisional matrix is assembled with a com-
position and structure different from that of
the uninjured stroma. Collagen types IV and
Inflammation
VII, laminin, fibronectin and tenascin not
Modulated by transforming growth factor-b
found in the healthy stroma are deposited in
(TGF-b), inflammatory responses are also
a less organised arrangement.32,46 A reduc-
triggered in the early phases of wound
tion in keratocan synthesis parallels a rise
healing in the normal cornea. The rapid
in the production of decorin and chon-
infiltration of macrophages, T-cells and
droitin. This modified extracellular matrix
polymorphonuclear (PMN) cells from the
encourages fibroblast migration; however,
limbal vasculature resolves approximately
its altered biochemistry and structure may
48 hours after wounding.9,32 This reaction
cause corneal haze. Gradually, the provi-
probably functions to remove apoptotic
sional matrix is replaced by normal stromal
debris and ensures adequate anti-microbial
constituents including collagen types I and
defences due to the relatively immuno-
III to restore the physiological function of
privileged status of the cornea to maintain
the cornea.
its clarity.
Keratocytes with a distinctive bipolar
Proteomic analysis of tears from kerato-
morphology sparsely populate the normal
conic patients has established the presence
corneal stroma, producing collagen and
of many elevated inflammatory markers,
proteoglycans at basal quantities to maintain
including TNF-a, interleukin-17 (IL-17),
the extracellular matrix.44 Keratocytes bor-
interleukin-6 (IL-6) and intercellular ad-
dering an injury site extend new dendritic
hesion molecule-1 (ICAM-1).3335 A key
processes and migrate towards the lesion
mediator of acute inflammatory responses,
prior to differentiating into and proliferat-
TNF-a induces the release of other pro-
ing as repair fibroblasts, driven by TGF-b
inflammatory cytokines from keratocytes
and platelet-derived growth factor from the
following a corneal insult.36 Produced prima-
epithelium.8,9,32 The reorganisation of non-
rily by Th17 T-cells, IL-17 is implicated
muscle myosin and a-actinin into cytoskel-
in corneal inflammation and stimulates
etal stress fibres distinguishes fibroblasts,
stromal cells to produce pro-inflammatory
which encircle the wound and proliferate
cytokines.37 Both TNF-a and IL-17 induce
into a linked network of cells.47,48 An
the expression of IL-6, a pleiotropic cyto-
enlarged fusiform morphology and rise in
kine and potent trigger of the acute phase
organelle size and number reflects the
response and cell differentiation.37 In the
enhanced synthetic activity of fibroblasts.8
injured cornea, fibroblasts express recep-
The raised production of matrix metallopro-
tors for IL-6, while this cytokine also acti-
teinases (MMPs) removes damaged areas
vates and encourages the migration of
of extracellular matrix and remodels newly
epithelial cells.38,39 Moreover, the involve-
assembled regions, while the deposition of
ment of TNF-a, IL-17 and IL-6 in systemic
repair type extracellular matrix components
chronic inflammatory conditions is well
not present in the normal stroma leads
established.40,41 The cell adhesion molecu-
to fibrosis.49 The expression of adhe-
le ICAM-1 also participate in inflammation.
sion molecules is also increased; notably,
With the expression of ICAM-1 induced by
up-regulation of a5-integrin to form a recep-
IL-17,42 ICAM-1 molecules present on the
tor for fibronectin occurs concomitant with
surface of the keratocytes mediates stromal
the deposition of fibronectin in the wound.
interaction with polymorphonuclear leuko-
This may facilitate the formation of focal
cytes following corneal injury.43
adhesions to the extracellular matrix for
cell migration.50 A subset of fibroblasts may
Extracellular matrix remodelling further differentiate into myofibroblasts,
The normal stromal extracellular matrix is characterised by the presence of smooth
comprised mostly of collagen types I, III, V muscle actin and an additional increase in
and VI regularly interwoven into lamellae, stress fibres and focal adhesions.51 These
Clinical and Experimental Optometry 96.2 March 2013
189
Pathobiology of keratoconus Cheung, McGhee and Sherwin
alterations may support contraction of the
wound edges.52 Cell number and phenotype
largely revert to normal upon resolution of
healing; the loss of fibroblastic markers pre-
cedes the gradual reappearance of cytoplas-
mic granules and cell morphology typical of
normal keratocytes.8
Major structural alterations of the stromal
matrix in corneas affected by keratoconus
have been discerned by microscopic analy-
sis. Diminished corneal rigidity may be
a culmination of disorganised lamellar
arrangement and a decline in quantity and
diameter;5356 the latter brought about by a
reduction in the number of crosslinks within
and between collagen fibrils.6 Ascertained
by immunohistochemical studies, selected
stromal constituents are irregularly syn-
thesised in keratoconic corneas. Affected
corneas contain less collagen XII, which
regulates cell-matrix interaction and matrix
organisation.5759 Alluding to active repair,
the expression of collagen type XVIII by
keratocytes in the anterior stroma of corneas
with keratoconus is akin to that in scarred
corneas.60 Moreover, stromal scars in kerato-
conic corneas contained raised amounts
of myofibroblast-derived collagen XIII,61
which usually enables cell-matrix and cell-
cell adhesion.62
The levels of collagen and total protein
are diminished in corneas affected by kera-
toconus and the non-protein fraction is
augmented,6365 including a rise in several
proteoglycans. The observed excess of
decorin and its dermatan sulphate side
chain and thickened dermatan sulphate-
containing filaments could account for the
alterations in collagen fibril spacing and
diameter, as decorin binds type I collagen
to orchestrate its fibrillogenesis.66,67 More-
over, decorin may tangentially impinge
on healing by associating with TGF-b and
fibronectin.68,69 In contrast, a decline in the
amount of keratan sulfate and thinning of
keratan sulphate-bound filaments is appar-
ent in keratoconus, although the quantity
of keratocana prime keratan sulphate-
associated proteoglycanis consistently
pronounced.66,70 Combined with its highly
hydrated nature, keratocan in surplus may
modify extracellular matrix configuration
by intensifying its hydration.71 Persistent
corneal remodelling is further corrobora-
ted by the synthesis of tenascin in the
keratoconic stroma,72,73 in consort with
its induction by TGF-b1 and capability
to bind integrin.74,75 In keratoconus, kerato-
cytes appeared more fibroblast-like and
Clinical and Experimental Optometry 96.2 March 2013
190
total gelatinase and collagenase activity is
amplified in affected corneas.65,76 Intense
inquisition of the quantities and activities
of proteases and protease inhibitors
matrix metalloproteinases and their corre-
sponding tissue inhibitor of MMPs (TIMPs)
in particularensued as a plausible ac-
count for the progression of keratoconus;
however, descriptions of peculiarities in
these molecules in keratoconus remain at
odds.77
REACTIVE SPECIES, ANTIOXIDANTS
AND OXIDATIVE STRESS IN THE
NON-DISEASED CORNEA
In addition to aberrations in repair
mechanisms, an expanding number of
investigations evince the association of free
radical-mediated damage in the develop-
ment of keratoconus.
Reactive species
Intense exposure of the cornea to ultravio-
let (UV) light and high oxygen tension
encourages the generation of reactive
oxygen species (ROS).78 These trigger cell
and tissue destruction, attenuating the
integrity and viability of the cornea. Both
exogenous (for example, irradiation) and
endogenous sources (predominantly the
mitochondrial electron transport chain and
oxidative protein folding in the endoplasmic
reticulum) yield reactive oxygen species
through the partial reduction of oxygen.7983
The electrophilic nature of these molecules
brings about oxidative injury to biological
nucleophilesprincipally lipids, proteins
and DNA. Of the reactive oxygen species,
hydrogen peroxide, singlet oxygen and
oxygen are central, while superoxide anions
and hydroxyl radicals are the core free radi-
cals. Peroxidation of polyunsaturated fatty
acids in membrane phospholipids and lipo-
proteins by these entities additionally gener-
ate immensely toxic aldehydes, stimulating
extra modifications of biomolecules.
Antioxidants
Oxidative stress transpires when the pres-
ence of injurious molecules surpasses the
capability to counteract them. Antioxidants
in copious measure preserve corneal con-
figuration and function. Metabolically active
and the initial barrier, the corneal epi-
thelium synthesises the preponderance of
these neutralising molecules constitutively
with a secondary upsurge succeeding an
Clinical and Experimental Optometry 2013 Optometrists Association Australia
oxidative affront. The corneal endothelium
likewise has augmented quantities of anti-
oxidants to additionally safeguard more
posterior ocular structures. The enzymes
superoxide dismutase (SOD), catalase, glu-
tathione peroxidise, nicotinamide adenine
dinucleotide phosphate (NADPH) cyto-
chrome P450 reductase, haem oxygen-
ase-1, aldehyde dehydrogenase-3A1 and
transketolase are the principal high mole-
cular weight antioxidants in the cornea,
along with the low molecular weight mol-
ecules a-tocopherol, ascorbate, ferritin,
serum albumin and proteoglycans which
deactivate reactive entities in a non-catalytic
manner.
Cytosolic, mitochondrial and extracellu-
lar isoforms of superoxide dismutase are
also found in the corneal stroma to con-
vert superoxide into hydrogen peroxide
and molecular oxygen.84 Catalase detoxifies
hydrogen peroxide by reducing it to water
and molecular oxygen;85 furthermore
this defends superoxide dismutase from
hydrogen peroxide-mediated inactivation.86
Glutathione peroxidase also transforms
hydrogen peroxide and other hydroperox-
ides, including lipid peroxides, into oxygen
and water;87 this reduction entails the oxida-
tion of glutathione. Moreover, glutathione
directly scavenges reactive species and is
present almost wholly in its reduced form on
account of NADPH-dependant modification
by NADPH cytochrome P450 reductase.88
NADPH itself bears antioxidant properties
by chemically reducing reactive molecu-
les, absorbing UV-A radiation and binding
macromoleculesfor instance catalase
to guard against their oxidative damage-
provoked dysfunction.90
Haem-derived iron can be a co-factor
in the breakdown of hydrogen peroxide
into the tremendously aggressive hydroxyl
radical, thus heme oxygenase-1 (in concert
with NADPH cytochrome P450 reductase)
catalyses the decomposition of haem into
free ferrous iron, carbon monoxide and
biliverdin.91 The latter is metabolised further
into bilirubin. Both of these bile pigments
are free radical scavengers.92 Additionally,
carbon monoxide may prompt the trans-
cription of other genes involved in neutral-
ising oxidants.93 Also localised to the stromal
keratocytes, aldehyde dehydrogenase-3A1
and transketolase generate NADPH.87 Plus,
the former directly absorbs UV-B radia-
tion further to the oxidation of aldehyde
substrates including lipid peroxidation
products. Moreover this averts proteasome
2013 The Authors

inhibition by oxidants, deterring protein
aggregation and ultimately light scattering
in the cornea.89
A constituent of cell membranes and
lipoproteins, a-tocopherol (vitamin E)
impedes lipid degradation by quenching
lipid peroxidation-derived peroxyl radi-
cals.94 a-tocopherol augments glutathione
peroxidase activity and exerts its cytoprotec-
tive effects synergistically with ascorbate
(vitamin C), which acts to scavenge free radi-
cals and absorb UV radiation.95 Free iron
ions are sequestered in particles of ferritin
to avert their utility in reactive species
formation96. In the stroma, serum albumin
filters UV irradiation and binds reactive
metabolites,97,98 while proteoglycans such as
hyaluronic acid and those possessing chon-
droitin sulphate, dermatan sulphate and
keratan sulphate side chains likely associate
with pro-oxidant transition metals.78
Oxidative stress and the effects
of antioxidants
Reactive species-mediated cellular damage
instigates lipid peroxidation and apoptosis
throughout the cornea.95,99102 Moreover, the
detachment of epithelial cells from one
another and their basement membrane in
addition to alterations to the thickness of
the epithelial layer may disrupt the integrity
of this permeability barrier.83,103,104 Nuclear
fragmentation is perceptible and mitochon-
drial DNA damage may diminish mitochon-
drial and subsequently cell viability by
lowering the efficiency of oxidative phos-
phorylation, intensifying reactive oxygen
species formation.104106 Stromal scarring
manifests from a reparative response and
causes haze from light scattering,107,108 while
covalent bonding disruptions in stromal
matrix collagen and proteoglycan molecules
may precede disorganisation and aggrega-
tion of the collagen fibres.87,107109 This
may alter collagen solubility and me-
chanical strength and collagen-fibroblast
interactions,6,110,111 bringing about corneal
oedema and ultimately corneal cloud-
ing.112,113 Hyaluronan accumulation is also
appreciable,114 as well as infiltrates of pro-
inflammatory polymorphonuclear cells.99
These release inflammatory mediators and
more reactive oxygen species, aggravating
the tissue destruction.115 Mitochondrial
DNA degradation is again apparent in kera-
tocytes,116 which exhibit modifications in cell
morphology.114 Nuclear DNA damage is also
observed in endothelial cells, which may
account for the decline in the proliferative
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Clinical and Experimental Optometry 2013 Optometrists Association Australia
Pathobiology of keratoconus Cheung, McGhee and Sherwin
capacity of these cells,117 possibly compro-
mising the barrier properties of this layer.
Exposure to free radicals lowers the activities
of the enzymatic antioxidants glutathione
peroxidise, superoxide dismutase, catalase
and aldehyde dehydrogenase in the cor-
nea;118,119 furthermore, an escalation of reac-
tive oxygen species-generating oxidase levels
may exacerbate reactive species-induced
pathology.120
A number of endogenous and exogenous
antioxidant molecules have demonstrated
protective qualities against corneal damage
brought about by reactive species. Aldehyde
dehydrogenase decreases light scattering by
precluding the aggregation of photolabile
proteins,118 while keratocyte apoptosis is
averted by vitamin E.121 Pirenoxine, a radi-
cal scavenger, attenuates reactive oxygen
species overproduction and extracellular
vacuolisation in the stroma and preserves
stromal morphology and cell and mitochon-
drial viability.103,122 Polymorphonuclear
cell influx and myeloperoxidase activity
is diminished by ascorbic acid, reducing
inflammatory damage.99,123 The quantity of
the endogenous antioxidants glutathione
peroxidase and superoxide dismutase are
induced by a-lipoic acid.124 Moreover, the
diminution of lipid peroxidation through-
out the cornea by the antioxidative vitamins
A, C and E has been ascertained.95,99
Effect of reactive species and
antioxidants in the injured cornea
The presence of oxidative stress in lesions
and its deleterious impact on healing have
been consistently verified in numerous
tissue and cell types, as has the favourable
outcomes of antioxidant exposure and
low level reactive oxygen species on lesion
repair. In the cornea, a fall in antioxidant
quantities, including aldehyde dehydroge-
nase and transketolase,125,126 alludes to an
upsurge in reactive oxygen species and a
predisposition to oxidative stress post-injury.
Biomolecules encompassing antioxidant
capabilities promote corneal wound heal-
ing, inferring an adverse impact of reactive
oxygen species on repair.
The amount of haem oxygenase and its
activity was shown to be enhanced in corneal
epithelial cells after an insult.127 Haem
oxygenase and its products biliverdin and
carbon monoxide are crucial to downgrad-
ing inflammatory sequelae and expediting
closure of the lesion through corneal epi-
thelial cell migration.127,128 Furthermore, a
dearth of haem oxygenase has been shown
to bring about oxidative stress.129 In corneal
endothelial cells ascorbic acid obstructed
cell loss and morphological alteration,
including the appearance of intracellular
vacuoles suggestive of organelle membrane
rupture succeeding injury.130
Conversely, the positive effects of low-level
reactive oxygen species on the healing of
lesions are becoming apparent. In corneal
epithelial cells, minute amounts of extracel-
lular hydrogen peroxide amplified cellular
viability, adhesion and migration and the
rate of wound healing.131 These phenotypes
may be mediated through the actions of the
epidermal growth factor receptor (EGFR),
as they were accompanied by a rise in EGFR
phosphorylation. Additionally, hydrogen
peroxide intensified the stimulation of
focal adhesion kinase and Src tyrosine
kinase. Thus, reactive oxygen species may
also mimic integrin engagement and insti-
gate focal adhesion assembly and rearrange-
ment of the cytoskeleton. Besides acting as a
stimulus itself, basal intracellular quantities
of reactive oxygen species may function as
second messengers in signal transduction
cascades in response to other stimuli in
wound healing-associated bio-actions. The
exposure of injured corneal epithelial cells
to epidermal growth factor prompted the
intracellular generation of reactive oxygen
species, critical to epidermal growth factor-
induced cell proliferation, adhesion, migra-
tion and wound healing via the activation of
Akt and mitogen-activated protein kinase
signalling.132
OXIDATIVE STRESS
IN KERATOCONUS
The overabundance of reactive oxygen
species in keratoconus is attested to by the
findings of a number of surveys. Compared
to normal cells keratoconic fibroblasts
generated more reactive oxygen species,
including superoxide,133,134 while immuno-
histochemical evaluation of affected corneas
detected 8-hydroxy-2-deoxyguanosine in all
epithelial cell layers. This product of oxida-
tive DNA damage was restricted in nor-
mal corneas to superficial epithelial cells.135
Similarly raised were various markers of oxi-
dative lipid disintegration. The lipid peroxi-
dation products 4-hydroxy-2-nonenal and
malondialdehyde were present throughout
the keratoconic cornea.136,137 Moreover, the
localisation of the latter signified oxidative
damage in the epithelium and stroma con-
tiguous to breaks and fibrosis in Bowmans
Clinical and Experimental Optometry 96.2 March 2013
191
Pathobiology of keratoconus Cheung, McGhee and Sherwin
layer.136,137 Lysosomal membrane disruption
was indicated by an upsurge in lysosomal
enzymes, including acid esterases, phos-
phatases, lipases and cathepsins.139142
Of particular interest, proteolytic cathep-
sins can increase synthesis of reactive oxy-
gen species and mitochondrial dysfunction
besides downgrading collagen synthesis,
destabilising the cell membrane and degrad-
ing extracellular matrix elements.143,144
Nitrogen-derived reactive species, princi-
pally peroxynitrite formed by the reaction
of nitric oxide (NO) with superoxide, also
cause injurious oxidative sequelae.145 Pro-
duced by NO synthase (NOS) nitric oxide
controls inflammation, oedema and angio-
genesis in the healthy cornea.146149 In kera-
toconus, an escalation in nitro-oxidative
interactions was indicated by the intensifica-
tion of several implicated factors. Kerato-
conic corneas had a higher total nitrite
content and nitrotyrosinea corollary
of protein alteration by peroxynitrite
was identified by immunochemistry in
Bowmans layer ruptures co-localised with
inducible NOS (iNOS).136,137 Corneal ex-
tracts and cultured fibroblasts from affected
corneas possess enhanced catalase activity;
the former being accompanied by aug-
mented transcript levels.141,150 These findings
uphold a surfeit of the catalase substrate
hydrogen peroxide in keratoconus.
On the other hand, a diminution in a
number of other antioxidants may contrib-
ute to the progression of keratoconus. A
marked fall in the amount of epithelial eno-
lase protein has been demonstrated,151153
principally in superficial and wing cells.152
Additionally, variants of enolase with atypi-
cal molecular weights and charges are dis-
tinguishable,152 suggestive of its augmented
degradation in keratoconus. A marker of
basal cell differentiation,154 enolase also pos-
sesses refractive qualities and has a role in
metabolic glycolysis.155 In consequence, less
enolase may bring about a dearth in these
cellular functions in affected corneas.
A drop in glutathione along with
the quantity and activity of superoxide
dismutase in keratoconic corneas is also
apparent.84,136 Typically associated with
proteoglycans in the stromal matrix and
basal membranes,84 diminution of the extra-
cellular form of superoxide dismutase is
noteworthy, as this is in agreement with the
destruction of extracellular matrix percepti-
ble in disease. Sequencing of SOD1 revealed
several deletions in both coding and non-
coding regions in affected patients.156158
Clinical and Experimental Optometry 96.2 March 2013
192
Alternative splicing, protein truncation or
deletion of the active site in the protein may
transpire from these mutations. A number of
non-synonymous and frame shift nucleotide
variations were also found in the mitochon-
drial complex I genes ND2, ND4 and ND5,159
likely compromising protein expression and
activity. Further implying a role of flawed
mitochondrial workings, the transcript
levels of reactive oxygen species-generating
apoptosis-prompting cytochrome c oxidase
was intensified in keratoconic fibrob-
lasts.133,159 As both the source and target of
reactive oxygen species, metabolic stress may
arise from malfunction of mitochondria,
subsequently lowering the efficiency of
oxidative phosphorylation and escalating
synthesis of reactive oxygen species.160
Interestingly, keratoconic fibroblasts re-
spond abnormally to oxidative assault.116
Compared with normal cells under identi-
cal conditions, contemporaneous metabolic
and oxidative stress instigates escalated
amounts of reactive oxygen species, reactive
nitrogen species and pro-apoptotic caspase-
3 enzyme activity along with a drop in cell
viability. An early hallmark of apoptosis,
a decline in mitochondrial membrane
potential is discernible, as well as mitochon-
drial DNA degradation. These observations
suggest that under metabolic stress, corneas
with keratoconus are more susceptible to
oxidative damage, strengthening a multifac-
torial mechanism of disease development
with oxidative sequelae secondary to meta-
bolic idiosyncrasies.
IS THE INTERACTION OF REPAIR
MECHANISMS AND REACTIVE
SPECIES-ASSOCIATED ACTIVITIES
LINKED TO THE PATHOGENESIS
OF KERATOCONUS?
Numerous investigations characterising the
pathological features of keratoconus attest
to the occurrence of superfluous and inces-
sant regenerative and reactive species-linked
activities. The interdependence of these
functions in the non-diseased cornea is also
apparent and we propose that these proc-
esses and their interactions are implicated
in disease pathophysiology. Constituents of
restorative mechanisms can directly influ-
ence those of oxidative pathways and vice
versa. Interestingly, this linkage may also be
deviant in keratoconic corneas.
A principal regulator of corneal wound
healing, interleukin-1 down-regulates extra-
cellular superoxide dismutase in repair
Clinical and Experimental Optometry 2013 Optometrists Association Australia
fibroblasts from corneas affected by kerato-
conus.161 This could dampen the anti-
oxidative defences of the stromal matrix
during active repair, increasing its vulner-
ability to reactive species-mediated damage.
In healthy keratocytes, elements of the nitric
oxide pathway disrupt their overall gelati-
nase capacity and the expression and func-
tion of MMP and TIMP.162 Nitro-oxidative
damage, higher gelatinase activity and pro-
tease levels and decreased TIMP in kerato-
conus have been demonstrated by selected
reports.65,136,137,141,163,164 In addition, markers
of oxidative damage, peroxynitrite, nitric
oxide and iNOS have been detected contigu-
ous to breaks and fibrosis in Bowmans
layer.136,137 Collectively, these observations
could support basement membrane rup-
ture, as an outcome of reactive oxygen
species-mediated structural destruction
and stimulation of localised regenerative
activities, including augmented enzymatic
digestion of extracellular matrix elements.
Moreover, our preliminary studies (Cheung
et al., 2012, unpublished data) comparing
stromal cells isolated from corneas with
and without keratoconus demonstrated that
the expression of stromal repair-regulating
factors is reduced in affected corneas
in response to low concentration hydrogen
peroxide compared with similarly treated
non-keratoconic corneas. Antioxidants are
also present at diminished levels in kerato-
conus after an incision injury compared with
wounded non-diseased corneas.
Following our conjecture that over-
active healing and oxidative processes and
their abnormal interaction are intimately
involved in the pathogenesis of keratoconus,
prospective efforts will endeavour to expand
this concept into a complete and corrobo-
rated hypothesis of disease mechanisms by
comprehensively characterising these cellu-
lar functions and their associations in the
diseased cornea. It is possible that the ectasia
in clinically recognised keratoconus mani-
fests from any amalgamation of manifold
inherent and environmentally induced
abnormalities in various biological proc-
esses. In reference to our suggestion, one
such scenario may encompass self perpe-
tuating repair anomalies and oxidative
imbalances which are exacerbated by their
abnormal interaction.
Of great consequence will be identifica-
tion of the origin of the initial injury or
the cause of the presence of excess reactive
oxygen species, namely, the aetiology of
keratoconus. Perhaps this may encompass
2013 The Authors

some of the current hypotheses not already
mentioned, such as atopy and mechanical
trauma associated with eye rubbing and
contact lens wear.165 NAPDH oxidase in
inflammatory polymorphonuclear cells is
the prime source of reactive oxygen species
during corneal wound healing.115,166 Hence,
in the context of our proposition, the role
of inflammation in the development of
keratoconus may be of even greater signifi-
cance than formerly considered. Through
an informed exploration and appreciation
of disease mechanisms, the application of
therapies presently used in other disorders
and the development of novel therapeutic
approachespossibly anti-oxidant and
anti-inflammatory measuresmay in due
course progress the clinical treatment of
keratoconus.
ACKNOWLEDGEMENTS
The authors wish to thank the Maurice
and Phyllis Paykel Trust, the New Zealand
National Eye Bank and tissue donors and
their families for supporting this project.
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