Received Date : 12-Sep-2016

Revised Date : 17-Oct-2016

Accepted Date : 18-Oct-2016
Article type : Special Issue Invited Review
Accepted Article
Damaging Effects of Ultraviolet Radiation on the Cornea

Naomi C. Delic1,2, J. Guy Lyons1,2,3, Nick Di Girolamo4, Gary M. Halliday1

1
Discipline of Dermatology, Bosch Institute, University of Sydney, Camperdown, New South

Wales 2006, Australia

2
Immune Imaging Program, Centenary Institute for Cancer Medicine and Cell Biology,

Camperdown, New South Wales 2042, Australia

3
Sydney Head and Neck Cancer Institute, Cancer Services, Royal Prince Alfred Hospital,

Camperdown, New South Wales 2050, Australia

4
Department of Pathology, School of Medical Sciences, University of New South Wales,

Randwick, New South Wales 2052, Australia

*Corresponding author email: gary.halliday@sydney.edu.au (Gary M. Halliday)

This article is part of the Special Issue honoring Dr. Hasan Mukhtar's 70th Birthday and his

outstanding contributions to various aspects of photobiology research, including

photocarcinogenesis and chemoprevention.

This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/php.12686
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 ABSTRACT

The cornea sits at the anterior aspect of the eye and additionally to the skin, is highly exposed
Accepted Article
to ultraviolet radiation (UVR). The cornea blocks a significant proportion of UVB from

reaching the posterior structures of the eye. However, UVA can penetrate the full thickness

of the cornea, even reaching the anterior portion of the lens. Epidemiological data indicate

that UVR is a contributing factor for a multitude of corneal diseases of the cornea including

pterygium, photokeratitis, climatic droplet keratopathy, (CDK) and ocular surface squamous

neoplasia (OSSN), although the pathogenic mechanisms of each require further elucidation.

UVR is a well-known genotoxic agent and its effects have been well characterized in organs

such as the skin. However, we are only beginning to identify its effects on the cornea, such as

the UVR signature CT and CCTT transversions identified by sequencing and increased

proliferative and shedding rates in response to UVR exposure. Alarmingly, a single low dose

exposure of UVR to the cornea is sufficient to elicit genetic, molecular and cellular changes,

supporting the consideration of using protective measures, such as wearing sunglasses when

outdoors. The aim of this review is to describe the adverse effects of UVR on the cornea.

The cornea

The human eye is a roughly spherical organ that measures approximately 2.5cm in diameter

which contains two internal cavities; the anterior and posterior chambers. The cornea is

located at the most anterior aspect of the eye, overlying the iris, pupil and anterior chamber,

and its health is essential for exquisite vision (Fig. 1a). Structurally, the mammalian cornea

consist of five layers (Fig. 1b); beginning at the most anterior position (a) a multilayered

corneal epithelium, (b) a basement membrane-like structure known as Bowmans layer, (c)

the corneal stroma, maintained by resident mesenchymal cells known as keratocytes, (d) a

second basement membrane, known as Descemet's membrane and (e) a mono-layer of

specialized corneal endothelial cells. Located at the periphery of the cornea is a narrow

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 transitional band of tissue approximately 1mm wide, known as the limbus, which separates

the cornea from the conjunctiva. The corneal epithelium is perpetually self-renewing,
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whereby the progeny of stem cells residing in the limbus replace terminally differentiated

cells that are constantly shed from the corneal surface into the tear pool (1-4). These limbal

epithelial stem cells (LESCs) are unipotent as they give rise to corneal, but not conjunctival

epithelia. In vivo studies combined with mathematical modeling, have described a striking

spoke-like pattern that forms as corneal epithelia migrate from the limbus, moving

centripetally towards the apex of the cornea (Fig.1c-d) (1, 5). Mathematical modeling of this

process has demonstrated that the development of this pattern requires no external cues;

rather it is purely population-pressure that driven (5). Lineage tracing experiments using a

mosaic mouse model have recently shown that a single precursor cell is responsible for a

unique spoke that forms, confirming the role of stem cells in the maintenance of the corneal

epithelium (1). The mosaic model (Confetti) used a tamoxifen inducible system so that any

one or two of four fluorescent proteins (mCFP, nucGFP, cytoYFP, cyto-tDimer2) could be

expressed in K14 expressing cells, including the limbal epithelial stem cells, giving each of

these cells a distinct color. These limbal stem cells give rise to progeny (stem cell and/or

corneal epithelial cell) that inherit the same fluorescent protein, therefore making this system

ideal for lineage tracing.

Exposure of the eye to UVR

The sun is the main source of UVR emitting a wide spectrum of electromagnetic radiation.

However, only the ultraviolet (UV), visible and infrared bands reach the Earths surface to

make up what is known as terrestrial sunlight, with UVR constituting approximately 5% (6)

(Fig. 2a). UVR can be further divided into 3 spectral regions; UVC (100-290nm), UVB (290-

320nm) and UVA (320-400nm). UVC is absorbed by ozone in the earths atmosphere(7), and

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 thus the UVR component of terrestrial light consists of approximately 4% UVB and 96%

UVA with irradiances differing depending on cloud cover (Fig. 2b) (8).
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The eyes and skin are the organs most highly exposed to UVR and its potentially damaging

effects. When discussing exposure to solar radiation, it must be acknowledged that there are

two components; direct and diffuse. Direct UVR exposure involves looking directly at the

sun, something that humans naturally avoid. However diffuse (or albedo) UVR exposure is

multidirectional due to photo scattering and reflectance by gas or aerosol molecules in the

atmosphere. Due to the directional incidence of diffuse UVR, most ocular UVR exposure is

due to this component, which poses a more difficult task in minimizing its exposure. In

Toowoomba, Australia (27.56 S, 151.95E), the proportion of diffuse UVB varies from 23%

in spring to 59% in winter, whereas diffuse UVA varies from 17% in spring to 31% in winter

(9). This change in proportion of diffuse UVR can be affected not only by the season, but

also by the time of day. For example, Parisi et al. (9) noted that cloud cover increases in the

afternoon, and therefore diffuse UVR readings are often higher during this time for both UV

wavelengths. In addition to the atmosphere and clouds, the physical environment (such as

snow, buildings, ground coverings) contributes to the scattering effects observed in diffuse

UV. Furthermore, eyelid opening must be considered when estimating the dose of UVR

reaching the ocular surface. Sliney (10) performed extensive measurements which were used

to design an algorithm that took into account the reflectivity of surfaces in the immediate

environment and the extent of eyelid opening, when estimating the dose of UVR that was

received by the ocular surface. The use of a mannequin dosimetry system allowed for the

evaluation that sunglasses could attenuate UVR irradiance to the corneal surface by 80%,

compared to the absence of sunglasses (10). Most impressively, UVB exposure was reduced

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 to 1% when wrap-around style sunglasses were employed (10), thus confirming the

importance of choosing the appropriate eyewear when in a sunny environment.

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In recent years, UVA exposure has also been developed for clinical treatments. UVA in

conjunction with riboflavin has been approved for cross-linking stromal collagen fibers as a

therapy for keratoconus, a degenerative eye disease that is often characterized by coning of

the cornea due to thinning of stromal collagen. (11)

In general, shorter wavelengths of UVR and visible light penetrate tissues less than longer

wavelengths. Thus, UVB is primarily absorbed by the corneal epithelium whereas UVA can

penetrate the entire thickness of the cornea (12) (Fig. 1c). The age of the eye is important to

its UVR penetrance. In humans, a cornea from a 24-year old blocks over 90% of UVB (290-

315nm) and 45% of UVA (315-400nm) (12). However, corneas from the elderly (80+ years),

are more efficient at blocking UVA (60%) but less efficient at filtering shorter wavelengths

(80%) (12). Furthermore, corneas from younger individuals (<24 years) transmit even more

UVA, therefore rendering them and the ocular medium behind this tissue more vulnerable to

UVR damage (12).

Medical conditions of the cornea associated with UVR exposure

Photokeratitis UVR has long been known to cause damage to the cornea, the acute reaction

to UVR-induced "burns" being referred to as photokeratitis. The most common accounts of

photokeratitis are snowblindness and welders flash. Snowblindness results from

excessive exposure to naturally occurring UVB in an environment of high reflectivity; such

conditions are encountered when skiing, at the beach or at high altitude. Conversely, welders

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 flash results from exposure to artificial UVB (and sometimes UVC) sources such as those

from a welders arc.

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Photokeratitis remains asymptomatic until several hours post-UVR exposure with the earliest

symptoms presenting as a gritty ocular sensation, followed by photophobia and tearing.

These initial symptoms are attributed to the loss and damage of epithelial cells in the

superficial layers of the cornea. Corneal edema results in haze and vision impairment. Further

exposure to UVR results in epithelial exfoliation, which leads to agonizing pain. In 1916,

Verhoeff and Bell calculated that a threshold dose of approximately 500mJ/cm2 of UVR from

an artificial source was needed to cause pathological changes in the cornea, and that the main

wavelengths responsible within the terrestrial sunlight band were in the UVB part of the

spectrum (13). Ren et al (14) showed that suprathreshold doses of UVB did in fact disrupt the

normally ordered process of epithelial desquamation from the corneal surface, by elevating it

to such a rate that the subepithelial nerve endings underlying the cornea are exposed, thus

giving rise to the characteristic pain associated with photokeratitis.

Climatic Droplet Keratopathy (CDK) An association of CDK with chronic exposure to UVR

has been confirmed by epidemiological studies (15, 16). This condition occurs when soluble

plasma proteins photochemically react with UVR and deposit beneath the corneal epithelium,

within the Bowmans layer and the superficial stroma, causing opacification of the cornea

and visual impairment (17, 18). Recent advances in confocal laser scanning microscopy for

studying CDK described further abnormalities in addition to those already defined. Patients

suffering from moderate to advanced CDK display an increased reflectivity of the superficial

corneal epithelium as well as alterations in the density and morphology of the sub-basal nerve

plexus (19). Tear fluid from patients with CDK has been analyzed and found to contain a

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 myriad of inflammatory cytokines including, interleukin (IL)-1, IL-5, IL-6, IL-7, IL-8,

monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1 beta

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(MIP-1) and tumor necrosis factor alpha (TNF-) (18). The matrix metalloproteinse (MMP)

proenzymes, proMMP-2 and proMMP-9, are found in higher quantity in the tear fluid of

CDK patients, while a higher level of active MMP-9 is found in the corneal epithelial

basement membrane, compared to healthy subjects (18). The significant increase in

inflammatory cytokines and MMPs in corneas of patients suffering from CDK indicates that

its pathology is attributable to a significant chronic inflammatory response and disruption of

the extracellular matrix (ECM) (18).

Pterygium Pterygia (Fig. 3a) are common benign lesions of the ocular surface (20),

characterized by local displacement of normal epithelium by abnormal epithelial cells.

Histologically (Fig. 3b-c), a demarcation zone exists that distinguishes the normal corneal

epithelium from the advancing edge of the pterygium head, which often displays regions of

elastotic connective tissue (21). Pterygia commonly arise from the nasal limbus, and form

characteristic wing-shape lesions, that can grow bilaterally, and if left untreated, restrict eye

movement and vision (22). The Coroneo Effect has aided in explaining the predominance

of nasally located pterygia (and cortical cataracts), by showing that the anterior chamber is

capable of focusing light that enters the eye temporally and is internally reflected onto the

opposing nasal side of the corneolimbal region. Epidemiological studies have indicated that

UVR plays a major role in the pathogenesis of pterygia (23, 24). This association is supported

by the increase in prevalence in equatorial regions and amongst individuals that work

outdoors in an environment with high reflectance compared to indoor workers (regardless of

latitude). This risk was increased 20-fold when the surface was sealed concrete, and several

hundred-fold when working on sand (23). Further evidence of the role of UVR in pterygia is

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 seen by the formation of elastic tissue in the pterygium stroma, similar to the solar elastosis

that is often seen in the dermis after prolonged UVR exposure (Fig. 3d) (25).
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Ocular Surface Squamous Neoplasms (OSSN) Ocular surface squamous neoplasms (OSSN)

are the most common form of non-melanocytic lesions in the conjunctiva and cornea, and

refers to a disease spectrum comprising mildly dysplastic to invasive lesions that originate

from squamous epithelium (26-28). Patients with these lesions present with irritation, red eye,

the pathological mass can be raised and gelatinous, and leukoplakia, originating from the

limbus before involving the cornea and conjunctiva (27, 29). Peak incidence of OSSN occurs

at a latitude of 16 south (30, 31). Two predominant and distinct main groups of patients are

affected. The first comprises of older males, who live in temperate climates and are not

associated with human immunodeficiency virus (HIV) or human papillomavirus (HPV)

infection (31). The second group includes younger individuals (both males and females) that

reside in the tropics, but are associated with HIV or HPV infection (31). The association with

HIV suggests that immunosuppression plays a role in predisposing these patients to UV

induced OSSNs, whereas the nature of the association with, and identities of specific

subtypes of HPV involved, are still unknown (31). OSSN and UVR are also linked via

patients suffering from xeroderma pigmentosa, a rare autosomal recessive genetic disorder in

which the DNA damage repair machinery is faulty (32). These patients often develop OSSNs

as well as skin cancer at a young age (32, 26).

UVR induced genetic changes

It is well established that solar radiation is the main culprit in the initiation and progression of

several types of skin cancers (squamous cell, basal cell carcinomas, melanomas) (23).

Specifically, UVR has been proven to be a highly genotoxic agent (33) by inducing DNA

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 damage in the form of two well described photoproducts; cyclobutane pyrimidine dimers

(CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4 PPs). UVB photons can be
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directly absorbed by DNA bases, largely by the pyrimidine constituents. This energetic

absorption induces the formation of covalent bonds between two adjacent pyrimidine bases,

with CPDs being the most prevalent photolesion. Because of their inefficient removal and

abundance, CPDs are considered pro-mutagenic. Misincorporation of adenine instead of

guanine opposite a cytosine in CPDs results in the UV signature CT and CCTT

transversions (12). Since the cornea is at the air interface, and absorbs approximately 90% of

the UVB that irradiates the surface of the eye, it is expected that it will sustain DNA damage.

Studies exploring DNA damage in the mammalian cornea have observed the presence of

photolesions. Experimental models using human and rabbit eyes described the rate of CPD

formation versus CPD distribution in the cornea, according to different UV wavelengths (34,

12). UVA penetrates as far as the corneal endothelium and superficial layers of the crystalline

lens; however, CPDs are induced at a much lower rate by UVA than by UVB (34, 12).

Conversely, although little UVB penetrates beyond the anterior third of the corneal stroma,

this region has a significantly higher rate of CPD formation (34, 12). Although

physiologically irrelevant, UVC induced CPDs are highly concentrated in the superficial

layers of the epithelium and are virtually undetectable in the remaining 90% of the stroma

(12). Those studies demonstrated a shield-like role the cornea plays in protecting the

posterior structures of the eye from UVR-induced damage. In addition to CPDs, UVB

induced 6-4 PPs were more abundantly in the epithelial layers of the rat cornea. However,

when broadband UVR (280-380nm) doses were used, equating to well over 2 minimal

erythemal doses (i.e. twice the dose needed to cause sunburn to exposed skin), evidence of 6-

4 PPs was detected throughout all ocular structures, including the corneal stroma, iris, lens

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 and retina (35). Notably, transmittance of UVR through various ocular structures is highly

dependent on age.
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Much remains to be learnt about genetic changes that occur in the corneal epithelium as a

result of overexposure to UV. Recent advances in understanding the effects of mutagenic

changes to specific genes in the cornea have come from Moloney et al (36) and Hassan et al

(37). In these investigations, BRM was found to be mutated or have reduced expression in

human non-melanoma skin carcinomas (NMSC) (38, 36). This gene encodes for one of two

ATPase subunits in the SWI/SNF complex involved in unraveling DNA for factors requiring

access to it to mediate their functions, such as repair and transcription. In a

photocarcinogenesis study, using a BRM knock-out mouse, it was shown that the absence of

this gene resulted in the increased incidence of both eye and skin tumors, implying that it

functions as a tumor suppressor (39). Both carcinomas and sarcomas of the eye were induced

by chronic UVR exposure in Brm and/or Tp53 deficient mice. Subsequent studies (37)

examined corneas of mice chronically exposed to UVR (i.e. beyond 25 weeks). Corneas of

Brm knock-out mice displayed increased thickening, hyperplasia and decreased atrophy when

compared to Brm wild-type mice, the damage further exacerbated when Brm knock-out mice

lost a p53 allele. It was also noticed that irradiated Brm knock-out mice demonstrated

increased proliferation and regeneration of the corneal epithelium, which was confirmed by

Ki-67 staining (37). Brm knock-outs showed increased Ki-67+ corneal epithelial cells

compared to Brm wild-types after only 2 weeks of irradiation, which was maintained after 25

weeks of irradiation (37). Of significance is that proliferation in the Brm deficient corneas

displayed disorganization, with proliferative cells observed in the suprabasal layer of the

epithelium, rather than being confined to the basal layer, which is typical in the Brm wild-

type corneas with or without irradiation (37). Taken together, the corneal thickening and

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 cellular proliferation data firmly suggest that Brm plays a significant role in preventing

hyperproliferation in response to acute or chronic UVR exposure, thus confirming its tumor
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suppressor status in the cornea.

As with NMSCs, TP53 mutations are observed in UVR induced corneal sarcomas. Studies in

the opossum found that these mutations were the hallmark UVR signature C T conversion

located in codons equivalent to those found in mutational hotspots of human UVR induced

SCCs (40). Similar studies were conducted in human conjunctival SCCs, where a high

prevalence of CC TT transversions, a signature mutation of UVR, were found (41). The

location of these mutations at sites of DNA photoproduct formation suggested that the TP53

mutations in these OSSN were a result of UVR directly acting on DNA (40, 41).

Although no TP53 mutations were identified, Galor et al (42) were first to perform whole

exome sequencing on OSSN. Of the seven samples included in their analysis, they identified

mutations in Titin (TTN), Neuron Navigator 2 (NAV2), FAT atypical cadherin 2 (FAT2),

hepatocyte growth factor (HGF), dynein axonemal heavy chain 8 (DNAH8) and CREB

Binding Protein (CREBBP)(42). The functional significance of these mutant genes remains to

be elucidated.

UVR induced molecular changes

Multiple molecular factors that have altered activity in response to UV have been identified

in the cornea, including nuclear factor-B (NF-B), cytokines, MMPs and PAX6. NF-B has

been identified as an initiator of cell death in the cornea, and is also known to be a key driver

of cytokine production in response to injury. Likewise, cytokines can upregulate MMP

production (43, 44). Although the current literature describes these independently of each

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 other, it is likely that these factors work in concert and in a cascade-type arrangement to

exhibit these identified molecular changes. The relationship of PAX6 to the NF-
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B/cytokine/MMP pathway in the cornea remains to be explored.

NF-B Although cell death does not appear to be the primary route for removal of UVR

exposed corneal epithelia (see the section on cellular changes in response to UV irradiation),

NF-B may play a role in initiating cell death. NF-B is a transcription factor that remains

inactive via ubiquitous expression of its inhibitor IB or contiguous latency peptide sequence

(45). Targeted phosphorylation and subsequent degradation of IB or latency peptide allows

activation of NF-B followed by its translocation to the nucleus where its dimers are free to

bind to its target DNA elements. This activates transcription of genes involved with

responses to inflammation or cell growth. Using the SV40 immortalized corneal epithelial

cell line T-HCEC, Lee et al (46) showed that cell death followed nuclear translocation of NF-

B after UVR irradiation. Furthermore, cell death could be blocked by treating cell lines with

the potent NF-B inhibitors sulfasalazine and SN-50 prior to UVR exposure.

Immunocytochemical analysis confirmed that NF-B was inhibited from translocating to the

nucleus after irradiation, and downstream cell death was blocked. Electrophoretic mobility

shift assays (EMSA) in conjunction with immunocytochemical staining showed that NF-B

translocated to the nucleus prior to cell death being observed, and its binding activity

remained increased for 2 hours post irradiation, suggesting that NF-B indeed plays an early

role in the cellular death cascade.

PAX6 PAX6 plays a major role in structural development and ocular cell differentiation in

the eye of vertebrates and has therefore been coined a master oculogenic control gene (47,

48). Its expression is essential for development, maintenance and repair of the eye. PAX6 is

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 transcriptionally regulated by CTCF, a zinc finger phosphoprotein that binds to the CCCTC

motif, located upstream of the PAX6 P0 promoter. Because overexpression of the CTCF gene
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in corneal epithelial cells resulted in a downregulation of PAX6 activity, Wu et al (49)

explored PAX6 expression regulation by CTCF in cultured rabbit and human corneal

epithelial cells that were stimulated with UV. Within 8 hours of exposure to UV, PAX6

expression increased whilst CTCF decreased, both detectable by western blot analysis. The

upregulation of PAX6 by decreased activity of CTCF was confirmed by EMSA that showed

that the binding activity of CTCF on PAX6 P0 upstream enhancers was significantly

decreased after UVR exposure. Finally, they showed that CTCF had a direct effect on PAX6

by using site directed mutagenesis on the 80bp PAX6 P0 enhancer sequence to genetically

alter the five binding motifs, resulting in decreased binding activity of CTCF.

MMPs MMPs are a family of proteolytic enzymes that are important for tissue remodeling

and wound healing. They are synthesized as inactive zymogens, and become activated once

proteolytically cleaved at their pro-peptide domain on the N-terminus. Once active, MMPs

are capable of either degrading or processing extracellular matrix components and other

bioactive proteins such as cytokines (50). In normal circumstances, MMPs are expressed at

low levels and their activation is tightly regulated by their natural inhibitors (TIMPs),

therefore an overexpression and activation of MMPs is associated with excessive degradation

of the ECM; which can be potentially devastating for the structure and clarity of the cornea

(51). The gelatinases, MMP-2 and -9, and two of the collagenases, MMP-1 and -8, have been

studied in the cornea in depth (52-56). MMP-2 and -9 were originally identified as enzymes

involved in the degradation by epithelial cells of type IV collagen, a principal component of

basal membranes (57, 58).

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 MMP-1 has been demonstrated to be one of the most abundant MMPs present in pterygia. Di

Girolamo et al (52) showed that UVB could significantly increase MMP-1 production in both
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pterygium epithelial cell lines and ex vivo pterygia explants. It is regulated via the UVR

inducible ERK1/2 pathway. MMPs -2, -9, -7, -8 and to a lesser degree -14 have all been

shown to be upregulated by UVB exposure in vivo (51, 59, 60), but their expression remains

unaltered when corneas are exposed to UVA (51). MMP-14 is active in multiple roles such as

the cleavage of ECM, activation of other MMPs, and activating signaling pathways for cell

migration during wound healing in the cornea (61-63), but interestingly, its expression is only

mildly increased after UVB exposure in rabbit corneal epithelium (51).

Cytokines UV-induced changes in the cornea can be heavily attributed to the upregulation of

pro-inflammatory cytokines including IL-1, IL-6, IL-8 and TNF- in the corneal epithelium

and stroma (64-67). Specifically, it was found that IL-6 and IL-8 were expressed in the

superficial epithelium, with some IL-8 expression in the vascular endothelium of pterygium

tissue that was exposed to low dose UVB (40mJ/cm2) (66). In cultured pterygium epithelial

cells that were irradiated with UVB, expression of IL-6 and IL-8 increased in a time and

dose-dependent manner (66). Normal limbal fibroblasts and corneal epithelial cells

upregulate pro-inflammatory and macrophage recruiting cytokines, TNF, interferon gamma

(IFN) and MCP-1 after low dose (20mJ/cm2) UVB (64). Conversely, Notara et al (64) also

showed that UVB treatment of limbal fibroblasts downregulates their production of soluble

factors usually involved in mediating lymphatic and blood endothelial activity, most likely a

regulatory mechanism to avoid neovascularization of the limbus and cornea.

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 Cellular changes in response to UV irradiation

Corneas of mice that are chronically exposed to low doses (equivalent to 2 minimal
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erythemal doses) of UVR display abnormalities from as early as 25 weeks with epithelial

hyperplasia, dysplasia, atrophy of the corneal epithelial center, significant hypocellularity of

the stroma, fibrosis, vascularization and inflammation (37, 68). These changes eventually

culminate in the formation of OSSNs (see above).

The acute histological changes to corneas exposed to UVR occur in a dose-dependent manner

(37, 68, 69). Pitts and colleagues (69) showed that UVR enhanced the premature

shedding/sloughing of the outermost epithelial cell layers, and increased cell death 24 hours

after radiant exposure. Gradually increasing the radiant exposure caused loss of corneal

epithelia; keratocytes became fragmented and the endothelium became disorganized,

vacuolated and detached from the stroma (69). Through the use of ex vivo organ cultivated

corneas, Ren et al. (14) showed that a single dose of 1.05J/cm2 of UVB increased the

shedding rate of epithelial cells from the ocular surface. They found that, after irradiation,

there was a delay of approximately 3h before shedding was increased, and maintained for the

ensuing 3h (14).

Using computer modeling, Lobo and colleagues (2016) recently demonstrated that the well

documented spoke pattern that corneal epithelial cells form as they move from the limbus (1,

70), requires no external physiological cues. Rather, this pattern can be simply explained by

population pressure from the periphery. However, the spoke formation does not rule out the

role of UV-regulated factors, such as signaling molecules, neurons and biophysical cues in

response to inflammation and injury (5).

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 Using the Confetti mosaic mouse model, it was shown that a single dose of 150mJ/cm2 UVB

increases the spoke growth rate from the control value of approximately 10m/day to
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approximately 50m/day after UVR exposure (1, 5). Mathematical modeling (5), together

with in vivo experiments (5, 68, 71, 72) has shown that, although apoptosis peaks at around

24 hours post UV-exposure, it is insufficient to account for the significant influx of cells in

response to UVR exposure without causing a substantial thickening of the corneal epithelium.

On the contrary, the corneal epithelium thins following UVR exposure (14).

A hypothesis to explain this lack of apoptotic cell death in response to UVB, comes from

Leerar et al. (73). Following UVB, human corneal epithelial cells lose potassium ions (K+)

that would normally activate caspases, triggering the apoptotic cascade (73-75). However,

tears are naturally high in K+, and therefore it was argued that they would reduce the loss of

intracellular K+, preventing apoptosis (73, 74, 76). Additionally, Ren and Wilson (14)

observed that cells which were shed in response to UVR, were smaller than that of a typically

differentiated cell, suggesting that the epithelial wing cells were failing to fully terminally

differentiate before leaving the epithelium.

Thus, although the cornea is anatomically simple, the mechanisms which it uses to maintain

its integrity are complex and remain relatively unexplained. While apoptosis and increased

cell shedding/terminal differentiation appear to be the main means of stabilizing the structure

after a UV insult, the current evidence indicates these mechanisms may not be wholly

responsible, and other regulatory mechanisms await discovery.

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 Immunogenicity and the cornea

The eye is considered an immune privileged site. It was originally hypothesized that the
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anterior chamber of the eye was lacking in lymphatic vessels, protecting this organ from an

overwhelming immune response if an antigen was detected. This was proposed to explain the

results of studies performed on transplanted cells from genetically different hosts, which

survived significantly longer in the anterior chamber than in other sites of the body (77, 78).

However, this hypothesis was challenged when Kaplan and Streilein (79) found the presence

of hemagglutinating antibodies in serum from rats, 4 days after being immunized

intracamerally with allogeneic lymphoid cells, concluding that antigens present in the

anterior chamber can indeed elicit a systemic immune response (78-80). Today it is well

established that the cornea contains resident antigen presenting cells of dendritic and

macrophage lineages that seem to be entrenched in the limbal transition zone, perhaps

playing a key role as first-line defense cells able to sample antigen at a sight prone to

infection, inflammation and tumorigenesis (81).

Although much is now known about the immune system in the eye, little is known about the

immune response following UVR irradiation. Immunosuppression by UVR has been well

studied in the skin, with these effects being attributed partially to cis-urocanic acid (UCA)

which is naturally abundant in the skin as the trans isomer (82). UV irradiation causes its

isomerization from trans to cis. Treatment with a topical form of cis-UCA on a corneal

allograft in BALB/c mice suppressed the immune system therefore prolonging the graft

survival time compared to untreated groups (83). This presence of cis-UCA inhibits the

expression of the cytokines, IL-6 and IL-8, and general cytotoxicity associated with

inflammation (84). In human corneal epithelial cells, cis-UCA is able to inhibit UVB induced

apoptosis and latent proliferation by blocking the binding of the transcription factors, c-Jun

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 and c-Fos, but not NF-B (84). It has also been proposed that UVR induced upregulation of

JunB is able to antagonize the pro-apoptotic activity of c-Jun (85). Additionally, in cultured
Accepted Article
spleen cells, cis-UCA increases the production of IL-10, an important factor in

immunosuppression since it inhibits antigen presentation and production of T helper 1 (TH1)

cytokines by epidermal antigen presenting cells (83).

Transplantation is often the only solution for treating patients with limbal stem cell

deficiency, which is frequently caused by chemical and thermal burns. Rama and colleagues

showed that grafts generated from the patients own limbal stem cell population were a

potential solution, although there was still a 10% failure rate, and cited inflammation as one

of the contributing complications (86). Unlike the skin, an influx of immune cells and chronic

inflammation in the cornea can potentially cause irreparable damage, and so understanding

the immunogenicity of the cornea is imperative for positive advancements in therapeutic

applications.

CONCLUSIONS AND FUTURE NEEDS

It is clear from the available data that the level of UVR required to provoke a response in the

cornea is minimal and, when provoked chronically, will result in clinical disease. UVR-

protective eyewear should be worn whenever outdoors, with the need being greater on days

with cloud cover and in surroundings with highly reflective surfaces, due to increased

reflectance and scatter.

There is currently a need for deeper understanding of the genetic, cellular and immunological

changes that occur in the cornea after UV exposure, compared to the greater amount of

literature on the skin. Galor and colleagues (42) have pioneered the area of somatic genetic

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 changes of the cornea by identifying mutational changes in human patients with OSSNs.

Although a very small number of samples was analyzed, they demonstrate the potential for
Accepted Article
identifying mutations and correlating them with efficient therapies. The mechanism for the

non-apoptotic cell loss that results in thinning of UVR-exposed corneas remains to be

elucidated. Identifying the molecular pathways involved might shed insights into novel

means of treating degenerative conditions involving the corneal epithelium. Studies

identifying the semi-immunosuppressive state of the eye and its correlation with OSSN

demonstrate the complexity of the immune system. This requires considerably more analysis.

There are many mouse models available in which immune cells are fluorescently tagged and

these would assist in teasing out mechanisms of the ocular immune response.

Mathematical models might prove to be useful in improving therapies such as UVA cross-

linking and limbal stem cell transplants to optimize locations of treatment and avoiding more

damage due to the treatment (87). Lobo et al. (5) demonstrated that their model was efficient

in replicating the biology of the epithelial cells under homeostatic and experimental

conditions. Mathematical simulations could potentially be programed to incorporate a vast

number of experimental parameters to identify appropriate targets. This could be used to help

design experiments, reducing costs associated with large scale experimental testing and

optimizing therapeutic strategies. Therefore, not only would mathematical modeling be

beneficial for developing treatment strategies, but also for planning research programs.

ACKNOWLEDGEMENTS N.C. Delic was supported by Australian Postgraduate

Award and Sydney Catalyst Top-Up Scholarship from Cancer Institute NSW. This work was

supported by Human Frontiers Science Program Grant RGP0041. The authors thank

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 Professor Minas Coroneo (Department of Ophthalmology, Prince of Wales Hospital,

Randwick, Sydney, Australia) for providing the clinical image of a pterygium.

Accepted Article
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Accepted Article

Naomi Delic is a PhD student at the University of Sydney. She received her Bachelor of
Science with first class honours from the University of New South Wales in 2006. Her
research interests include cancer biology of the skin; however, her current research is focused
on understanding the response of the cornea to ultraviolet radiation exposure, using intravital
lineage tracing techniques.

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Accepted Article

Guy Lyons received his PhD from the University of Sydney in 1988. He is a cancer biologist
with a long-term interest in epithelial carcinogenesis. Squamous cell carcinomas of the skin,
oral mucosa and ocular surface are of particular interest. He is currently located at the
Centenary Institute, where he is using intravital lineage tracing and genetic manipulation
techniques to study clonal evolution during development and carcinogenesis.

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Accepted Article

Professor Nick Di Girolamo is Director of the Ocular Diseases Research and Head of the
Mechanisms of Disease and Translational Research at the School of Medical Sciences,
University of New South Wales, Australia. His research interests include diseases that arise
on the ocular surface, predominantly the cornea. His team recently developed a novel
technique to deliver stem cells to cornea using a therapeutic contact lens. His team also
developed a transgenic mouse model to study the activity of corneal stem cells from
development to aging.

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Accepted Article

Professor Gary Halliday is a Professor of Dermatology at the University of Sydney. He

obtained his PhD from Monash University in 1981 and his Doctor of Science from the
University of Sydney in 2001. His research is directed towards understanding the role of
sunlight in skin carcinogenesis; particularly ultraviolet radiation suppression of immunity,
induction of gene mutations, skin cancer cell biology and development of effective
prevention. His research group is part of the Sydney Cancer Centre, the Bosch Institute, the
department of Dermatology of Royal Prince Alfred Hospital, and the Central Clinical School
of the University of Sydney.

FIGURE CAPTIONS

Figure 1. Schematic of the human eye (a) the limbus is a narrow area, located at the

periphery of the cornea, where the stem cells reside; (b) cellular structure of the cornea,

depicting the five distinct layers and the depth of penetration of each ultraviolet (UV)

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 wavelength; (c) colored arrows depict the direction that the epithelial progeny move from the

limbus into the epithelium; (d) A K14CreERT2-Confetti murine eye, 50 weeks post tamoxifen
Accepted Article
injection (1, 5) with fluorescently labeled corneal epithelial cells, derived from stem cells in

the limbus. The bright halo in the center is auto-fluorescence of the crystalline lens. Scale bar

is 200m.

Figure 2. (a) Schematic demonstrating the wide spectrum of electromagnetic radiation

(EM), with ultraviolet radiation (UVR) spanning 100-400nm; (b) an example of solar

irradiance measured on a sunny day (red line) compared to an overcast day (black line).

Spectrums were measure on the roof of The Centenary Institute, Camperdown, Australia

using an Optronics OL-754 spectroradiometer. The boundaries of the UVC, UVB, UVA and

visible (VIS) regions are shown.

Figure 3. (a) clinical image of a patient with a pterygium; (b) a low power histological

micrograph demonstrating the encroaching edge of the pterygium (hatched box); (c)

magnified micrograph of the hatched box from b, BV- blood vessels; (d) demonstrates

stromal elastosis, a feature often seen in tissue chronically exposed to UVR.

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Accepted Article

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Accepted Article

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Accepted Article

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