PII: S1350-9462(01)00008-8
`
`The Corneal Wound Healing Response:
`Cytokine-mediated Interaction of the Epithelium, Stroma,
`and Inflammatory Cells
`
`Steven E. Wilson*, Rahul R. Mohan, Rajiv R. Mohan, Renato Ambro´ sio Jr,
`JongWook Hong, and JongSoo Lee
`The Department of Ophthalmology, University of Washington School of Medicine, Box-356485 Seattle, WA
`98195-6485, USA; The Department of Ophthalmology, Korea University, Seoul, Korea and Department of
`Ophthalmology, Research Institute of Medical Science, College of Medicine, Pusan National University,
`Pusan, South Korea
`
`CONTENTS
`
`Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 625
`Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`625
`1.
`2. Evolutionary context . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`626
`3. Overview of the wound healing response . . . . . . . . . . . . . . . . . . . . . . . . . . .
`626
`4. Master regulators of the wound healing response . . . . . . . . . . . . . . . . . . . . . . .
`626
`5. The corneal wound healing cascade . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`628
`6. Keratocyte apoptosis and necrosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`628
`7. Lacrimal-gland-derived cytokine mediators and epithelial healing . . . . . . . . . . . . . .
`630
`8. Keratocyte proliferation and migration-myofibroblasts . . . . . . . . . . . . . . . . . . . .
`631
`9.
`Inflammatory cell infiltration and function . . . . . . . . . . . . . . . . . . . . . . . . . . . 634
`10. Return to normalcy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 635
`Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 636
`References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 636
`
`AbstractFThe corneal wound healing cascade is complex and involves stromal–epithelial and stromal–epithelial–immune
`interactions mediated by cytokines. Interleukin-1 appears to be a master modulator of many of the events involved in this
`cascade. Keratocyte apoptosis is the earliest stromal event noted following epithelial injury and remains a likely target for
`modulation of the overall wound healing response. Other processes such as epithelial mitosis and migration, stromal cell
`necrosis, keratocyte proliferation, myofibroblast generation, collagen deposition, and inflammatory cell
`infiltration
`contribute to the wound healing cascade and are also likely modulated by cytokines derived from corneal cells, the lacrimal
`gland, and possibly immune cells. Many questions remain regarding the origin and fate of different cell types that contribute
`to stromal wound healing. Over a period of months to years the cornea returns to a state similar to that found in the
`unwounded normal cornea. r 2001 Elsevier Science Ltd. All rights reserved
`
`*Corresponding author. Tel.: +1-206-543-7250; fax: +1-
`206-543-4414; e-mail: sewilson@u.washington.edu.
`
`The corneal wound healing response is an exceed-
`ingly
`complex
`cascade
`involving
`cytokine-
`mediated interactions between the epithelial cells,
`
`1. INTRODUCTION
`
`Progress in Retinal and Eye Research Vol. 20, No. 5, pp. 625 to 637, 2001
`r 2001 Elsevier Science Ltd. All rights reserved
`Printed in Great Britain
`1350-9462/01/$ - see front matter
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`
`keratocytes of the stroma, corneal nerves, lacrimal
`glands, tear film, and cells of the immune system.
`As complex as the response is, it is relatively simple
`in contrast to wound healing that occurs in skin
`and organs that contain blood vessels and other
`components that participate in the process. This
`response is very similar
`in different
`species,
`although there appear to be some quantitative
`and qualitative variations in specific processes that
`comprise the cascade. Within a species there is
`variability depending on the inciting injury. For
`example, incisional, lamellar, and surface scrape
`injuries are followed by typical wound healing
`responses that are similar in some respects, but
`different in others. This review article will provide
`an overview of
`the cellular
`interactions
`that
`contribute to the corneal wound healing response
`with emphasis on cytokine regulation of these
`interactions.
`
`2. EVOLUTIONARY CONTEXT
`
`to view the corneal wound
`is important
`It
`healing response in the context of the types of
`injuries that were most likely to place selective
`pressure during evolution. Clearly vision was
`essential to the survival of most animals and
`responses would likely have evolved to maintain
`vision following injury. It seems probable that
`abrasions from branches, projectiles, and other
`sources would have been the most common
`injuries to the vertebrate cornea. The wound
`healing responses to these variable injuries must
`retain integrity of the eye, restore the protective
`epithelial surface, while at the same time main-
`taining sufficient corneal clarity to provide vision.
`Infection from ubiquitous viral pathogens such as
`herpes simplex virus, smallpox virus, adenovirus,
`and their ancestors may have posed significant
`threats to the vision and survival of the evolving
`animals. Thus, pathogens that had the potential to
`permanently blind would likely have placed great
`selective pressure on animal
`species. Systems
`designed to impede the spread of viral pathogens
`until the immune response could eradicate the
`invader could have provided an advantage (Wilson
`et al., 1997). The responses that occur in the
`
`cornea appear to be well designed to accomplish
`these objectives.
`
`3. OVERVIEW OF THE WOUND HEALING
`RESPONSE
`
`The epithelium, stroma, and nerves participate
`in homeostasis of the anterior cornea and ocular
`surface. The lacrimal glands and tear film also
`contribute to the maintenance of surface smooth-
`ness and integrity important to function of the eye.
`Following injury, these components participate in
`an orchestrated response that efficiently restores
`corneal structure and function in most situations.
`Many cytokines and receptors modulate the
`process. Activation of these systems also attracts
`immune cells that function to eliminate debris and
`microbes that may breach the injured surface and
`gain entry to the corneal
`stroma. Only by
`considering the individual contributions of each
`of these components and their interactions can one
`begin to truly appreciate the beauty and efficiency
`of the overall response.
`
`4. MASTER REGULATORS OF THE WOUND
`HEALING RESPONSE
`
`The cascade of responses to injury to the cornea
`is initiated very rapidly regardless of the type of
`injury. For example,
`the keratocyte apoptosis
`response detected by electron microscopy is so
`rapid that if one euthenizes a mouse, enucleates
`the eye, and performs a single scrape across the
`epithelium prior to plunging the eye into fixative,
`the anterior keratocytes already show chromatin
`condensation and other morphologic changes
`consistent with apoptosis. Thus, the cornea is
`primed and ready to respond immediately to
`injury. This would make sense if one of
`the
`functions of this response was to retard dissemina-
`tion of viral pathogens until other defense
`mechanisms can be rallied.
`What are the key modulators that regulate the
`early events in the wound healing cascade? After
`working on these problems
`for many years
`we have come to believe that there are a few
`key cytokine modulators that act as ‘‘master
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`627
`
`regulators’’ of the response. Some of these master
`regulators and their receptors are likely to be
`constitutively produced and, therefore, constantly
`available to initiate the wound healing response.
`These initiators are likely to be sequestered until
`needed so that their mischief is not unleashed
`unduly, at least in the normal cornea. Thus, these
`master regulators serve as ever-vigilant guardians,
`silently waiting and on the ready to unleash the
`cascade at the first sign of trouble.
`Interleukin (IL)-1 seems to fit the requirements
`of a master regulator with regards to expression
`and function. First the expression of this cytokine–
`receptor system will be discussed and then the
`many functions that are regulated in the kerato-
`cytes by IL-1 will be reviewed.
`Both IL-1 alpha (Fig. 1) and IL-1 beta mRNAs
`and proteins are expressed constitutively in the
`corneal epithelium (and endothelium)
`(Wilson
`et al., 1994b; Weng et al., 1996). IL-1 receptor
`(binds both IL-1 alpha and IL-1 beta) is also
`constitutively produced in keratocytes and corneal
`fibroblasts (cultured keratocytes will be referred to
`as corneal fibroblasts if they are cultured in serum
`and cultured keratocytes if
`they are cultured
`without serum) (Bereau et al., 1993; Fabre et al.,
`1991; Girard et al., 1991; Wilson et al., 1994c;
`Beales et al., 1999; Jester et al., 1999b). Neither
`IL-1 alpha or
`IL-1 beta are detectable by
`
`Fig. 1. Interleukin 1-alpha is constitutively produced in
`the corneal epithelium. This immunocytochemical locali-
`zation was performed in normal human cornea. Note
`there is no detectable expression of IL-1 alpha in the
`keratocytes in the unwounded cornea. Reprinted with
`permission from Wilson et al., (1994), Exp. Eye Res. 59,
`63–72, r 1994 by permission of the publisher, Academic
`Press.
`
`immunocytochemistry in keratocytes in the un-
`wounded cornea. However, Fini and coworkers
`have demonstrated that exposure to IL-1 can
`induce keratocytes to produce IL-1 via an auto-
`crine loop (Strissel et al., 1997a,b; West-Mays
`et al., 1995). Thus, IL-1 protein is detectable in
`keratocytes or myofibroblasts in the wounded
`cornea. IL-1 (alpha or beta) does not appear to
`be released from the epithelium into the stroma in
`significant amounts in the normal unwounded
`cornea. Both forms of IL-1 lack signal sequences
`for transport from the cell and released via cell
`injury or death (Dinarello, 1994). IL-1 is released
`from apical
`epithelial
`cells undergoing pro-
`grammed cell death as a part of the normal
`maturation and turnover of the epithelium and
`may be present in tears at increased levels in
`conditions associated with ocular surface injury
`such as keratoconjunctivitis sicca.
`(Pflugfelder,
`1998; Pflugfelder et al., 1999). However, tear IL-
`1 probably does not pass into the anterior stroma
`in the absence of epithelial injury or death because
`of the barrier provided by the intact epithelium.
`IL-1 is dumped onto the exposed stroma immedi-
`ately following epithelial injury that is of sufficient
`magnitude to break down epithelial barrier func-
`tion or directly damage basal epithelial cells. In
`some cases, epithelial debris could be tracked into
`the stroma, for example with a microkeratome cut
`into the cornea. Once IL-1 penetrates the stroma,
`it can bind IL-1 receptors on the keratocyte
`cells and modulate
`the
`functions of
`these
`cells. Thus, IL-1 is sequestered within the epithe-
`lium separated from the stromal cells in the
`normal unwounded cornea until epithelial injury
`triggers its release.
`important effects on
`IL-1 has an array of
`keratocyte cells related to wound healing. It has
`been shown to modulate apoptosis of keratocytes
`and corneal fibroblasts (Wilson et al., 1996),
`although the effect appears
`to be mediated
`indirectly via the Fas/Fas ligand system through
`autocrine suicide (Mohan et al., 1997). Since IL-1
`alpha also triggers NF-kappa B activation (Mohan
`et al., 2000) it also has negative apoptotic effects
`on the keratocyte cells and myofibroblast cells that
`appear during the stromal wound healing re-
`sponse. Thus, the overall effect of IL-1 could be
`related to the
`specific milieu of
`the
`cell
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`628
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`
`and the overall input by a number of different
`cytokines.
`IL-1 has negative chemotactic effects on kera-
`tocytes and corneal fibroblasts and may have a
`role in maintaining corneal tissue organization (the
`morphologic separation of epithelium from stroma
`in the normal cornea) through this effect (Wilson
`et al., 1996; Kim et al., 1999a,b; Wilson and Hong,
`2000).
`IL-1 is the primary regulator or hepatocyte
`growth factor (HGF) and keratinocyte growth
`factor (KGF) production by keratocytes (Weng
`et al., 1996). HGF and KGF are classical
`mediators of stromal-epithelial
`interaction that
`are produced by keratocytes and myofibroblasts to
`regulate proliferation, motility, and differentiation
`of epithelial cells (Wilson et al., 1994a). Thus, IL-1
`released by injured corneal epithelial cells triggers
`production of HGF and KGF by the keratocytes
`to regulate the repair process of
`the corneal
`epithelial cells.
`IL-1 upregulates expression of collagenases,
`metalloproteinases, and other enzymes by kerato-
`cytes (Strissel et al., 1997a,b; West-Mays et al.,
`1995). These enzymes have an important role in
`remodeling of collagen during corneal wound
`healing. IL-1 and TNF alpha also upregulate the
`expression of some chemokines such as IL-8,
`RANTES, and monocyte chemotactic protein
`(MCP)-1 in keratocytes and corneal epithelial cells
`(Tran et al., 1996; Takano et al., 1999). IL-1 also
`potentiates
`the chemotactic effect of platelet-
`derived growth factor (PDGF) on corneal fibro-
`blasts.
`Thus, IL-1 directly regulates several critical
`processes that contribute to wound healing. This,
`along with the distribution of expression of IL-1
`and IL-1 receptor in the cornea and the sequestra-
`tion of IL-1 in the absence of injury, suggest a role
`for IL-1 as a master regulator of the corneal
`wound healing response.
`There may be other master regulator cytokines
`that function to initiate the early wound healing
`response. For example, PDGF is expressed by
`corneal epithelial cells and the keratocytes express
`the PDGF receptors (Denk and Knorr, 1997;
`Andresen and Ehlers, 1998; Kamiyama et al.,
`1998; Kim et al., 1999a,b). PDGF is found at very
`high levels in the epithelial basement membrane.
`
`This localization and sequestration is likely related
`to the heparin-binding property of PDGF. PDGF
`is released into the stroma after damage to the
`epithelium and underlying basement membrane.
`PDGF modulates corneal fibroblast proliferation,
`chemotaxis, and possibly differentiation (Denk
`and Knorr, 1997; Andresen and Ehlers, 1998;
`Kamiyama et al., 1998; Kim et al., 1999a,b).
`Tumor necrosis factor (TNF) alpha could also
`participate (Mohan et al., 2000). Less is known
`about
`the function of TNF alpha and other
`cytokines that could also serve as master regula-
`tors in the corneal wound healing response.
`
`5. THE CORNEAL WOUND HEALING
`CASCADE
`
`Studies performed over the past decade have
`revealed numerous processes that comprise the
`overall wound healing cascade in the cornea. It is
`helpful to outline the steps in this response prior to
`discussing individual components of the response.
`It must be appreciated that many of these events
`occur
`simultaneously
`and,
`therefore,
`the
`‘‘cascade’’ should be viewed as such only in rough
`terms. It is clear that some events proceed others.
`For example, keratocyte apoptosis is the earliest
`stromal response that can be detected following
`epithelial
`injury and other components of the
`cascade appear to follow. Figure 2 provides the
`rough framework of the cascade. It is not intended
`to be comprehensive and some processes that are
`clearly important to the overall wound healing
`response are not depicted in the figure. Subsequent
`sections will concentrate on individual processes in
`the wound healing cascade with emphasis on
`cytokine mediation.
`
`6. KERATOCYTE APOPTOSIS AND
`NECROSIS
`
`Work in our laboratory demonstrated that the
`disappearance of keratocytes that followed epithe-
`lial
`injury was mediated by apoptosis (Wilson
`et al., 1996). Studies have suggested that this
`regulated cell death is mediated by cytokines
`released from the injured epithelium such as IL-1
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`629
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`Fig. 3. Keratocyte apoptosis detected with the TUNEL
`assay at 4 h after epithelial scrape injury in the human
`cornea. The scrape injury was produced 4 h prior to
`enucleation for intraocular melanoma. Note staining of
`the superficial keratocytes (small arrows) beneath Bow-
`man’s layer (large arrows). This experiment was approved
`by the Institutional Review Board at the University of
`Washington, Seattle, WA. Magnification 200 .
`
`IL-1 system on
`the
`effect of
`the
`example,
`keratocyte apoptosis may be mediated indirectly
`through the Fas–Fas ligand system via autocrine
`suicide (Mohan et al., 1997). Many types of
`epithelial injury will induce keratocyte apoptosis.
`Some of the triggers include mechanical scrape
`(Wilson et al., 1996), corneal surgical procedures
`like photorefractive keratectomy (PRK) and laser
`in situ keratomeliusis (LASIK) (Helena et al.,
`infection (Wilson et al., 1997),
`1998), viral
`incisions (Helena et al., 1998), and even pressure
`applied with an instrument on the epithelial
`surface (Wilson, 1997). Recent experiments have
`confirmed that apoptosis occurs in the keratocytes
`underlying Bowman’s layer in the human eye when
`the epithelium has a scrape injury (Fig. 3,
`Ambrosio et al., unpublished data).
`It was previously noted that keratocyte apopto-
`sis is the first observable stromal response follow-
`injury (Wilson et al., 1996). The
`ing epithelial
`earliest changes are noted at the electron micro-
`scopic level. It takes a few minutes longer (up to
`30 min) before apoptosis can be detected with the
`TUNEL assay. Thus, DNA fragmentation de-
`tected by the TUNEL assay takes somewhat
`longer to develop and has been found to be most
`prominent at approximately 4 h after the scrape
`injury in mice and rabbits (Wilson et al., 1996,
`1998).
`
`Fig. 2. Schematic diagram indicating some of the events
`that occur in the corneal wound healing response that
`occurs following corneal epithelial
`injury or surgical
`procedures such as PRK or LASIK. Note that this is a
`simplified scheme and not all events
`that may be
`important are included. While there is some indication
`of sequence (for example keratocyte apoptosis is the first
`observable event following injury) many of the events
`occur simultaneously in the cornea.
`
`and TNF alpha (Wilson et al., 1996; Mohan et al.,
`1998, 2000). It is largely unknown how these
`different pro-apoptotic cytokine pathways interact
`to determine whether signaling dictates cell death
`or some other response in individual cells. Likely
`there is redundancy of the systems and possibly a
`requirement for context before a death signal is
`recognized and acted upon. In some cases, more
`than one of these systems may be involved. For
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`S. E. Wilson et al.
`
`Keratocyte apoptosis appears to continue for a
`period of time extending for at least 1 week
`following injuries such as epithelial scrape, epithe-
`lial scrape followed by PRK, or a microkeratome
`cut into the cornea (Gao et al., 1997; Mohan et al.,
`2001). Apoptosis detected with the TUNEL assay
`appears, however, to diminish with time past 72 h.
`Thus, while many keratocytes near the surface die
`immediately after epithelial injury by apoptosis,
`other cells derived from keratocytes that were
`stimulated to proliferate and migrate may con-
`tinue to undergo apoptosis for days. Other cell
`types that arrive later could also undergo apopto-
`sis. Thus, it is likely that the inflammatory cells
`that begin to arrive approximately 12–24 h after
`the injury eventually undergo apoptosis, but the
`time course over which this occurs is presently
`unknown. The disappearance of myofibroblasts
`over time following injury could also be mediated
`by apoptosis, rather than through a transition
`back to a keratocyte cell. There is currently no
`data regarding the mechanism of myofibroblast
`disappearance as the cornea returns to morphol-
`ogy and function more like that
`in the pre-
`wounding cornea.
`Virtually all of the anterior keratocytes that
`disappear in the early post-wounding period seem
`to undergo apoptosis at the electron microscopic
`level. As the wound healing process continues,
`however, there appear to be some cells recogniz-
`able as keratocytes that have hallmarks of necrosis
`rather than apoptosis (Mohan et al., 2001). It can
`be difficult to ascertain with certainty that these
`necrotic appearing cells noted between 12 h and 1
`week are keratocytes rather than inflammatory
`cells. If some of these cells are keratocytes, then it
`is possible that this cell death is mediated via the
`inflammatory cells that arrive at the site of stromal
`wound healing during this same time period
`following the initial epithelial
`injury. More in-
`vestigation needs to be performed to elucidate the
`complex processes occurring at these later points
`in the wound healing process.
`Studies have demonstrated that keratocytes in the
`unwounded cornea are connected by cellular
`processes called gap junctions to form a synsytium
`(Watsky, 1995; Spanakis et al., 1998). It may be that
`cytokines released from the injured epithelium only
`bind to receptors on the most superficial keratocytes
`
`and that the signal to undergo apoptosis is directed
`to deeper cells via these intercellular communication
`channels. Once keratocyte cells begin to proliferate
`and migrate into the area of the wound healing
`response they do so as individual cells. At some
`point, however, the synsytial connections of the
`normal cornea appear to be restored.
`The location of the keratocyte apoptosis and
`necrosis response varies with the type of corneal
`epithelial injury. This in turn tends to influence the
`location and effect of the subsequent events in
`the cascade. Thus, injuries such as scrape of the
`epithelium, mechanical pressure on the epithelium,
`and viral
`infection of
`the epithelium trigger
`keratocyte apoptosis and necrosis in the superficial
`stroma (Fig. 4A). In contrast, a lamellar cut across
`the cornea produced by a microkeratome induces
`keratocyte apoptosis and necrosis at the site of
`epithelial injury and anterior and posterior to the
`lamellar interface (Fig. 4B). This localization is
`thought to be attributable to tracking of epithelial
`debris, including pro-apoptotic cytokines, into the
`interface by the microkeratome blade. Cytokines
`from the injured peripheral epithelium could also
`diffuse along the lamellar interface and into the
`central
`stroma. This can be of considerable
`importance because it influences the localization
`of other events such as proximity between myofi-
`broblasts and wound healing fibroblasts
`that
`produce increased HGF. HGF has effects on
`corneal epithelial cells
`that
`tend to promote
`epithelial hyperplasia (Wilson et al., 1994a). Thus,
`superficial keratocyte apoptosis and necrosis such
`as that triggered by PRK could be more likely to
`result in epithelial hyperplasia than deeper kerato-
`cyte apoptosis and necrosis noted in LASIK. This
`could be of clinical significance and may, at least in
`part, explain differences between the two proce-
`dures when they are used to correct high myopia.
`
`7. LACRIMAL-GLAND-DERIVED
`CYTOKINE MEDIATORS AND EPITHELIAL
`HEALING
`
`Expression of several growth factors such as
`HGF and epidermal growth factor (EGF) that
`modulate epithelial healing increase in the lacrimal
`gland shortly after epithelial injury (Steinemann
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`Fig. 4. Localization of keratocyte apoptosis depends on the type of injury. (A) A lamellar cut produced by a microkeratome
`in preparation for lamellar in situ keratomeliusis (LASIK) triggers keratocyte apoptosis at the site of blade perforation of
`the epithelium (not shown) and along the lamellar interface (arrows). (B). Epithelial scrape injury in photorefractive
`keratectomy (PRK) triggers superficial stromal keratocyte apoptosis (arrows) detected by the TUNEL assay in a rabbit
`cornea. Magnification 200  .
`
`et al., 1990; Wilson et al., 1999) (Fig. 5). This
`correlates with an increase in the bioavailability of
`HGF in the tears (Tervo et al., 1997). This
`upregulation is likely mediated via a reflex arc
`involving the trigeminal sensory nerves of the
`cornea connecting via the brainstem to the
`facial nerve fibers that
`innervate the lacrimal
`gland. Keratocyte
`cells,
`that are a source
`of growth factors such as HGF, undergo immedi-
`ate apoptosis
`in the anterior
`stroma. Thus,
`the lacrimal gland could serve as the primary
`source of HGF and other epithelium-modu-
`lating
`cytokines
`that
`regulate proliferation,
`migration, and differentiation during the early
`wound healing period until myofibroblasts or
`corneal fibroblasts repopulate the anterior stroma.
`Lacrimal-gland-derived cytokines may continue to
`modulate differentiation and other functions in
`superficial epithelial cells once epithelial integrity
`and barrier function is restored. Thus, the lacrimal
`gland appears to have an important role in
`the
`corneal wound
`healing
`response
`and
`likely continues to modulate functions of
`the
`superficial epithelium once homeostasis is re-
`stored. The accessory lacrimal glands may also
`contribute to this response, but this has not been
`examined.
`
`There are also likely to be localized effects of a
`variety of modulators on corneal epithelial wound
`healing. These include autocrine growth factor and
`receptor effects such as those that might be regulated
`by the EGF-transforming growth factor (TGF)
`alpha-EGF receptor systems within the epithelium
`itself (Wilson et al., 1994b, Mohan and Wilson,
`1999). In addition, there are clearly important effects
`of other biomolecular modulators such as integrins,
`fibronectin, and other matrix components. The
`functions of these systems have been discussed in a
`recent review (Alio´ et al., 2000). Recently identified
`discoidin domain receptors expressed in the corneal
`epithelium that are activated by collagen could also
`have a role in epithelial healing (Mohan et al., 2001).
`Variations in proliferative and migration-related
`activity occur across the surface of the epithelium
`with some cells migrating to close the defect and
`others proliferating to provide additional cells.
`Cytokines are likely involved in regulating these
`processes (Zieske, 2000; Zieske et al., 2000).
`
`8. KERATOCYTE PROLIFERATION AND
`MIGRATION-MYOFIBROBLASTS
`
`Apoptosis and necrosis result in an area of the
`cornea being relatively devoid of keratocytes with
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`S. E. Wilson et al.
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`Fig. 5. Growth factor expression increases in the lacrimal gland in response to corneal epithelial scrape injury. HGF mRNA
`(A), KGF mRNA (B), and EGF mRNA (C) levels were monitored in rabbit lacrimal glands following corneal epithelial
`wounding using an RNAse protection assay. Levels of mRNA were monitored in lacrimal glands of unwounded control
`animals and in the lacrimal glands of animals at 1 h, 8 h, 1, 3, and 7 days after corneal epithelial scrape injury. Beta actin
`mRNA was also monitored as a control for RNA loading in each lane. Note that beta actin mRNA levels are similar in most
`samples within an experiment. Sizes of the protected RNA sequences based on the base pair (BP) lengths of the cDNA
`sequences used to generate probes are indicated. HGF mRNA, KGF mRNA, and EGF mRNA levels increase in the
`lacrimal gland after corneal epithelial wounding. The levels appear to peak at 3 days for each growth factor. Reprinted with
`permission from Invest. Ophthalmol. Vis. Sci. 1999, 40, 2185–2190. r 1999 Association for Research in Vision and
`Opthalmology.
`
`the region within the cornea being related to the
`type of wound. Zieske and coworkers (2001) have
`demonstrated that approximately 12–24 h follow-
`ing the original injury remaining keratocytes begin
`
`to proliferate (Fig. 6). For example, with a corneal
`epithelial scrape injury, the anterior stroma is
`relatively devoid of keratocytes and the remaining
`keratocytes in the posterior and peripheral cornea
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`The corneal wound healing response
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`633
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`Fig. 6. Keratocyte proliferation monitored by detection of the mitosis specific Ki-67 antigen in rabbit corneas using
`immunohistochemistry. Cells undergoing mitosis stain green for the mitosis-associated antigen Ki-67 (arrows). Propidium
`iodide stains other cell nuclei orange/red: (A) is the unwounded cornea. Note no keratocytes stain for mitosis. (B) shows the
`cornea 8 h after epithelial scrape injury. Note there are no cells detected in the anterior stroma due to keratocyte apoptosis.
`No mitosis is detected. (C) shows the cornea 24 h after scrape injury. The epithelium has healed and there are some posterior
`keratocytes now beginning to undergo mitosis (arrows). (D) shows the cornea at 48 h after epithelial scrape. Note the broad
`band of keratocytes in the posterior stroma undergoing mitosis. Keratocyte or myofibroblast cells have repopulated the
`anterior stroma. (E). At 5 days after epithelial scrape injury the anterior stroma is fully repopulated. A few cells in the
`anterior stroma continue to undergo mitosis (arrows). (F) At 10 days after wounding no keratocytes undergoing mitosis are
`detected. Magnification 200 . Figure kindly provided by SR Guimaraes, AEK Hutcheon, and JD Zieske from their studies
`presented at the Association for Research in Vision and Ophthalmology annual meeting in Ft. Lauderdale, FL in 1999.
`
`begin proliferation and migration. The factors
`modulating the beginning and ending of prolifera-
`tion are not understood. We have hypothesized
`that platelet-derived growth factor (PDGF) and
`PDGF receptors have a role in this process (Kim
`et al., 1999b). PDGF is sequestered via its heparin-
`binding properties in high levels in the basement
`membrane in the unwounded cornea. Once epithe-
`lial injury occurs there is likely release of PDGF
`into the stroma. Vesaluoma and coworkers (1997)
`also reported that PDGF is detected in the tears
`following corneal
`injury. Binding of PDGF to
`PDGF receptors on viable keratocytes
`likely
`stimulates proliferation and chemotaxis of the
`keratocytes, based on studies of PDGF in vitro
`(Denk and Knorr, 1997; Andresen and Ehlers,
`1998; Kamiyama et al., 1998; Kim et al., 1999a,b).
`There may be other cytokines that contribute to
`
`this regulation of keratocyte apoptosis that have
`yet to be identified.
`Keratocytes proliferation continues for several
`days (Zieske et al., 2001; Mohan et al., 2001).
`Recent data has demonstrated that keratocyte
`apoptosis and necrosis also continue for at least a
`week after the initial injury and, therefore, it may
`be that some of
`the new cells derived from
`keratocyte proliferation themselves undergo apop-
`tosis or necrosis (Mohan et al., 2001). After a few
`days, apoptosis, necrosis, and mitosis wind down
`and a relatively quiescent state is restored. It is
`unknown how this overall process is regulated.
`One possible signal is healing and restoration of
`the normal differentiated state of the epithelium
`leading to restoration of the homeostatic levels of
`key cytokines including IL-1 and PDGF, but this
`has not been tested.
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`What cell types are derived from the mitotic
`keratocytes? In vitro studies have suggested that
`myofibroblasts are an important cell type gener-
`ated during the first few days following injury
`(Masur et al., 1996; Jester et al., 1990, 1999b;
`Moller-Pederson et al., 1998). These
`studies
`suggest that transforming growth factor beta has
`an important
`role in the generation of
`the
`myofibroblasts. Myofibroblast cells are character-
`ized by alpha smooth muscle actin expression.
`They also have altered transparency related to
`corneal cystallin production (Jester et al., 1999a).
`They may have additional differences relative to
`keratocytes that include increased production of
`growth factors such as HGF and KGF (Weng
`et al., 1996), collagen (Kaji et al., 1998; Jester,
`et al., 1999b; El-Shabrawi et al., 1998), glycosa-
`minoglycans, collagenases, gelatinases, and metal-
`loproteinases associated with remodeling of the
`collagen and the stroma (Girard et al., 1991;
`Strissel et al., 1997a,b; West-Mays et al., 1995; Ye
`and Azar, 1998; Ye et al., 2000).
`A question that remains unanswered is whether
`all of the cells derived from keratocytes prolifera-
`tion following corneal injury are myofibroblasts.
`This could be the case, but it is also possible that
`all of the cells are originally typical keratocytes
`and some are modulated to transform into
`myofibroblasts once they migrate to the area
`involved in the healing process.
`The e