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   Table of Contents      
CURRENT OPHTHALMOLOGY
Year : 2003  |  Volume : 51  |  Issue : 1  |  Page : 5-15

Corneal epithelial wound healing.


Leelavati Hospital and Research Centre, Mumbai (VBA), India

Correspondence Address:
Vinay B Agrawal
Leelavati Hospital and Research Centre, Mumbai (VBA)
India
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Source of Support: None, Conflict of Interest: None


PMID: 12701857

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  Abstract 

One of the important functions of the cornea is to maintain normal vision by refracting light onto the lens and retina. This property is dependent in part on the ability of the corneal epithelium to undergo continuous renewal. Epithelial renewal is essential because it enables this tissue to act as a barrier that protects the corneal interior from becoming infected by noxious environmental agents. The renewal process also maintains the smooth optical surface of the cornea. This rate of renewal is closely maintained by an integrated balance between the processes of corneal epithelial proliferation, differentiation, and cell death. Attempts to understand this complex cascade make it evident that the appropriate integration and coordination of corneal epithelial renewal depends on the actions of a myriad of cytokines. We have attempted in this review to collate the receptor and cell signaling events and cytokine studies that are responsible for mediating corneal wound healing.

Keywords: Corneal epithelium, renewal, cyotkines, corneal wound healing


How to cite this article:
Agrawal VB, Tsai RJ. Corneal epithelial wound healing. Indian J Ophthalmol 2003;51:5-15

How to cite this URL:
Agrawal VB, Tsai RJ. Corneal epithelial wound healing. Indian J Ophthalmol [serial online] 2003 [cited 2020 Aug 11];51:5-15. Available from: http://www.ijo.in/text.asp?2003/51/1/5/14743



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The corneal epithelium is exposed to a continuously changing external environment and serves as the frontal barrier of the entire eyeball. For clear vision, it is important that the surface of the corneal epithelium remain smooth, so that light is uniformly refracted, and that an active repair process resurfaces defects. Epithelial wound healing depends on a complex interaction of various cellular components that interact via a network of interactive, signaling molecules. Cell-cell and cell-matrix interactions play important roles in the maintenance of the stratified structure of the corneal epithelium. Any damage to the corneal epithelium elicits a series of events that help in covering the area of injury.

Wound healing

Corneal epithelial wound repair can be divided into three overlapping phases.[1] In the first phase, the hemidesmosomes[2] (which normally attach the epithelium to its underlying matrix, and other anchoring structures, such as desmosomes,[3] and anchoring fibres of type VII collagen[4]) are lost, and a provisional attachment complex is formed, called focal contacts . During this phase, the epithelial cells flatten and migrate as an intact sheet to cover the wound. This phase is independent of cellular proliferation.[5] During the second phase, cells distal to the original wound proliferate to repopulate the wound area, and cell stratification and differentiation occur. In the third phase, the hemidesmosomes are reformed, and extra- cellular matrix synthesis and reassembly occur.

Interaction

The corneal epithelial wound healing response is brought about by a complex cascade of events involving cytokine (soluble factors)-mediated interactions between the epithelial cells, keratocytes of the corneal stroma, corneal nerves, lacrimal glands, and cells of the immune system. This interaction of the various components is crucial to restoring the function of the epithelium as a barrier and as the cornea's refractive surface. The level of interaction is dependent on the inciting injury. For example, lamellar, incision, and surface scrape injuries are followed by typical wound healing responses that are similar in some respects, but different in others.[6] 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 access the corneal stroma. Thus it is an orchestrated response of these components that efficiently restores corneal structure and function in most situations.

"Cross-talk"

The epithelial-mesenchymal interactions play a vital role in wound healing. These interactions can be mediated by signals transmitted from the mesenchyme to the epithelium or vice versa in a reciprocal manner. Such signals can be extracellular matrix components, cell membrane-associated molecules, and cytokines. These three types of signals are not mutually exclusive. The action of one may be dependent on or mediated by the expression of the other. Four patterns of cytokine dialogue have been studied.[7] Three of them constitute the basic network of the potential epithelial-mesenchymal cytokine dialogue system [Table - 1].


  Importance of Corneal Epithelial Renewal to Function and Transparency Top


In addition to the healing response to injury, a renewal process is needed to maintain the protective function of the epithelium. This process is important to the two major properties of corneal epithelium needed for normal vision. The first is the formation of a smooth refractive surface via interaction with tear film. The second is to form a tight protective junctional barrier. This barrier prevents alterations in net fluid transport from the corneal stroma and prevents penetration by pathogens. Wounding of the corneal surface epithelium leads to breakdown of the tight junctional integrity due to loss of the outer limiting epithelium. This loss can lead to a breakdown of cell membrane permeability and selectivity. This is not restored until after epithelial migration from the periphery has resulted in resurfacing of the denuded corneal epithelium. During this period of injury the cornea thus becomes susceptible to infection by microbes and the attendant consequences. The alteration in the healing process caused by such invasion can cause derangement of cytokine-mediated control of the healing. This in turn decreases the endothelial fluid transport, and increases stromal hydration, causing corneal opacity.

The normal renewal of the corneal epithelium is an ongoing process. First the surprabasal cells in the limbal and corneal epithelia are terminally differentiated and appear to have exited the cell cycle. At the other end are the basal cells of the corneal epithelium where approximately 40% of the cells are actively proliferating.[8] Between these two extremes are the basal cells of the limbal epithelium, where approximately 20% of the cells are actively cycling. This group of cells contains a mix of slow-cycling stem cells and cycling transient amplifying cells.[9],[10] The superficial cells are specialised to form tight adhesions between one another and to the basal lamellae. This provides the epithelial barrier function. As the superficial cells are lost, the underlying basal cells first move into differentiating wing cells and then migrate upwards as superficial cells. The formation of the tight epithelial barrier is of paramount importance as a physical barrier to noxious agents. The corneal epithelium is thus well maintained through a process of mitosis, migration, and shedding. The last step of superficial cell desquamation is aided by eyelid blinking.[11] The interaction of the superficial cells with the tear film is supported by the presence of numerous microvilli and microplicae, which facilitate transport of metabolites and tear film adhesion.[12] This allows formation of a smooth refractive surface for the cornea.


  Corneal Epithelial Stem Cells Top


Location of stem cells

The limbal location of corneal epithelial stem cells was first described by Davanger and Evensen in 1971.[13] The experimental work of Schermer et al[14] and Costarelis et al [15] later confirmed that the source of cell proliferation and migration after corneal epithelial defect is the sclero-corneal limbus. The existence of corneal epithelial stem cells has proven by several elegant studies, but all the current evidence uses only indirect methods. No direct biological marker for stem cells has been established. The various properties of the stem cells are outlined in [Table - 2].

The specific location of the corneal epithelial stem cells in the limbus provides several functional advantages. As the central corneal epithelium has to be transparent, its basal cells are devoid of pigment and consequently, are highly susceptible to solar damage. Basal cells in the limbal region do not have this constraint; they are heavily pigmented and thus well protected.[9],[15] The transparency of the cornea dictates a smooth epithelial-stromal junction. This minimal anchorage renders corneal epithelium susceptible to physical shearing. In contrast, limbal epithelium is very resistant to shearing forces and displays a highly undulating epithelial-stromal junction.[16] This gives a natural advantage for retention of stem cells in the face of any environmental damage.

All the same, in the absence of a definite biological marker, the stem cells concept awaits final proof. It is commonly believed that corneal epithelial stem cells constantly generate corneal epithelial basal cells and transient amplifying cells with a life span of a few months. However, we do not have definite evidence to support this view. The other common belief is that all corneal epithelial stem cells reside in the limbus. While this may be true, it is not out of question that some corneal epithelial stem cells may exist in the peripheral cornea, in much the same way that peripheral blood contains a few stem cells.[17]

Significance of properties

The corneal epithelial stem cells are normally slow cycling in vivo . They can self-renew and are responsible for the longterm maintenance of the tissue. They can be activated by wounding or by in-vitro culture conditions to proliferate and regenerate the tissue. Additionally, they have a high proliferative potential. The slow-cycling attribute is particularly important because it conserves the cell's proliferative potential and minimises DNA replication-related errors.[18] Epithelial stem cells in the limbus have a longer life span than those of the central cornea.[19] They are thus able to provide longterm maintenance of corneal epithelial homeostasis. These properties thus enable the limbal stem cells to effectively replace the central corneal epithelial cells that are continuously shed and thus lost from the corneal epithelial surface.

In a normal cycle one stem cell gives rise to one stem cell that remains in the stromal "niche" and one transient amplifying cell (TA), which replicates rapidly but has only limited proliferative potential.[20] These TA cells are known to exist in a hierarchy, the cells in the periphery are capable of multiple divisions and the central TA cells have only one remaining division.[21] This is consistent with the fact that cells derived from the central part of the cornea have poor division capacity. This suggests that as a cell migrates from its point of origin in a stem cell-enriched region it becomes closer to the post-mitotic state.

A large fraction (up to 50%) of the limbal stem cells can be induced to proliferate in the event of corneal epithelial injury.[21] In addition, the TA cells are capable of multiple rounds of division. This capability may not be utilised in the maintenance of normal homeostasis, but is significant for response to wound healing. The average cell cycle time for corneal TA cells is 60 hours under normal physiological conditions. This can be shortened to 24 hours upon stimulation. Thus, in addition to up-regulation of stem cells, increasing the proliferative capacity of the TA cells achieves the following objectives: (a) amplifying each stem cell division and minimising the need for stem cell proliferation; (b) minimising the chance of introducing replicative DNA errors into the stem cell population; and (c) providing new cells that are much closer to the terminally differentiated compartment.


  Phases of Wound Healing Top


The healing of corneal epithelium can be divided into three distinct but continuous phases: (1) sliding of superficial cells to cover the denuded surface; (2) cell proliferation and stratification; and (3) reassembly of adhesion structures. These steps are preceded by the Lag phase during which the epithelial cells alter their metabolic status.

Lag phase

The time between wounding and the onset of cell migration is the lag phase. This phase sees a great deal of cellular reorganisation and protein synthesis. Several cytoskeletal proteins[22] such as, vinculin, actin, talin, and integrin are synthesised, as are other cell surface receptors (e.g. hyaluronan [HA] receptor CD44). Cell surface glycoprotines and glycolipids are also synthesised.[23]

Integrin a6β 4 is an integral membrane glycoprotein which anchors extracellular proteins to cytoskeletal components. It is a heterodimer composed of a and β subunit that is specific for binding to laminin in the corneal basement membrane. In its normal distribution it is located at the base of the basal cells. It is responsible for the adhesion of these cells to the underlying basement membrane. During the lag phase this integrin dissociates from the desmosomes and hemidesmosomes [Figure - 1] and distributes evenly on the cellular surface. Thereafter this serves as an adherens molecule to the adjacent extracellular matrix (ECM).[24] It is also believed that these surface glycoproteins are responsible for a bidirectional signaling between the ECM and the cytoskeleton.[25]

Cell migration

Once the lag phase remodeling of the epithelial cells is complete, cells adjacent to the wound commence migration to re-establish the integrity of the ocular surface epithelium. It has been postulated that the basal and wing cells participate in the formation of the leading edge.[26] Re-epithelialisation does not occur one layer at a time. It is now understood that there is a gradient of cells from opposite directions that meet in the center of the wound. The multiple layers of cells extending over the regenerating surface result from migration of epithelial cells that originated adjacent to the wound margin. In case of the suprabasal cells some of the cells have been shown to have "stalks" extending to the basement membrane[9] indicating that the second layer too comprises of displaced basal cells. It is thus postulated that progressive movement of both, basal and suprabasal cell layers that are adjacent to the wounded surface, resurfaces the corneal epithelial wound.[27]

After about 5 hours of wounding, cells begin to migrate at a constant rate of 60 - 80 mm/hr until wound closure is completed.[28] The process of cell migration is achieved by synthesis of an elaborate cytoplasmic array of actin - rich stress fibers. Blocking these stress fibres is known to inhibit epithelial cell migration and adhesion. Proparcaine, a topical anaesthetic, inhibits corneal epithelial migration partly through alteration of the actin cytoskeleton.

Cell proliferation and differentiation

Flattening and elongation of cells during migration covers the wound area. Cell proliferation then occurs to repopulate the wound area.[1] The migratory and proliferative responses are compartmentalised in that limbal and peripheral epithelial cells exhibit an enhanced proliferative rate following wounding, while cells at the leading edge of migrating epithelium do not proliferate. Corneal epithelial debridement stimulates a 4.5-fold and 3.2-fold increase in cell proliferation in limbal and peripheral corneal epithelium respectively.[29]

The amount of cellular proliferation is demand dependent. The corneal epithelium meets this demand in various ways. In the normal situation, the stem cells located in the limbus, cycle infrequently with a relatively long cell cycle time. Upon division, stem cells give rise to regularly cycling transient amplifying (TA) cells located in the peripheral and central corneal epithelium. Young TA cells with multiple division capacity are preferentially located in the peripheral cornea, whereas the more mature TA cells having little proliferative reserve reside in the central cornea. These mature TA cells may divide only once before becoming terminally differentiated.

Under normal circumstances not every TA cell utilises its full capacity to divide. Stimulated by an injury, self-renewing epithelium can adopt 3 strategies to expand the cell population. It may recruit more stem cells to divide with a more rapid cell cycle time producing more TA cells. It may induce the young TA cells in the peripheral cornea to exercise their full replicative potential thereby generating more mature TA cells. Finally, it may increase the efficiency of TA cell replication by shortening the cell cycle time. Taken together these three strategies result in the production of a large number of post-mitotic terminally differentiated cells.[21] All the cell proliferation and differentiation even during the wound-healing phase is similar to that which occurs during normal homeostasis. During normal homeostasis, continuous centripetal movement of peripheral corneal epithelium toward the visual axis maintains corneal epithelial mass. This balances the cellular loss resulting from anterior movement of basal epithelial cells to the surface and into the tear film. This is the classic X, Y, Z hypothesis first proposed by Thoft and Friend.[30] This has been further supported by the mathematical model demonstrating that the rate of exfoliation of epithelial cells is consistent with their production from the limbal cells.[31] The half-time of corneal epithelial replacement is 9 weeks, while the time required for 95-99% replacement is 9-12 months.


  Basement Membrane Changes Top


Events immediately following injury

Inflammatory cells (polymorphonuclear neutrophils) bind to the surface of the exposed basement membrane after debridement wounding.[32] These release proteases capable of degrading the basement membrane. The tear film may also play a role, as it contains proteases, especially after corneal wounding.[33],[34] In addition, corneal epithelial cells release gelatinase B and matrilysin,[35],[36] metalloproteinases capable of degrading components of the basement membrane. Protease digestion of the basement membrane over a period of time after injury alters both its structure and function

In addition to the proteases, simple mechanical unraveling of the lamina densa over time after debridement is a possible mechanism.[37] Given sufficient time after injury and the absence of integrins and other molecules to permit their stabilisation and organisation, the basement membrane proteins within the lamina densa may diffuse into the tear film as a result of mechanical friction caused by the blinking eyelid.[37] This suggests that there is a regulated disassembly of basement membrane after injury to the corneal epithelium.

Importance of basement membrane to epithelial wound healing

Basement membrane disassembly can affect re-epithelialisation in one or more of the following ways:

1.Exposure of leading epithelial cells to underlying stromal extracellular matrix (ECM): This induces new integrin expression or activation in migrating epithelial cells. The demonstration of integrin-mediated migration and collagenase-I induction in keratinocytes exposed to native collagen I supports this concept.[38]

2. Modification of intracellular signaling pathways in migrating epithelial cells: Cytokines like Transforming growth factor- β (TGF-β ), and basic Fibroblast growth factor (bFGF) are found in the basement membrane (BM) after injury and are held there by binding to molecules in the matrix.[39],[40] Thus partial disassembly of BM releases molecules involved in modulation of cell proliferation, cell differentiation, and/or apoptosis.

3. Formation of a stable adhesion complex: Studies on both animal and human corneas show that the structure and composition of the epithelial basement membrane affects the adhesion of the cells sitting on it.[37] This is well documented in diabetic corneas. Corneas of patients with diabetes mellitus basement membrane abnormalities, and these losses correlate with reductions in integrin localisation within the epithelial cells.[41] This is further supported by studies to determine the most effective treatments for recurrent epithelial erosions. It is concluded that the need of the epithelial cells to resynthesize a new basement membrane is one common parameter in successful treatment.[42]

A better understanding of the role of basement membrane in corneal epithelial wound healing would help us treat the patients with recurrent corneal erosions. Although the cause of these erosions in patients without blistering diseases is unclear, all studies point to a poorly formed basement membrane as a feature common to all patients.

Reassembly of basement membrane

Basement membrane functions as a dynamic structure of the tissue morphology, differentiation, and maintenance. Remodeling of the basement membrane thus constitutes an integral part of the corneal epithelial wound healing process. Migrating epithelial cells synthesize and deposit laminin-1 beneath themselves within 24 hours after photoablation.[43] Laminin-1 is a major constituent of the basement membrane and regulates various cell processes, including adhesion, proliferation, and differentiation.[44],[45] The interaction of Laminin-1 and 5 with a6β 4 integrin can mediate cell migration. Expression of connexin[42] and desmoglein 1 and 2 increases at approximately the same time as that of laminin-1. The observation that the re-establishment of basement membrane coincides with the formation of two of the four types [Table - 3] of intercellular junctions suggests that the basement membrane may affect the expression of junctional adhesion proteins in corneal epithelial cells.

Role of integrins

The integrins are a family of cell surface glycoproteins, that mediate several cellular adhesive functions. The primary function of integrins is adhesion of cells to the extracellular matrix. These interactions influence many aspects of cell behaviour including cell morphology, adhesion, migration as well as cellular proliferation and differentiation. They are fundamentally important in forming a connection between the extracellular matrix on the exterior and the cytoskeleton on the interior. In fact, they have been so named for the perceived integration of the cell surface with the cytoskeleton.[46] The integrins function in bidirectional transduction of signals across the plasma membrane.

Following injury an elaborate chain of events is triggered; integrins play an integral part in this process. One of the first changes involves the cells that have migrated beyond the basement membrane. These cells are exposed to the extracellular matrix. The resulting interaction of fibrillar collagen with epithelial a2β 1 integrin rapidly triggers MMP-1 (collagenase -1) expression.[38] which is sustained by subsequent cross talk with activated epidermal growth factor (EGF) receptor. MMP-1 denatures the fibrillar collagen I by cleaving the a1 and a2 chains, thereby reducing its adhesive function and providing a more favorable environment for migration.[47]

The corneal epithelium adheres to its basal lamina through hemidesmosomes and a6β 4 localizes in the hemidesmosomes.[48] When an injury occurs, cells disassemble their hemidesmosomes and migrate along the substrate. This disassembly of hemidesmosomes is achieved by a redistribution of the a6β 4 integrin molecules such that they move away from the basal area of the cells.[49] This suggests that the various integrins have a dynamic relationship with the plasma membrane and are altered depending on the condition of the surrounding matrix and ligands.

The presence of selective integrin subunits on the plasma membrane and the interaction of these subunits with their ligands plays a role in the formation of the cell matrix adhesion complexes. This response is sensitive to changes in the extracellular environment in vitro and in vivo . Thus a better understanding of these heterodimers may allow us to manipulate the wound-healing cascade. If we can create a set of the normal ligands (e.g. fibronectin) that allow these integrins to localise to the slots that they occupy during normal homeostasis, we would probably be able to enhance the wound healing rate several fold.


  Growth Factors and Cytokines Top


Cytokines are multipotential (glyco) proteins, which act non-enzymatically in picomolar to nanomolar concentrations to facilitate intracellular communications. It is now known that cytokines, including growth factors, are very much involved with corneal tissue remodeling. Recent evidence also suggests that programmed cell death (apoptosis), also plays an important role during wound healing.

EGF, KGF, and HGF

A number of different growth factors are involved in the regulation of corneal wound healing. Among them, epidermal growth factor (EGF), keratinocyte growth factor (KGF), and hepatocyte growth factor (HGF) are strong mitogens of corneal epithelial cells.

EGF exists in various body fluids. In the eye it exists in tears (main source lacrimal galnd) [50],[51],[52] and to increase corneal epithelial cell proliferation both in-vitro and in vivo . When epithelial erosions occur suddenly, EGF already present in tears can act immediately on the corneal epithelium to promote cell proliferation. KGF and HGF specifically promote proliferation in cells of epithelial origin in a paracrine fashion. KGF mRNA is expressed predominantly in corneal stromal fibroblasts, while KGF receptor mRNA is expressed in corneal epithelial cells but not in stromal fibroblasts.[53] Like EGF, KGF stimulates corneal epithelial cell growth both in vivo and in vitro .[54] HGF mRNA is also expressed in cultured human stromal fibroblasts, and its receptor mRNA in corneal epithelial cells.[54] Both KGF and HGF play important roles in corneal epithelial cell growth as paracrine stimulators from stromal fibroblasts.

Based on several pieces of evidence, it is now thought that EGF acts as the basic facilitator for epithelial proliferation, whereas KGF and HGF are additionally up-regulated depending on the amount of damage. Additionally, interesting differences are apparent between the regional specificity of these growth factors. For example, topical KGF predominantly affects the limbal epithelial cells after wounding, whereas EGF acts on epithelial cells both in the limbus and in the peripheral cornea.[55] Since corneal epithelial stem cells are present in the limbal region, KGF may act by increasing their proliferation. It is likely that KGF plays a role in the proliferation of limbal and conjunctival epithelial cells, while HGF is involved with the proliferation of central and peripheral corneal epithelial cells. The dominant growth factor (KGF or HGF) may depend upon the site of the epithelial defect.

These growth factors also elicit different responses from corneal epithelial cells, particularly with regard to their motility and differentiation. For example, EGF and double chain mature HGF (DC-HGF) induce changes in epithelial cell morphology and motility that depend on cell density as well as inhibited keratin 3 expression, whereas KGF does not affect motility and differentiation appreciably.[56]

TGF-b

The transforming growth factor-β (TGF-β ) family of proteins comprises three closely related isoforms in mammal - TGF-β 1, -β 2, and -β 3. Various isoforms have been detected in the human tears and in human corneal epithelium. TGF-β s have multifunctional regulatory activity, and are known to be intimately associated with the regulation of wound healing. It has been shown that TGF-β antagonises the actions of EGF on corneal epithelial cells, and that both TGF-β 1 and -β 2 inhibit the corneal epithelial cell proliferation promoted by KGF and HGF, [57] and weakly inhibit the corneal epithelial cell proliferation promoted by EGF. It is now thought that TGF-β 1 or -β 2 may be activated at the leading edge of the epithelium, where it might antagonise the proliferative effects of EGF, KGF, and/or HGF. Also, it is possible that TGF-β 1 and -β 2 may play important roles as negative modulators against cell proliferation in the wound healing process.

Cytokines

Inflammatory cytokines in corneal wound healing

Cytokines play an important role during corneal wound healing. Various cytokines are up-regulated after injury to the corneal epithelium. Of particular interest are Interleukin 1a (IL-1a) and IL6. The levels of these are significantly up-regulated after injury. More importantly, the levels of IL-1a and IL-6 correlate with the severity of injury.[58] This increase in the levels of IL-1 and IL-6 in injured cornea can initiate the cascade of events that constitute epithelial wound healing. IL-6 for example stimulates epithelial migration in the cornea by up-regulation of integrin,[59] and the IL-1 accelerates epithelial wound closure in vitro and acts synergistically with EGF.[60] It is also possible that increased expression of IL-1 in the cornea may act in conjunction with EGF in tears to stimulate epithelial wound healing, and may also induce KGF and HGF expression in corneal fibroblasts,[61] causing epithelial cell proliferation.[62]

It is noteworthy that all the effects of IL-1 are not beneficial. IL-1 induces the matrix metalloproteinase (MMP) family of enzymes that may cause corneal stromal melting,[63] and IL-1 released from injured corneal epithelium mediates keratocytes apoptosis. IL-1 also induces IL-6 production and this has a role in inflammation because it induces lymphocyte differentiation. In addition, IL-1 induces the corneal epithelial and stromal cells to express IL-8, a strong chemotactic factor of neutrophils,[64] as well as a strong angiogenic factor.[65] As such, IL-1, IL-6, and IL-8 expression in injured cornea can induce stromal melting, cell infiltration, and neovascularisation.

Inflammatory cytokines in human tears

Ocular surface cells themselves express and produce some inflammatory cytokines. These include IL-1, -6, and IL-8. Investigations to detect the level of these inflammatory cytokines in basal and reflex human tears have shown that IL-1β , IL-6, IL-8 levels in reflex tears are significantly lower than those in basal tears.[66] It is thus a possibility that these cytokines are principally products of the ocular surface cells in their quiescent state, while IL-1a is provided by lacrimal tears. Since IL-6 and IL-8 are present even in tears of eyes with no signs of inflammation it is possible that they play a major role in ocular surface maintenance and homeostasis.

Cytokine networks during corneal wound healing

The interaction of various cytokines that leads to a network-like action is crucial to the final outcome of wound healing. This cytokine network governs proliferation of corneal cells, and degradation of denatured collagens. It acts as a defense against foreign organisms such as bacteria and fungi [Figure - 2]. However, the excess of these cytokines that are needed for corneal wound healing can also severely damage the cornea. They can induce corneal ulceration, melting, and neovascularisation. It is speculated that the degraded corneal collagens induce expression of inflammatory cytokines, and that the suppression of stromal degradation by the matrix metalloproteinase inhibitor results in a curtailed cytokine expression. The initial expression of the inflammatory cytokines immediately after corneal injury induces a "secondary" injury.[67] It is possible that some amount of corneal infections, immune reactions, and inflammatory reactions, occur due to the cascade of the inflammatory cytokines. Thus, specific therapies designed to control the cytokine network could well form the basis for treatment of a variety of corneal problems.


  Extracellular Matrix and Corneal Epithelial Wound Healing Top


There are two main modalities in which the extracellular matrix (ECM) can affect cell behavior. One of these is through harboring growth factors or growth factor-binding proteins. Rather than passively sequestering such factors, we now realise that the ECM plays an active role in their mobilisation. Indeed, remodeling enzymes are pivotal in decisions to release matrix-bound growth factors and thereby control differentiation. Additionally, the cell-ECM interactions can directly regulate cell behavior, either through receptor-mediated signaling or by modulating the cellular response to growth factors. In terms of cell differentiation they need to interact with specific types of matrix protein so that appropriate intracellular signal transduction cascades are triggered.

ECM remodeling and differentiation in corneal epithelial repair

Matrix Metalloproteinases (MMPs) play an important role in ECM remodeling. Most MMPs are secreted from cells as zymogens and become activated only following cleavage of their amino-terminal pro-domains. In order that MMP proteolysis does not lead to widespread destruction of the ECM, activation is closely orchestrated adjacent to the cell surface.[68]

Basal epithelial cells reside on basement membrane, which separates them from the underlying stroma. Following injury an elaborate chain of events is triggered to ensure that the basal epithelial cells migrate to re-epithelialise the wound. One of the first changes is an altered basal epithelial cell- ECM interaction at the wound edge, where cells that have migrated beyond the basement membrane become exposed to the stromal collagen. The resulting interaction of fibrillar collagen with basal epithelial cells rapidly triggers MMP-1 expression.[38] This is sustained by further cross talk with activated epidermal growth factor (EGF).

In addition to triggering the healing response, ECM remodeling is required to provide stop signals for epithelial cell migration and to trigger subsequent re-epithelialisation. The basement membrane underlying the basal epithelial cells contains laminin-5, which provides essential structural integrity to the epithelium by linking hemidesmosomes within the basal epithelial cells to collagen-VII containing anchoring fibrils of the subtending stroma. Laminin-5 secreted by a motile transformed epithelial cell line contains an unprocessed form of its a3 subunit, which fails to support hemidesmosome assembly but permits migration. Plasminogen binds to this laminin subunit and upon activation by tissue-type plasminogen activator, cleaves laminin to a shorter form that promotes the assembly of hemidesmosomes and thereby impedes motility.[69] Thus, remodeling laminin can convert it from an ECM protein that directs motility to an adhesive one that promotes differentiation. This is further supported by the fact that corneal epithelial cells migrate to a greater extent on unprocessed laminin-5 than on processed laminin-5.[70]

An interesting finding has been the detection of the cell cycle kinase inhibitor p15INK4b to epithelial cells migrating over the provisional wound matrix.[71] This may in part explain the findings that cells migrating over the original wound area do not proliferate, whereas cells distal to the wound show an enhanced proliferation rate.


  Inflammatory Cells and Their Function Top


Beginning approximately 12-24 hours after injury to the cornea there is an influx of inflammatory cells into the corneal stroma. These inflammatory cells are thought to move in from the limbal blood vessels and possibly from the tear film. A surprisingly large type of inflammatory cells can be detected using immunocytochemistry and electron microscopy. The signals that draw these inflammatory cells into the cornea are, most likely, soluble cytokines or chemokines released following injury. Corneal fibroblasts secrete monocyte chemotactic protein-1 (MCP-1) in vitro when stimulated by IL-1 or TNF a.[72] Interestingly this protein is expressed in vivo only after epithelial scrape injury or some other injury to the overlying epithelium.

It seems likely that one important function of these inflammatory cells is scavenging of cellular components released during programmed cell death. Thus, apoptotic bodies that are distributed throughout the stroma are engulfed by some these inflammatory cells. If the corneal injury is associated with tissue invasion by microorganisms then these cells function to eliminate these pathogens. These inflammatory cells may also be responsible for some of the fibroblastic appearing cells that repopulate the stroma after epithelial injury.[73]

After the immediate healing of the injured area, the inflammatory cells are gradually eliminated from the site. The exact mechanism is poorly characterised. It is likely that the majority of inflammatory cells eventually undergo apoptosis since this is the process through which these cells are eliminated in many other organs.


  Conclusions Top


Studies of corneal wound healing have yielded useful information for our understanding of the roles of cytokines in the maintenance of corneal epithelial functions. However, little information is available about the precise roles of individual cytokines in corneal morphogenesis during development and homeostasis in adults. Also, in most cases the information has not been translated successfully to clinical therapies.

In the past few years a number of studies, some of them quoted in the review, have established that several proteins are up-or down-regulated during the corneal epithelial wound healing process. However, even though the players have been identified it is still not clear how the healing processes are organized. Identification of the signaling components will continue to be an interesting area of corneal wound-healing research.

Identification of genes that play important roles in maintaining the corneal epithelium remains critical for understanding and treating many devastating corneal diseases. Molecular biology techniques like DNA chip technology, macro arrays and real time PCR will greatly facilitate this analysis.[17] With regard to surgical therapies, corneal epithelial cells from the limbus have been used as a source of in vivo epithelial transplantation and can be cultured on amniotic membrane and then transferred to the surface of a diseased or injured cornea in order to treat devastating ocular surface diseases. This new technique has just begun at several institutes and the short-term results are exciting.[74],[75] It seems likely that the transplantable epithelial sheet modified in conjunction with in vitro gene transfection may hold the potential to cure someof the corneal dystrophies.

 
  References Top

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    Figures

  [Figure - 1], [Figure - 2]
 
 
    Tables

  [Table - 1], [Table - 2], [Table - 3]


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