Adhesion of migrating corneal epithelial cells to a fibronectin matrix is mediated by integrins, which constitute a family of cell surface receptor proteins. To date, this family has been shown to include 24 different α subunits and nine different β subunits, with the selective combination of these α and β subunits determining the specificity of binding to ECM proteins. The integrin subunits α2, α3, α5, α6, αv, β1, β4, and β5 have been detected in the human cornea.^[136] The binding of integrins α5β1, αvβl, and αIIβ3 to fibronectin is mediated by the RGD sequence. The appearance and disappearance of the integrin β1 chain and fibronectin during corneal epithelial wound healing are well coordinated (Fig. 1.13).^[83] Immediately after an incision to the rat cornea, fibronectin was detected on the surface of the V-shaped defect in the stroma. Epithelial cells expressing the integrin β1 subunit then began to migrate over and to fill in the defect. With the exception of that in basal cells, expression of the integrin β1 chain in epithelial cells was down-regulated coincident with the completion of wound healing. The abundance of fibronectin at the interface between the new epithelium and the stroma also markedly decreased at this time.

The integrin α6β4 heterodimer is a component of hemidesmosomes and is not related to fibronectin-mediated cell adhesion and migration. In response to wounding of the corneal epithelium, hemidesmosomes in the basal cell layer are disassembled. They eventually reappear after migration of the remaining epithelium has resulted in a recovering of the denuded area.^[137]

Hyaluronan

Hyaluronan is also recognized as a biological signaling molecule and, like fibronectin, plays an important role in inflammation and wound healing.^[138] Hyaluronan is not present in the normal cornea. Unlike other glycosaminoglycans, a core protein for hyaluronan binding has not yet been identified. Hyaluronan is transiently expressed in the rabbit cornea during wound healing.^[139],[140] These observations suggest that hyaluronan plays a key role in the late stages of corneal wound healing.

Exogenous hyaluronan also increases the rate of corneal epithelial wound healing. The administration of hyaluronan eyedrops thus promotes corneal epithelial wound closure after epithelial debridement in rabbits^[141] and in diabetic rats.^[142],[143]

Proteolytic enzymes

Proteolytic enzymes also play an important role in wound healing. Cellular motility depends not only on the interaction of cells with the underlying ECM but also on the termination of such interaction by degradation of matrix proteins. Proteases, including plasminogen activator, have been detected in tear fluid.^[143–146] MMPs are also up-regulated in the migrating corneal epithelium.^[147]

Cytokines and growth factors

Epidermal growth factor

The roles of various cytokines and growth factors in the regulation of corneal epithelial migration have also been investigated.^[148] In general, these molecules modulate corneal epithelial wound healing by regulating the various healing-related systems described above.

Epidermal growth factor (EGF) was first isolated from the mouse submaxillary gland as a factor that stimulates eye opening and incisory tooth eruption in newborn mice.^[149] This 53-amino acid polypeptide is a potent stimulator of proliferation in a variety of cell types, including corneal epithelial cells.^[150],[151] It was also found to promote corneal epithelial wound closure in animals.^[152],[153]

The continuous exposure of the corneal epithelium to EGF present in tear fluid suggests that the stimulatory effect of this growth factor on epithelial cell proliferation must be counteracted if the normal thickness and function of the epithelium are to be maintained. In addition to its stimulatory effect on cell proliferation, EGF exerts a variety of other actions in corneal epithelial cells, including promotion of cell adhesion to a fibronectin matrix.^[154],[155]

Transforming growth factor-β

Corneal epithelial cells express transforming growth factor (TGF)-β1.^[156] The stimulatory effects of EGF on corneal epithelial cell proliferation, attachment to fibronectin, and migration are modulated by TGF-β.^[157],[158] Although TGF-β alone inhibits corneal epithelial cell proliferation, it has no effect on cell attachment to a fibronectin matrix in the absence of EGF. Endogenous TGF-β also promotes corneal epithelial cell migration.

Basic fibroblast growth factor and platelet-derived growth factor

Basic fibroblast growth factor (bFGF) is another polypeptide growth factor that stimulates the proliferation of various cell types of mesodermal or neuroectodermal origin.^[159] The application of recombinant human bFGF (200–500 ng, twice a day) was shown to accelerate corneal epithelial wound closure in rabbits.^[160] Rabbit corneal epithelial cells also express both the β and α type of receptors for platelet-derived growth factor (PDGF) at a density of 4.3 × 10⁴ and 2.8 × 10³/cell, respectively.^[161] Consistent with this observation, the BB and AB isoforms of PDGF increased the cytosolic concentration of free Ca²⁺ in these cells. Although Ca²⁺ is implicated as a second messenger in regulation of a variety of functions in many cell types, a role for this ion in corneal epithelial wound healing remains to be determined.

Interleukins

Interleukins are cytokines that regulate the function of the immune system, inflammation, and other reactions of tissue to external stimuli.^[162] They modulate the activities of immune or inflammatory cells both locally in tissue as well as systemically in the circulation and in bone marrow. Although 35 members of the interleukin family (IL-1 to IL-35) have been identified to date, the roles of many of these proteins in corneal wound healing remain to be investigated. For example, the corneal epithelium expresses IL-1, and exogenous IL-1 promotes the healing of corneal epithelial wounds.^[163] Corneal epithelial cells also express IL-6.^[164] Exposure of rabbit corneal epithelial cells in culture to IL-6 resulted in a marked increase in the number of cells that attached to a fibronectin matrix. IL-6 stimulates the expression of integrin α5β1 in corneal epithelial cells, suggesting that this cytokine may regulate corneal epithelial migration through modulation of the fibronectin–integrin system.^[53,165,166]

Neural regulation

The physiological role of corneal innervation in corneal epithelial wound healing remains to be fully clarified. The loss of corneal sensation, however, often results in breakdown of the normal integrity of the cornea. Persistent corneal epithelial defects or delayed epithelial wound healing are frequently observed in individuals with a reduced corneal sensation, such as those infected with herpes simplex or herpes zoster virus as well as those with diabetes mellitus. Abuse of topical anesthetics also impairs corneal epithelial migration in an organ culture system.^[167] Furthermore, frank corneal ulceration has been shown to develop in anesthetized eyes. These various observations thus implicate neural regulation in maintenance and repair of the corneal epithelium.

As discussed earlier, the cornea is densely innervated by sensory nerve fibers of trigeminal nerve origin which contain the sensory neurotransmitter substance P. Substance P is thought to regulate various physiological processes, including plasma extravasation, vasodilatation, and the release of histamine from mast cells.^[168–171] Exposure of rabbit corneal epithelial cells in culture to the combination of substance P and insulin-like growth factor-1 (IGF-1) resulted in a marked increase in the number of cells that attached to a fibronectin matrix.^[154] Substance P has also been implicated in neuronal responses to various stimuli in the eye as well as in other tissues.^[20,172,173]

Trigeminal denervation correlates with a reduction in the abundance of substance P in the cornea.^[174] Sectioning of the trigeminal nerve results in trophic or degenerative changes in the cornea as well as in the depletion of substance P.^[175] Substance P may thus contribute to maintenance of the normal integrity of the corneal epithelium. The combination of substance P and IGF-1 synergistically promotes corneal epithelial migration, with neither agent alone having an effect on this process. Furthermore, the administration of eyedrops containing IGF-1 and either substance P or a tetrapeptide derived from its carboxyl terminus has been shown to be an effective treatment for persistent corneal epithelial defects in individuals with neurotrophic keratopathy or diabetic neuropathy.^[176–178]

Nerve growth factor (NGF), first discovered by Levi-Montalcini in the early 1950s,^[179] is a polypeptide that stimulates the regeneration of peripheral nerve fibers.^[180] Furthermore, eyedrops containing NGF promote resurfacing of persistent corneal epithelial defects in animals and humans.^[181–185]

Stromal maintenance

Extracellular matrix and stromal repair

Structural and biochemical homeostasis of the ECM in the corneal stroma is thus maintained by a balance in the keratocyte regulation of the synthesis and degradation of ECM components. In response to corneal injury, keratocytes transdifferentiate into myofibroblasts and actively produce matrix components for healing of the injured stroma, with each newly expressed macromolecule appearing to play an important role in the repair process.